Plasmodium falciparum biology

Plasmodium falciparum
Blood smear with Plasmodium falciparum
Scientific classification
Domain:	Eukaryota
Kingdom:	Chromalveolata
Superphylum:	Alveolata
Phylum:	Apicomplexa
Class:	Aconoidasida
Order:	Haemosporida
Family:	Plasmodiidae
Genus:	Plasmodium
Species:	P. falciparum
Binomial name
Plasmodium falciparum Welch, 1897

Plasmodium falciparum has been the focus of much research due to it being the causative agent of malaria. This article describes some of the recent findings surrounding the unique biology of this organism.

Overview of life cycle

Plasmodium falciparum has a complicated life-cycle, requiring both a human and a mosquito host, and differentiating multiple times during its transmission/infection process.

Plasmodium life cycle

Genome

The genome of Plasmodium falciparum (clone 3D7) was fully sequenced in 2002. The parasite has a 23 megabase genome, divided into 14 chromosomes. The genome codes for approximately 5,300 genes. About 60% of the putative proteins have little or no similarity to proteins in other organisms and thus currently have no functional assignment. It is estimated 52.6% of the genome is a coding region, with 53.9% of the putative genes containing at least one intron.

Haploid/diploid

It is haploid during nearly all stages of its life-cycle, except for a brief period after fertilization when it is diploid from the ookinete to sporogenic stages within the mosquito gut.

AT richness

The P. falciparum genome has an AT content of roughly 80.6%. Within the intron and intergenic regions, this AT composition rises to roughly 90%. The putative exons contain an AT content of 76.3%. The parasite's AT content is very high in comparison to other organisms. For example, the genomes of Saccharomyces cerevisiae and Arabidopsis thaliana are considered AT rich but have AT contents of 62% and 65%, respectively.

Promoters

Although promoters are present in the genome, very little is known about them.

Recombination

The overall recombination rate is 9.6 kilobase per centimorgan and 54 candidate recombination hotspots have been identified. The centromeres are found in chromosome regions largely devoid of recombination activity like other organisms. Within the hotspots a number of motifs were enriched including a 12 base pair G/C-rich motif with 3 base pair periodicity that may interact with a protein containing 11 predicted zinc finger arrays.

Subtelomeric regions

Throughout the eukaryotic kingdom, the overall structure of chromosome ends is conserved and is characterized by the telomeric tract - a series of short G-rich repeats. This is succeeded by an extensive subtelomeric region consisting of various types and lengths of repeats — the telomere associated sequences (TAS). In general transcription of genes located next to telomeres is repressed, a phenomenon termed the telomere position effect. This effect is somewhat misnamed as it appears to be due to the sequences found in this region rather than the position of the gene.

Subtelomeric regions in general are low in gene density, low in transcription, low in recombination, late replicating, are involved in protecting the end from degradation and end-to-end fusions and in completing replication. The subtelomeric repeats can rescue chromosome ends when telomerase fails, buffer subtelomerically located genes against transcriptional silencing and protect the genome from deleterious rearrangements due to ectopic recombination. They may also be involved in fillers for increasing chromosome size to some minimum threshold level necessary for chromosome stability; act as barriers against transcriptional silencing; provide a location for the adaptive amplification of genes; and be involved in secondary mechanism of telomere maintenance via recombination when telomerase activity is absent. The repressive histone 3 lysine 9 tri-methylation mark and heterochromatin protein 1 are found throughout the TAS region and adjacent gene families on all chromosomes. These heterochromatic marks are important in telomere proximal gene silencing.

In parasitic species genes involved in antigenic variation are commonly located in these regions.

The chromsomes of P. falciparum conform to this basic pattern with the ends of the chromosomes consist of a stretch of telomeric GGGTT(T/C)A repeats with an average size of 1.2 kilobases (kb). This is followed by an extensive 20 to 40 kb TAS domain. These show a high degree of conservation within the genome and contain significant amounts of repeated structure. Telomere repeats are followed by a mosaic of six distinct telomere associated repetitive elements (TAREs 1-6) which are always found in the same order but vary in size.

Many genes involved in antigenic variation are located in the subtelomeric regions of the chromosomes. These are divided into the var, rif, and stevor families. Within the genome, there are 59 var, 149 rif, and 28 stevor genes along with multiple pseudogenes and truncations.

The chromosome ends form clusters of 4–7 telomeres that localize around the nuclear periphery. Within this location the telomeric areas undergo frequent recombination which seems to increases antigenic variation.

Transcriptome

Phaseogram of Plasmodium falciparum intraerythrocytic development cycle transcriptome

Transcription in P. falciparum appears to have significant differences from that found in most other eukaryotes examined to date with the chromatin undergoing dramatic unheavals during the cell cycle.

A transcriptome analysis has been conducted on the intraerythrocytic development cycle of P. falciparum. Roughly 60% of the genome is transcriptionally active during this portion of the parasite's life cycle. Whereas many genes appear to have stable mRNA levels throughout the cycle, many of the genes are transcriptionally regulated in a continuous cascade.

The transition from early trophozoite to trophozoite to schizont correlates with the ordered induction of genes related to transcription/translation machinery, metabolic synthesis, energy metabolism, DNA replication, protein degradation, plastid functions, merozoite invasion, and motility. Closely adjacent genes along the chromosome do not exhibit common transcription characteristics. Thus, genes are likely individually regulated along the parasite chromosome. Conversely, the apicoplast genome is polycistronic and most of its genes are coexpressed during the intraerythrocytic development cycle.

Introns

The intron splicing has been examined experimentally. The 5' and 3' splice sites agree with the canonical sequences (GT and AG respectively). The 5' consensus motif is weakly conserved and tolerates nucleotide substitution including the fifth nucleotide in the intron. This fifth position, typically a G nucleotide in most eukaryotes, is frequently an A in P. falciparum. The 3' splice site has a strong eukaryotic consensus sequence and a conserved adjacent polypyrimidine tract. The branch point is less well conserved with multiple branch points per intron with some at U instead of the typical A residue. A weak branch point consensus has been identified.

Proteome

There are 5,268 predicted proteins in Plasmodium falciparum and roughly 60% share little or no similarity to proteins in other organisms and thus are without functional assignment. Of the predicted proteins, 31% contain at least one transmembrane domain and 17.3% have a signal peptide or signal anchor.

It is estimated that 10.4% of the proteome is targeted to the apicoplast and 4.7% to the mitochondria.

The parasite has different subsets of its proteome expressed during various stages of its developmental cycle. In one study, of the 2,415 proteins were identified in four stages(sporozoite, merozoite, trophozoite, gametocyte), representing 46% of the theoretical number of proteins. Only 6% of the proteins were found in all of the four stages. Of the proteins found, 51% were annotated as hypothetical proteins.

Merozoites contained high levels of cell recognition and invasion proteins. Trophozoites contained proteins implicated in erythrocyte remodeling and hemoglobin digestion. Gametocytes contained high amounts of gametocyte-specific transcription factors and cell cycle/DNA processing proteins. The gametocytes had low levels of polymorphic surface antigens. Sporozoites contained large amounts of proteins related to invasion, as well as members of the var and rif families.

Translation initiation

This has been examined experimentally for the heat shock protein 86. Like other eukaryotes purines at the -3 and +4 positions are essential for efficient translation. Uracil at the -1 position resulted in 2.5-fold higher reporter activity compared to wild type.

Mosquito bite

P. falciparum is transmitted to humans by the females of the Anopheles species of mosquito. There are about 460 species of Anopheles mosquito, but only 68 transmit malaria. Anopheles gambiae, found in Africa, is one of the best malaria vectors. It is long-lived, prefers feeding on humans, and lives in areas near human habitation.

Prior to transmission, Plasmodium falciparum resides within the salivary gland of the mosquito. The parasite is in its sporozoite stage at this point. The Pumilio-FBF family member Puf2 appears to be critical for its survival in the mosquito salivary gland.

As the mosquito takes its blood meal, it injects a small amount of saliva into the skin wound. The saliva contains antihemostatic and anti-inflammatory enzymes that disrupt the clotting process and inhibit the pain reaction. Some of the details of this process are known. A salivary protein - anophelin - is a powerful thrombin inhibitor. This protein occupies thrombin's active site but is highly resistant to cleavage by the enzyme.

Typically, each infected bite contains 5-200 sporozoites which proceed to infect the human vector. Once in the human bloodstream, the sporozoites only circulate for a matter of minutes before infecting liver cells.

~1900 proteins have been reported from sporozoites infecting the salivary glands.

Liver stage

After circulating in the bloodstream, the P. falciparum sporozoites enter hepatocytes. Before entering a hepatocyte the sporozoite typically engages in traversal of several cells. The reason for this behavior is not clear but it appears to reduce clearance of the sporozoites by Kupffer cells.

Once within a hepatocyte the parasite loses its apical complex and surface coat and transforms into a trophozoite. Within the parasitophorous vacuole of the hepatocyte, P. falciparum undergoes schizogonic development. In this stage, the nucleus divides multiple times with a concomitant increase in cell size but without cell segmentation. This exoerythrocytic schizogony stage of P. falciparum has a minimum duration of roughly 5.5 days. After segmentation, the parasite cells differentiate into merozoites.

Invasion of the hepatocytes appears to involve at least 2 proteins: sporozoite invasion-associated proteins (SIAP)-1 and -2. These proteins bind heparin sulfate and chondroitin sulfate type membrane receptors on host cells.

Productive invasion of the hepatocyte results in the creation of a digestive vacuole, while merely passing through to reach another hepatocyte does not. Invasion of the liver cell changes the properties of the cell itself. The cell membrane becomes rougher and the cell itself becomes significantly stiffer. The mechanism of these changes is currently unknown.

During this stage of development the sporozoite selectively discards organelles unnecessary for growth at this stage of the life cycle. Among these are the micronemes and the inner membrane complex.

The division of the liver stages into thousands of merozoites is a complex process. In parallel with nuclear division, the apicoplast and mitochondrion become two extensively branched and intertwining structures. The organelles subsequently undergo morphological and positional changes prior to cell division. Finally to form merozoites, the parasite undergoes cytokinesis.

After maturation, the merozoites are released from the hepatocytes and enter the erythrocytic portion of their life-cycle. Note that these cells do not reinfect hepatocytes.

Molecular biology of liver stages

Plasmodium possess only a single pyruvate dehydrogenase enzyme (PDH) complex. This is localized to the plastid-like organelle known as the apicoplast. Unlike most eukaryotes, Plasmodium lacks a mitochondrial PDH. The PDH catalyzes the conversion of pyruvate to acetyl-CoA, an important precursor for the tricarboxylic acid cycle and type II fatty acid synthesis.

The process of maturation within the liver is still being investigated. In the species Plasmodium yoelli the exit from the liver appears to be dependent on type II fatty acid synthesis. Deletions in either the pyruvate dehydrogenase E1 alpha and E3 subunits produce a phenotype similar to that found in mutants of the type II fatty acid synthesis pathway. These mutants appear normal in blood stage development, mosquito stage development and early liver stage development but fail to exit the liver cells.

Plasmodium is unable to synthesize sterols they must obtain these from the host. However manipulation of cholesterol metabolism does not impede the development of the merozoites.

Invasion of the hepatocyte induces the production of CD81 - a member of the tetraspanin superfamily. CD81 also appears to play a role in liver invasion by Plasmodium species. It is required for Plasmodium vivax sporozoite entry into human hepatocytes and for Plasmodium yoelii sporozoite entry into murine hepatocytes.

The protein UIS3 is an essential protein for liver stage development. It is thought to be localised to the membrane of the parasitophorous vacuole of the infected cell.

Latency of sporozoites is controlled by the eIF2 alpha kinase IK2, a general inhibitor of protein synthesis. Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. In contrast Puf1 appears to be dispensable.

The RNA binding protein family PUF member Pumilio-2 (Puf2) appears to be involved in transformation of sporozoites into the hepatic stage of the life cycle. Knock out mutants of this gene exhibit genome wide transcriptional changes resulting in loss of gliding motility, cell traversal ability, reduction in infectivity and trigger metamorphosis typical of early Plasmodium intra-hepatic development.

Type II fatty acid biosynthesis is vital for this stage in the life cycle. This pathway may be inhibited by the antibiotic triclosan.

The host iron regulatory hormone hepcidin which is synthesised in the liver and spleen, appears to be able to inhibit growth of the liver stages. Levels of this hormone are elevated during infection and seem to correlate with the anaemia often found in malaria. Erythrocytic parasitaemia, above a minimum threshold, impairs the growth of subsequent liver cell sporozoite infection. The production of hepcidin leads to the redistributes iron away from hepatocytes thus slowing the development of the iron dependent liver stage.

Liver hepcidin expression is upregulated and downregulated during the early and late stages of malaria infection respectively. Inflammation and erythropoietin, rather than the iron sensing pathway, are involved in the regulation of hepcidin expression. Treatment of malaria infected mice with anti hepcidin neutralizing antibodies increased parasitemia and mortality rates. Overexpression of hepcidin improves the outcome.

Lipocalin 2, a host protein that sequesters iron, is upregulated during infection and appears to be involved in the host response. This protein increases both host macrophage function and granulocyte recruitment and decreases reticulocytosis.

Expression of the iron sequestering protein ferritin (ferritin H chain in mice) is associated with decreased tissue damage. The mechanism appears to be via prevention of activation of the proapoptotic c-Jun N-terminal kinase.

Invasion of the hepatocyte seems to require the RON4 protease.

Within the liver actin reorganization is a dynamic process in part controlled by the actin severing and capping protein - gelsolin. The hepatocyte cytoskeleton may contribute to parasite elimination.

Within the genome is encoded a homolog of macrophage migration inhibitory factor. This gene appears to be important for parasite development in the liver.

In Plasmodium bergei a protein - liver specific protein 2 (LISP2) - is expressed 24 hours after infection and rapidly increases during the liver stage schizogony. LISP2 is carried first to the parasitophorous vacuole and subsequently transported to the cytoplasm and nucleus of host hepatocytes. Mutations in this gene result in arrested development of the merozoites.

Two other proteins (p52 and p36)in Plasmodium bergei appear to be important in the formation of the parasitophorous vacuole membrane in the liver.

Infection of the liver induces apotosis in some of the liver cells. Blocking this apototic response seems to increase the number of parasites in the liver suggesting that this may be a host defense mechanism.

Erythrocytic stage

File:IEcycle.PNG

Plasmodium erythrocytic cycle

Effects on erythrocyte

Infection of the erythrocyte induces a series of changes in the host cell's membrane. These changes depend on the stage of infection and are due to a subset of parasite derived proteins that are exported across the parasic vaculole membrane into the host cell's cytosol where they interact with the host cell cytoskeleton or are exposed at the erythrocyte surface.

Among these changes are a loss of deformability, an increase in rigidity and a novel propensity to adhere to vascular endothelial cells and unparasitized erythrocytes. Several proteins including knob-associated histidine rich protein, Plasmodium falciparum erythrocyte membrane protein 3, mature parasite infected erythrocyte surface antigen and ring parasite infected erythrocyte surface antigen are known to bind to the cytoskeleton and increase the erythrocyte's rigidity. Others including the Plasmodium falciparum erythrocyte membrane protein 1 - the product of the var gene - are located on the surface of the erythrocyte and cause and are responsible for its new tendency to adhere.

Another induced change is an alteration in the zeta potential - an electrochemical property of cell surfaces that is determined by the net electrical charge of molecules exposed at the surface of cell membranes - of the cell. The normal zeta potential of the erythrocyte is -15.7 millivolts (mV). Much of this potential appears to be contributed by the exposed sialic acid residues in the membrane: their removal results in zeta potential of -6.06 mV. Infection of the erythrocyte decreases the mean to -14.6 mV at the trophozoite stage. Again removal of sialic acid in the infected cells increased the potential to -4.64 mV.

A further change is the appearance of knobs - localised swellings of the erythrocyte membrane. The number of knobs, their diameter and height vary between isolates. They are thought to be important in the pathogenesis by contributing to blockage of capillaries. A number of parasite proteins are associated with these knobs including erythrocyte membrane protein 1 (the var gene product) and knob-associated histadine rich protein. Knobs first appear ∼20 hour post invasion and increase in number to ∼35 hours post-invasion. They are more common in ex vivo isolates than in culture maintained strains.

Singlet oxygen is generated during the parasite's life cycle. Its production is least in the ring stages, maximal in the schizonts and intermediate in the trophozoites. Its production may be related to the formation of haemozoin.

In normal erythrocytes the proteins ankyrin, band 3, band 4.1, glycophorin and spectrin are phosphorylated. After invasion by the merozoite these proteins become dephosphorylated. As the parasite matures selective phosphorylation of ankyrin, band 3 and spectrin occurs.

Tyrosine phosphorylation of band 3 at the ring stage appears to be under the control of Syk kinase. Phosphorylation of additional cytoskeletal, trans-membrane and membrane associated proteins occurs as the parasite matures. These include actin, adducins, band 4.2 and catalase. During the late schizont stage widespread protein dephosphorylation occurs. The erythrocyte kinases may be involved in this process.

Aggregation of the erythrocytes is known to occur during infection. This effect can be caused by culture supernatant suggesting a soluble product is responsible. Part of this mechanism appears to be the externalization of phosphatidyl-serine residues in the erythrocyte membrane. Methaemoglobin has been identified as the main causative agent of this alteration. Its mechanism of action appears to be via the generation of reactive oxygen species: this action may be reversed with the addition of antioxidants.

Metalloproteinase 9 is released from human microvascular endothelium after contact with infected erythrocytes.

Significant changes occur in the erthrocyte's cytoskeleton during infection. Among these are the accumulation of spectrin around the knobs and a decrease elsewhere.

The change in the shape of the erythrocyte induced by the parasite depends on the parasite species. Infection with P falciparum induces an increase in the cell volume of 80% in the ring stage. The erythrocyte subsequently becomes spherical in the trophozoite stage and remains so in the schizont stage. In contrast the volume increase induced by P vivax is only 30% and the erythrocyte remains biconcave throughout the infective cycle.

Merozoite

After release from the hepatocytes, the merozoites enter the bloodstream prior to infecting red blood cells. At this point, the merozoites are roughly 1.5 µm in length and 1 µm in diameter and use the apicomplexan invasion organelles (apical complex, pellicle and surface coat) to recognize and enter the host erythrocyte.

The apicoplast measures 0.5 µm × 0.15 µm in the merozoite and is anchored to a band of 2-3 subpellicular microtubules. Within the merozoite the mitochondrion and the apicoplast are aligned asymmetrically along the same side as the microtubules.

There are up to 40 micronemes per merozoite shaped like longnecked bottles. They are ~160 nanometers (nm) long and 65 nm at their widest diameter. On their external surfaces, they bear bristle like filaments, each 3-4 nm thick and 25 nm long. The micronemes are translocated from a single Golgi like cisterna near the nucleus along a band of two or three subpellicular microtubules to the merozoite apex where they dock with the rhoptry tips.

Unlike species in the genus Toxoplasma which have multiple rhopteries, Plasmodium species typically only have two. These may be referred to in the literature as the 'paired organ'. Several proteins have been localised to the rhopteries including asparagine rich parasite protein encoded on chromosome 4. Another is the Apical rhoptry neck protein whose expression is confined to the schizont stage.

After release from the erythrocyte the merozoites of the rodent malaria parasite Plasmodium yoelii change their shape from flat elongated ovals to spherical bodies. This process takes ~60 seconds. During this time the merozoites were able to attach to and deform the erythrocyte membrane but were not able to reorient and invade. This morphological change may be related to the secretion or activation of invasion related proteins.

The parasite first binds to the erythrocyte in a random orientation. It then reorients such that the apical complex is in proximity to the erythrocyte membrane. A tight junction is formed between the parasite and erythrocyte. As it enters the red blood cell, the parasite forms a parasitophorous vesicle, to allow for its development inside the erythrocyte.

Five separate invasion paths have been delineated. The most common pathway is neuraminidase resistant, trypsin sensitive and chymotrypsin resistant invasion. Some parasites have a neuraminidase- and trypsin-sensitive phenotype indicating a dependence on the erythrocyte binding antigen 175/glycophorin A pathway(s). Most isolates appear to be dependent on a trypsin sensitive pathway.

Erythrocyte invasion

This is a complex and poorly understood process. The merozoite initially contacts the erythrocyte and rotates until the rhoptery containing part is adjacent to the erythrocyte membrane. A tight contact is then established and the parasite enters the erythrocyte. This happens within seconds making the invasion process difficult to analyse. In Plasmodium yoelii the serine type serine repeat antigen (SERAs) are non essential for blood stage development of the parasite but appear to be an important factor in enabling the parasite to fully utilize the whole age repertoire of circulating erythrocytes.

In culture invasion of the erythrocytes can be prevent with the use of heparin. Heparin binds only at the apical tip of the merozoite surface and multiple heparin binding proteins are found preferentially in the apical organelles. The Duffy and reticulocyte binding-like families bind to heparin with diverse affinities suggesting that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

Another protein that is bound by heparin is the merozoite surface protein 1.

262 open reading frames show sharp induction of expression during late schizont stages. At least 28 of these are proteins that are known to be involved in the invasion process. The functions of over 190 of these remain unknown.

Initial adhesion

The rhopties are an ancient organelle within this group of protozoa while the micronemes appear to be a more recent development. The rhopteries are involved in cell invasion in other species in this clade. It would appear that the micronemal proteins have become adapted to recognise a cell potentially suitable for invasion and that the rhoptry proteins facilitate the invasion itself. It is likely that there is overlap of these functions particularly in the invasion step and that other proteins are also involved.

Exposure of the merozoites to a low potassium medium - at a concentration similar to that found in blood - induces a rise in the merozoite's cytosolic calcium concentration. The low potassium levels in the blood (3.5-5.0 millimoles per liter) activates a phospholipase C enzyme.

The calcium dependent protein kinase 1 appears to play a role in micronene discharge. The drug purfalcamine is a specific inhibitor of this kinase and it also inhibits micronene discharge and erythrocyte invasion. These kinases typically have an N terminal kinase domain and C terminal calmodulin like domain with calcium binding EF hands. The N and C terminals are joined by a junction domain. The C terminal appears to interact with the junction domain in the process of binding calcium.

Calcineurin a Ca²⁺ dependent protein phosphatase is also involved in this process. The dephosphorylation and depolymerization of apical actin also seems to be involved in this process.

Phospholipase C initiates a pathway that leads to a rise in the intracellular calcium. This rise in calcium triggers secretion of microneme proteins including the 175 kD erythrocyte binding antigen and apical membrane antigen 1 to the merozoite surface where they can bind to erythrocyte surface proteins including glycophorin A. The interaction of EBA175 with glycophorin A, its receptor on erythrocytes, restores basal cytosolic calcium levels. This in turn triggers the release of rhoptry proteins including the thrombospondin related apical merozoite protein (TRAMP).

GAP45 is phosphorylated in response to Phospholipase C and calcium signaling. It is phosphorylated by the P. falciparum kinases Protein kinase B and Calcium dependent protein kinase 1, both of which are calcium dependent enzymes, at Serine89, Serine103 and Serine149. Phosphorylation of these sites is differentially regulated during parasite development.

Two families of proteins are known to be involved in the invasion process: the reticulocyte binding like homologues (PfRh or PfRBP) and erythrocyte binding like (EBL) proteins. The EBL family are principally located in the micronemes and the Reticulocyte binding Homolog (PfRH) family are principally located in the rhopteries. Ligands from the EBL family largely govern the sialic acid dependent pathways of invasion and the RH family ligands (except for RH1) mediate sialic acid independent invasion. During the invasion process these ligands are localized at the apical tip of the merozoite and are able to bind erythrocytes.

Microneme proteins

All known micronemal proteins are type I integral membrane proteins that contain a C-terminal transmembrane domain and a short cytoplasmic domain.

The EBL family of proteins includes EBA-165 (also known as PEBL), EBA-175 (also known as PfF2), EBA-181 (also known as JESEBL), EBA-140 (also known as BAEBL) and EBL-1. Whilst these parasite ligands function in merozoite invasion by binding to specific receptors on the erythrocyte, they appear also to have a central role in activation of the invasion process. Binding of EBA-175 to its receptor, glycophorin A, restores the basal cytosolic calcium levels after interaction of the merozoite with the erythrocyte and triggers the release of rhoptry proteins.

These proteins have several domains. Region II which is responsible for ligand erythrocyte interaction during invasion, consists of two homologous F1 and F2 domains.

Several of the receptors for the proteins are known: glycophorin A for EBA-175, glycophorin B for EBL-1 and glycophorin C for EBA-140.

The ebl family share a common intron/exon structure suggesting a common origin. These proteins also share a common organisation into several regions. Of these regions II and VI are the cysteine-rich regions of the DBL-EBP extracellular domains that have numerous conserved cysteine and hydrophobic amino acid residues, suggesting a conserved, functionally important three-dimensional structure. Despite this there is little nucleotide identity between the proteins.

The EBL proteins have a Duffy like binding domain (DBL) unique to Plasmodium species. The crystal structure of the binding domain of EBA-175 has been solved. It consists of a dimer of the beta fingers of the F1 and F2 regions.

The crystal structure of EBA-140 has been solved. The two domain binding region is present as a monomer. Both domains are required for binding to occur. Its electrostatic surface has a basic patch spanning both DBL domains that is important in the binding mechanism.

EBA-175 mediates adhesion to erythrocytes through binding of the Duffy binding like domains in its extracellular domain to Neu5Acα2-3Galactose displayed on the O-linked glycans of glycophorin A..

A member of the EBL family of proteins (MAEBL) has been shown to be present in Plasmodium gallinaceum. This protein is now known to be conserved in the primate, rodent and avian infecting species suggesting that it may play an important role in erythrocyte invasion. The duplicate extracellular binding domains of MAEBL are responsible for erythrocyte binding. MAEBL is a type I transmembrane protein with a carboxyl cysteine rich region.

A GPI-anchored micronemal antigen (GAMA) also appears to be essential in the process of erythrocyte invasion.

A microneme associated antigen (PfMA: PF3D7_0316000, PFC0700c) binds erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner. This gene is expressed only in the late blood stages. It is a 307 amino acid protein (~ 37.1 kiloDalton) that contains an N-terminal stretch of hydrophobic residues, a C-terminal single transmembrane domain and a short cytoplasmic tail. It is moderately (~40%) conserved between several Plasmodium species.

Rhoptery proteins

The PfRh family consists of five proteins and a pseudogene: PfRh1, PfRh2a, PfRh2b, PfRh3, PfRh4 and PfRh5. PfRh3 is a transcribed pseudogene in all strains examined to date. All the other members of this family bind to erythrocyes and antibodies to them inhibit invasion.

The Pfh1 protein binds a sialic acid containing erythrocyte receptor.

PfRh1 and PfRh2 are located in the neck of the rhopteries.

The genes PfRh2a and PfRh2b encode large proteins of about 3200 amino acids in length. They differ only in the last 500 amino acids of the C terminal and have clearly arisen by a process of gene duplication and mutation. The 500 amino acid region includes an ectodomain, a transmembrane domain and a cytoplasmic domain. PfRh2b is essential for a well-defined invasion pathway while PfRh2a is not required or sufficient for this pathway. It has been shown that the reason for this difference lies in the cytoplasmic domain.

Reticulocyte binding like protein homologue 2a (PfRH2a) is processed both in the schizont as well as during invasion resulting in proteins with different erythrocyte binding properties. It also moves from the rhoptry neck to the moving junction during merozoite invasion. PfRh2a undergoes a cleavage event in the transmembrane region during invasion consistent with activity of the membrane associated PfROM4 protease. Both PfRh2a and PfRh2b bind to red blood cells. The erythrocyte-binding domain lies within a 15 kDa region at the N-terminus of each protein.

PfRh2b appears to play an important - if not dominant - role in the binding and invasion process.

PfRh4 binds to the complement receptor 1 (CR1; CD35). Complement receptor 1 is a ∼190- to 280-kDa single-chain transmembrane glycoprotein and carries the Knops blood group antigen. The binding site lies within the three N-terminal complement control protein modules (CCPs 1-3) of CR1. This region also accommodate binding and regulatory sites for the key complement activation specific proteolytic products, C3b and C4b. The binding of Rh4 to CR1 does not inhibit the binding of C3b/C4b but it does inhibit their dissociation from the erythrocyte. The critical site for the binding of Rh4 appears to lie within the CCP-1 module.

PfRh4 is responsible for the majority (50–80%, depending on the parasite strain used) of the sialic acid independent invasion pathway.

PfRh4 binds to a second protein P. falciparum Rh5 interacting protein (PfRipr). PfRipr has a molecular weight of 123 kiloDaltons with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein.

PfRh5 is located within the rhoptries and appears to be an essential gene.

The receptor for the PfRh5 protein appears to be the Ok blood group antigen, basigin. Blocking access to this protein on the erythrocyte surface appears to inhibit erythrocyte invasion completely. Binding of the Rh5 protein appears to be critically dependent on a single residue within the Rh5 protein. Antibodies to this protein inhibit the parasite's growth in vitro and appear to be correlated with protection against infection with it.

There seems to be some overlap between the functions of these proteins. Loss of EBA-175 can be compensated by increased expression of PfRh4.

The rhoptry associated, leucine zipper-like protein 1 (RALP1) is specifically expressed in schizont stages and localized to the rhoptry of merozoites. It appears to be an essential gene. It translocates from the rhoptry neck to the moving junction during merozoite invasion. Anti-RALP1 antibodies disrupt the tight junction formation. There is an erythrocyte binding domain in the C terminal.

Another protein localised to the apical ends of the rhoptries is an asparagine rich parasite protein (PfAARP;PFD1105w). This protein has a predicted signal sequence, a C-terminal transmembrane region and its transcription and translation patterns are similar to other merozoite surface proteins.

The rhoptry neck protein PfRON4 - a homologue of Toxoplasma gondii rhoptry neck protein TgRON4 - forms a complex with the protein PfAMA1 during its secretion in the course of merozoite invasion.

Other rhoptery proteins include the rhoptry associated protein 1 (RAP1) and rhoptry associated membrane antigen (RAMA).

The RhopH complex proteins localize to the basal bulb of the rhoptries and are involved in erythrocyte binding and in establishment of parasitophorous vacuole.

Apical membrane antigen 1 (AMA-1) is a type I transmembrane protein located in the neck of the malaria merozoite rhoptries and later on the surface of the invasive merozoite. Its ectodomain is defined by three cysteine-rich domains characterised by disulfide bond patterns.

Merozoite surface protein family.

Merozoite surface protein 1 (MSP-1; P195; PMMSA; MSA 1) is a protein found on the surface of the merozoites. During invasion of the new red cell most of the MSP1 molecule is shed from the parasite surface except for a small C-terminal fragment which can be detected in ring stages. Within this fragment are two epidermal growth factor-like domains. This protein has been found in all Plasmodium species studied to date suggesting it has an important role in the life cycle.

It appears that the merozoite surface protein 1 (MSP1) binds to heparin like molecules on the surface of the erythrocyte and that is binding is an essential step in the invasion process.

Merozoite surface protein 2 is one of the most abundant proteins on the surface of the merozoite and plays a role in the invasion process. Mediated by hdrophobic residues in the N terminal 1-25 residues it forms aggregates in vitro and may be present on the merozoite as aggregates.

In P. vivax there are a family (10) of merozoite surface protein 3 termed MSP3α to MSP3λ arranged in a head to tail fashion on chromosome 10. These proteins have a predominant central alanine rich domain containing heptad repeats predicted to form α-helical secondary and coiled-coil tertiary structures. They lack transmembrane domain or GPI-lipid modification site.

Merozoite surface protein 8 is a single open reading frame of 1791 base pairs which encodes a polypeptide of 597 amino acids. At the N-terminus there is a secretory signal peptide while at the and C-termini there is a GPI attachment sites. There are two EGF-like domains located near the C-terminus.

Merozoite surface protein 9 is conserved between species and appears to be under purifing selection.

Merozoite surface proteins 8 and 10 which are thought to be involved in the invasion process appear to be under purifying selection.

Other proteins

Another family of proteins involved in the invasion process are the thrombospondin related anonymous protein (TRAP) family. These proteins are type I cell surface proteins with one or more extracellular thrombospondin type-I repeats (TSR) domains and/or von Willebrand factor like (vWF) A domain(s) and an acidic cytoplasmic tail with a subterminal tryptophan residue. The cytoplasmic tails of TRAP, CTRP, TLP and MTRAP interact with the enzyme aldolase.

One protein thought to be involved in the invasion process is the merozoite specific thrombospondin related anonymous protein homolog (MTRAP). The receptor for this protein has been identified as the GPI-linked protein semaphorin-7A (CD108). The MTRAP monomers interact via their tandem TSR domains with the Sema domains of a Semaphorin-7A homodimer.

The motile forms have their own stage specific cell surface TRAP family member: TRAP and S6 (also known as TREP) occur on the sporozoites; CTRP is found on the ookinetes; MTRAP is expressed in the merozoites; and TLP is present on both sporozoites and merozoites. Other members of this family are the proteins CSP, SPATR, TRSP, WARP and PTRAMP. Roles for several of these proteins has been discovered: TRAP is critical for sporozoite invasion of the mosquito salivary glands, infection of mammalian liver and sporozoite gliding motility; CTRP is required for invasion of the mosquito midgut; and S6 is important for both sporozoite gliding motility and invasion of mosquito salivary glands. TLP has a role in sporozoite cell traversal. The cytoplasmic tail of TRAP is essential for gliding motility and invasion of the mosquito's salivary glands. Both the TSR and A domains of TRAP are required for the invasion of the mosquito salivary glands. Penetration of the mammalian hepatocytes however requires the TSR, the A domain and the cytoplasmic tail. In contrast only the A domains of CTRP are essential for infectivity by the ookinete.

PfTRAMP is localised to the base of the rhopteries.

The protein PfTCTP causes the release of histamine in the host. This protein also activates the basophils.

The cysteine rich protective antigen appears to play a role in this process.

The ribosomal phosphoprotein P0 also seems to be involved in the invasion process.

A double C2 domain (DOC2) protein appears to be involved in the invasion of the erythrocyte. DOC2 proteins recruit the membrane fusion machinery an essential part of the Ca²⁺-dependent exocytosis mechanism. These proteins have a Munc13-interacting domain and tandem C2s (designated C2A and C2B) which are connected by a short polar linker. The C2 domains bind phospholipids in a Ca²⁺-dependent manner. Elucidating their precise role in erythrocyte invasion requires further work.

In Plasmodium vivax a number of tryptophan rich antigens are involved in erythrocyte invasion. Homologs of these proteins are found in P. falciparum - tryptophan-threonine rich antigen (PfTryThrA) and merozoite associated tryptophan rich antigen (PfMaTrA) and Plasmodium yoelii. These proteins also seem to be involved in the invasion process.

In P. vivax the asparagine rich protein has been cloned. It is a 281-residue-long molecule, which is encoded by a single exon and has an N-terminal secretion signal in addition to a tandem repeat region. This protein is expressed in mature schizonts and is located on the merozoite surface and appears to accumulate towards the apical pole.

Another protein that appears to be involved in the invasion process is PfDBLMSP (PF10_0348). This protein has a predicted signal sequence, a central Duffy binding-like (DBL) domain and a secreted polymorphic antigen associated with merozoites (SPAM) domain in its C-terminal half. The transcription and translation of this gene is up regulated specifically in schizonts, similar to other merozoite proteins involved in invasion of erythrocytes. This protein seems to be under selection pressure.

Invasion

The process of invasion is partly understood. The merozoite proteins forms a tight junction (the moving junction complex) with some of the erythrocyte membrane proteins. The attached merozoite proteins are then moved posteriorly by an actin-myosin motor. The net effect of this process is to drive the merozoite into erythrocyte.

Some details of the invasion process are known. The rhoptery protein RON2 is inserted into the erythrocyte membrane. The protein AMA1 secreted from microneme then binds to RON2. RON2 forms part of a macromolecular complex which includes RON2, RON 4, RON5 and RON8. The protein PfRON2 via its C-terminal as well as its central cysteine rich domain interacts with PfAMA1.

The membrane proximal domain of the AMA1 protein is responsible for direct binding to erythrocytes.

The invasion process appears to be ATP dependent and may involve a purogenic signalling pathway.

A protein that appears to be unique to the genus Plasmodium - RON12 has been described. RON12 lacks membrane anchors and is a major soluble component of the nascent parasitophorous vacuole. Most of the secretion of RON12 occurs late during invasion (after parasite internalisation) thus allowing accumulation in the fully formed parasitophorous vacuole. A small proportion of RON12 appears to be present in the moving junction. RON12 does not appear to be essential but its deletion reduces parasite proliferation.

The motor behind the invasion process is an actinomyosin motor complex that is assembled below the parasite's plasma membrane. This complex includes myosin, myosin tail domain interacting protein and glideosome associated proteins 45 and 50. It is anchored on the inner membrane complex which underlies the cell membrane. Myosin, myosin tail domain interacting protein and GAP45 first form a complex that then associates with GAP50. GAP45 is phosphorylated by calcium dependent protein kinase 1 on a number of serine residues. Removal of these residues does not appear to affect the assembly of this complex. This complex may have other function in addition to its role erythrocyte invasion.

The invasion process requires a coupling of the actin-myosin motor to the surface receptors. The myosin molecule involved belongs to the single-headed class XIV myosin. For the thromobospondin related anonymous protein on the sporozoites, aldolase which can bind actin forms this connection. This connection requires tryptophan and negatively charged amino acids in the ligand's cytoplasmic tail. PfRH2b also binds aldolase with its cytoplasmic tail. This binning requires an aromatic amino acid (phenylalanine or tyrosine) rather than tryptophan again also in the context of negatively charged amino acids. PfLRH2a does not bind aldolase. A second protein glyceraldehyde-3-phosphate dehydrogenase can also bind actin. It is capable of biding the cytoplasmic tails of some of the PfRh and Duffy binding ligands in an aromatic amino acid dependent manner.

Trophozoite

After invading the erythrocyte, the parasite loses its specific invasion organelles (apical complex and surface coat) and de-differentiates into a round trophozoite located within a parasitophorous vacuole in the red blood cell cytoplasm. The young trophozoite (or "ring" stage, because of its morphology on stained blood films) grows substantially before undergoing schizogonic division.

During asexual development the parasite increases in size to ~50% of the uninfected erythrocyte volume: the infected erythrocyte volume remains relatively constant. Haemoglobin content gradually decreases but its concentration remains constant until the early trophozoite stage when it decreases by 25%. It then remains constant again until just prior to rupture. During early sexual development the gametocyte has a similar morphology to a trophozoite but subsequently undergoes a dramatic shape change.

The parasite's presence within the erythrocyte induces changes in the properties of the host cell. Relative membrane deformability is less than 10% of uninfected erythrocytes. This change may contribute to the capillary occlusions that occurs in this disease. The deformability of the membrane is also dependent on the temperature and decreases with increased temperature. Deformability is reduced by a factor of 3-4 between 37 and 41 degrees Celsius. The fever that is commonly found in malaria may also contribute via this mechanism to capillary occlusion. The stiffness of the erythrocyte membrane increases as the parasite matures. The overall effect of these chances are to transform the erythrocyte from its normal biconcave shape into a parachute like structure. This change is apparent at high pressure rather than at low. Transition occurs at flow rates of ~65 µm per second. The mechanism of these changes are not known but changes in ATP consumption or alterations to the erythrocytes' spectrin framework may be important.

Within the red blood cell, the parasite metabolism depends greatly on the digestion of hemoglobin. A set of enzymes known as plasmepsins which are aspartic acid proteases are used to degrade hemoglobin. The parasite digests 70-80% of the erythrocyte's haemoglobin but utilizes only ~15% in de novo protein synthesis. Intraerythrocytic Plasmodium falciparum utilizes only a fraction of the amino acids derived from the digestion of host cell cytosol for the biosynthesis of its proteins. The excess amino acids are exported from the infected erythrocyte by new transport pathways created by the parasite. The reason proposed for this apparently excessive digestion of haemoglobin is the colloid-osmotic hypothesis which suggests that the digestion of haemoglobin increases the osmotic pressure within the infected erythrocyte leading to its premature rupture and subsequent death of the parasite. To avoid this fate much of the haemoglobin is digested and exported from the erythrocyte. This hypothesis has been experimentally confirmed.

Infected erythrocytes are often sequestered in various human tissues or organs, such as the heart, liver and brain. This is caused by parasite derived cell surface proteins being present on the red blood cell membrane and it is these proteins that bind to receptors on human cells. Sequestration in the brain causes cerebral malaria, a very severe form of the disease, which increases the victim's likelihood of death.

The parasite can also alter the morphology of the red blood cell causing knobs on the erythrocyte membrane.

Erythrocyte invasion and growth leads to activation of several distinct anion channels and a non-selective Ca2+-permeable cation channel. The non-selective cation channel's activation allows entry of Ca2+ and Na+. Absence of the channels is incompatible with pathogen survival. Although the mechanism of activation of these channels is not know it is presumed to be due to oxidation stree generated by the parasite because similar or identical channels are activated by oxidation of non-infected erythrocytes. Ca2+ entry stimulates an intraerythrocytic scramblase that facilitates bi-directional phospholipid migration across the bilayer. This results in an alternation of the cell membrane's phosphatidylserine asymmetry. Exposure of phosphatidylserine at the outer surface of the cell membrane is followed by binding to phosphatidylserine receptors on macrophages and the subsequent phagocytosis of the affected erythrocyte. It appears that the parasite because of its growth requirements is in a race to complete its life cycle before the infected erythrocyte is phagocytosed.

Schizont

At the schizont stage, the parasite replicates its DNA multiple times without cellular segmentation. These schizonts then undergo cellular segmentation and differentiation to form roughly 16-18 merozoite cells in the erythrocyte. The rhoptries are formed mainly between second and fourth nuclear divisions; the micronemes between the end of the fourth nuclear division and merozoite separation from the residual body. The dense granules are formed mainly after the micronemes.

The merozoites burst from the red blood cell, and proceed to infect other erythrocytes. The parasite is in the bloodstream for roughly 60 seconds before it has entered another erythrocyte.

This infection cycle occurs in a highly synchronous fashion, with roughly all of the parasites throughout the blood in the same stage of development. This precise clocking mechanism has been shown to be dependent on the human host's own circadian rhythm. Specifically, human body temperature changes as a result of the circadian rhythm, seem to play a role in the development of P. falciparum within the erythrocytic stage.

The synchronicity of the erythrocytic cycle is at least in part dependent on melatonin secretion by the host. A mechanism for this has been proposed. Melatonin can activate phospholipase C which acts to generate inositol trisphosphate (IP3) which opens IP3 sensitive calcium channels in the endoplasmic reticulum. The released calcium in its turn controls the cycle through mechanisms that have yet to be understood.

Gametocyte differentiation

During the erythrocytic stage, some merozoites develop into male and female gametocytes - a process is called gametocytogenesis. The specific factors and causes underlying this sexual differentiation are largely unknown. The base rate appears to be ~10% in laboratory strains but this is subject to many influences. One of the earliest suggestions in this area was made by Sinton who proposed that 'stress' might influence the rate of gametogensis. There is some evidence from other workers to support this hypothesis. Factors that have been reported to influence this rate include host erythrocyte age, hypoxia and exposure to schizonticidal drugs.

Gametocytes take roughly 8–10 days to reach full maturity and are metabolically active. The gametocytes remain within the erythrocytes until taken up by the mosquito host. The osmiophilic bodies, present in both male and female gametocytes but more abundant in the latter, are involved in the parasites' escape from the red blood cell during gametogenesis.

There is considerable variation in the ratio of female to male gametocytes between strains. Typically there are four female gametocytes to each male gametocyte. Mathematical modelling suggests that this may be due to differences in male fecundity.

The protein Pfg377 has been shown to be localised to the osmophilic bodies of the gametocytes.

The development of gametocytes is associated with down regulation of the erythrocyte membrane protein 1 (the product of the var genes) and loss of cytoadhesion.

The CPW-WPC protein family, named after the unique WxC repeat domains, is highly conserved among Plasmodium species. It is transcribed in gametocytes and CPW-WPC proteins are expressed in zygote/ookinete-stage parasites. These do not appear to be essential genes.

Sexual differentiation

Sexual parasite development is controlled by a DEAD box RNA helicase of the DDX6 family, termed DOZI.

The Puf2 gene, a member of the Puf family of transcriptional regulators, has been shown to be involved in gamete formation.

FACT (facilitates chromatin transcription) is a dimeric complex of two proteins - SPT16 and SSRP1 - which acts as a histone chaperone in the (dis)assembly of nucleosome (and chromatin) structure during transcription and DNA replication. It is an essential gene in Plasmodium. Changing its promoter to one expressed only in the blood stages leads to changes in the male gametocytes. The mutant gametocytes have delayed DNA replication and gametocyte formation. Male gamete fertility is strongly reduced. Female gametocytes appear to be normal. When successful fertilization is achieved, the ookinetes generate oocysts that arrest early in development and fail to enter sporogony.

The proteins cell division cycle protein 20 and its homologue, CDC20 homologue 1 are central to the cell cycle activating the anaphase-promoting complex/cyclosome (APC/C) in mitosis and facilitating degradation of mitotic APC/C substrates. A single homolog of this gene has been identified in Plasmodium berghei. It appears to be essential in male gametogensis but not for asexual reproduction. Blockage occurs at the nuclear spindle/kinetochore stage.

A gametocyte development 1 gene (Gdv1) which encodes a perinuclear protein has been identified. Its mechanism of action is not known. Homologues of this gene have been found in Plasmodium vivax, Plasmodium knowlesi and Plasmodium gallinaceum.

Egress from the erythrocyte

This is an essential step in the life cycle. The calcium dependent protein kinase PfCDPK5 which is expressed in the merozoite is essential for this process. Deletion mutations of this gene result in cell arrest in the late schizont stages. Merozoites released from these schizonts are capable of invasion.

The mechanics of release from the host membrane are partly known. Before release a pore opens in the membrane. A distortion of the usual arrengement of the lipids and proteins of the host cell membrane occurs around the pore. This area of altered protein and lipid eventually ruptures.

Large holes appear in the cytoskeleton ~35 hours post invasion. This occurs at the same time as the loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/β-adducin and tropomyosin. This is followed by the proteolysis of many cytoskeletal proteins during egress at ~48 hours post infection. This later proteolysis is mediated by the erythrocyte's own calpain-1.

Infected erythrocytes release microvesicles from their surface. They contain a number of parasite antigens associated with host cell membranes and proteins involved in parasite invasion which have potent immunomodulatory properties affecting macrophages and neutrophils. Their uptake by infected eythrocytes stimulates the production of transmission stage parasites in a dose dependent manner. Their release increases during the asexual parasite cycle particularly prior to parasite egress. A protein - PTP2 - appears to be essential in this process but its role is not yet clear.

A Gα(q) coupled signaling pathway that results in protein kinase C mediated loss of the host cytoskeletal protein adducin and weakening of the cellular cytoskeleton has also been implicated in the egress mechanism. This weaking of the cytoskeletal induces catastrophic Ca²⁺ influx mediated by the mechanosensitive cation channel TRPC6. This in turn which activates host cell's calpain which proteolyzes the host cytoskeleton allowing parasite release.

Along with the release from the erythrocyte of the merozoites, the now functionless digestive vacuole is also released. These can active complement and are rapidly taken up by the polymorphs. On ingestion the digestive vacuoles induce a vigorous respiratory burst which drives the cells into a state of functional exhaustion, blunting production of reactive oxygen species and microbicidal activity upon challenge with bacterial pathogens.

The serine repeat antigen (SERA) multigene family encode a series of proteins with a putative papain-like cysteine protease motif. One of these SERA5 (120 kiloDaltons) is produced at the late trophozoite/schizont stage. It is secreted together with other SERAs into the parasitophorous vacuole in an infected erythrocyte where it is cleaved into three fragments: an N-terminal domain (47 kDa), a central domain containing putative papain-like cysteine protease motifs (56 kDa) and a C-terminal domain (18 kDa). This N-terminal fragment is then cleaved in turn into two 25 kDa fragments. These fragments become covalently linked to the C-terminal 18 kDa fragment via disulfide bonding and attach to the merozoite surface. The central fragment is further cleaved to 50 kDa and 6 kDa fragments before being shed to the medium. These proteolytic cleavages are carried out by a subtilisin-like serine protease called PfSUB1 and the inhibition of this processing, likewise, results in blockade of merozoite release. SERA6 may also be involved in schizont rupture and merozoite release from the erythrocyte. Both SERA5 and SERA6 are essential for blood stage parasite viability. SERA6 is found in parasitophorous vacuole where it is activated by cleavage by the serine protease PfSUB1 just prior to egress. The release of PfSUB1 may be controlled by a calcium flux within the exomemes - storage vesicles within the parasite - of the merozoites. The release may be under the control of a phospholipase C.

PfSUB1 - which is encoded by the gene PF3D7_0507500 - is released from the exomemes into the parasitophorous vacuole before the merozoite egresses from the erythrocyte. Its release is inhibited by a cyclic GMP dependent protein kinase (encoded by the gene PF3D7_1436600). Substrates of PfSUB1 include MSP1, MSP6, MSP7, MSRP2, SERA5 and SERA6 and the rhoptry protein RAP1. PfSUB1 acts on MSP1 at three sites to produce 4 smaller proteins. It acts on SERA5 - located within the parasitophorous vacuole - a at one site to produce two smaller proteins. One of these is subsequently cleaved by a second protease. Inhibition of the cyclic GMP dependent protein kinase also inhibits microneme discharge.

A protein - gamete egress and sporozoite traversal - has been identified that appears to be involved in the egress of male and female gametes from the erythrocyte. It is also involved in sporozoite migration.

A perforin-like protein (PPLP2) is involved in egress of male gametocytes from the erythrocytes. This protein does not appear to be involved in the rupture of the parasitophorous vacuole. Instead of parasites with mutations in this gene produce gametocytes with only one, shared thicker flagellum rather than usual pattern of each male gametocyte forming 8 flagellated gametes.

Perforin-like protein 1 and perforin-like protein 2 are both transcribed in the blood stage. Perforin-like protein 1 localizes to the red blood cell membrane and parasitophorous vacuolar membrane in mature schizonts following its Ca²⁺-dependent discharge from micronemes. Perforin-like protein 1 has Ca²⁺-dependent permeabilization and membranolytic activities.

The internal calcium levels rise for two hours pre egress. This rise is dependent on internal stores of calcium probably from the endoplasmic reticulum. Inhibition of this rise prevents parasitophorous vacuole swelling and erythrocyte membrane poration.

Phosphorylation of the reticulocyte homologue protein 2b on residue on serine 3233 occurs prior to egress. This is the sole phosphorylation site on this protein. This action is carried put by casein kinase 2 and is cyclic AMP independent, utilizes ATP as well as GTP as phosphate donors and is inhibited by heparin and tetrabromocinnamic acid. This phosphorlation most likely occus most likely occurs before the protein is translocated from the rhoptry neck to the plasma membrane.

Mosquito stage

P. falciparum is taken up by the female Anopheles mosquito as it takes its bloodmeal from an infected human. Within the midgut human complement remains active for ~1 hour. To protect itself the parasite binds host complement regulator factor H. Specifically the gamete surface protein PfGAP50 binds to Factor H and uses the surface bound Factor H to inactivate the complement protein C3b.

How the parasite evades the mosquito's immune system is not understood. The protein Pfs47 appears to be involved in this process.

Gametogenesis

Upon being taken up by the mosquito, the gametocytes leave the erythrocyte shell and differentiate into gametes. The female gamete maturation process entails slight morphological changes, as it becomes enlarged and spherical. On the other hand, the male gamete maturation involves significant morphological development. The male gamete's DNA divides three times to form eight nuclei. Concurrently, eight flagella are formed. Each flagellum pairs with a nucleus to form a microgamete, which separates from the parasite cell - a process known as exflagellation.

Gametogenesis has been shown to be caused by:

• a sudden drop in temperature upon leaving the human host
• a rise in pH within the mosquito
• xanthurenic acid within the mosquito

An ATP-binding cassette (ABC) transporter encoded by the gene Pf14_0244 (PfABCG2) on chromosome 14 appears to have some role in the asexual stages, gametocyte stages and in the oocyst.

The process of the formation of flagella is unusual in that they are formed with in the cytoplasm before export. This pattern is also found in Chromera velia.

The movement of the flagellae has been studied. The flagellae alternate their direction of beating on each stroke: a left to right beat is followed by a right to left beat.

Fertilization

Fertilization of the female gamete by the male gamete occurs rapidly after gametogenesis. The motility of the male gametocytes is powered by glycolysis. The fertilization event produces a zygote. The zygote then develops into an ookinete. Of all the macrogametes present in the gut only ~2% develop into ookinetes. The zygote and ookinete are the only diploid stages of P. falciparum.

A gene - PBANKA_113980 - essential for the production of viable progeny after meiosis has been identified. This gene is the ortholog of the gene GEX1 in the plant Arabidopsis thaliana, the gene Cre06.g280600 in Chlamydomonas and the gene KAR5 in the yeast Saccharomyces cerevisiae. It appears that these genes are involved in nuclear fusion during fertilization.

Ookinete

The diploid ookinete is an invasive form of P. falciparum within the mosquito. It traverses the peritrophic membrane of the mosquito midgut and cross the midgut epithelium. Once through the epithelium, the ookinete enters the basal lamina, and forms an oocyst. Unlike the other invasive stages the ookinete lacks rhopteries. Only 1-2% of ookinites develop into oocyts.

The processes of maturation and invasion of the mosquito gut are under investigation. Both chitinase and plasmepsin 4 (an aspartic acid protease) are known to be involved in the invasion process.

During the ookinete stage, genetic recombination can occur. This takes place if the ookinete was formed from male and female gametes derived from different populations. This can occur if the human host contained multiple populations of the parasite, or if the mosquito fed from multiple infected individuals within a short time-frame.

A G-actin binding protein has been implicated in this process. These proteins - probably acting as dimers - bind actin monomers just before sporogony.

Azithromycin has been shown to suppress apicoplast replication at the period of sporozoite production in oocysts.

The proteins enolase and actin are present on the surface of ookinetes but their function there, if any, is unknown.

The myosin gene (MyoA) class XIV is essential for ookinete motility in the mosquito. Mutants placed under a different promotor active in the blood stages failed to complete their life cycle. Disruption serine repeat antigen 5 blocks parasite inhibits egress of sporozoites from an oocyst.

Within the genome is encoded a protein phosphatase with a domain architecture known only otherwise to occur in Plantae: protein phosphatase containing kelch-like domains (PPKL). The kelch motif normally occurs as a series of four to seven repeats forming a beta propeller tertiary structure and may occur at either terminus of the protein. In Plasmodium PPKL has five full and a one half kelch domains with 5 inserts unique to Alveolates. This protein is produced in schizonts and female gametocytes and is maternally inherited. Absence of this protein results in leads to the development of a malformed, immotile, non infectious ookinetes with extended apical protrusions. This protein is localised at the ookinete apical tip. Secretion of microneme contents is unaffected.

Development of the motile and invasive ookinete within the mosquito midgut is dependent upon two NIMA-related kinases, NEK2 and NEK4.

Two potassium channel have been identified in the genome. At least one of these is critical for the development of the ookinete in the mosquito.

The meiotic specific recombinase Dmc1 - a bacterial RecA homolog - appears to be essential for normal oocyst development in Plasmodium berghei.

Sporogony

Over the period of a 1–3 weeks, the oocyst grows to a size of tens to hundreds of micrometres. During this time, multiple nuclear divisions occur. After oocyst maturation is complete, the oocyst divides to form multiple haploid sporozoites. The number of sporozoiter produced per oocyst varies but the mean is ~500-700 per oocyst. Immature sporozoites break through the oocyst wall into the haemolymph. The sporozoites then migrate to the salivary glands and complete their differentiation. Once mature, the sporozoites can proceed to infect a human host during a subsequent mosquito bite.

Cell biology

Diagram of the major organelles including the rhoptries, micronemes and polar rings near the apical end.

Morphology

The nucleus, mitochondrion, apicoplast and the microtubules of Plasmodium sporozoites are linked to the parasite pellicle via long tethering proteins. The tethers originate from the inner membrane complex and are arranged in a periodic fashion following a 32 nanometer repeat. The tethers pass through a subpellicular structure that encompasses the entire parasite, probably as a network of membrane associated filaments.

The volume of the nucleus of the asexual stages (~5 µm³) is approximately constant in size in the ring and trophozoite stages. The nucleus is divided into several subcompartments including the nucleolus and the nuclear periphery which appear to be involved in transcription control.

The pellicle is a structure unique to the Apicomplexa and is made up of four components: the plasma membrane, the inner membrane complex, the subpellicular network and the subpellicular microtubules. The subpellicular network consists of a two-dimensional network of intermediate filaments located on the cytoplasmic side of the inner membrane complex and acts as a membrane skeleton. The proteins - the inner membrane complex proteins (IMCs) - that compose this structure are functional homologs of the articulins, the membrane skeleton proteins of free-living protists.

The subpellicular network contains alveolins - a group of proteins that determine the shape of the parasite. Another protein - G2 - which is structurally unrelated to alveolins appears to be involved in the organisation of the subpellicular microtubules.

Cell division

Cell division occurs through a process known as schizogony. This is a type of mitotic division in which multiple rounds of nuclear divisions occur before the cytoplasm segments.

DNA synthesis begins in the relatively small trophozoites but nuclear subdivision, which leads to the formation of multinucleate cells, occurs only during schizogony. Whether or not any gap phases exist between each round of DNA synthesis and mitosis is unknown. Eventually, a schizont composed of 8–32 nuclei undergoes segmentation, which culminates with the formation of individual merozoites that burst from the erythrocyte into the blood stream.

Apical complex

The rhopteries appear to have subcompartments allowing for differential secretion during the life cycle. Two of these are known as the neck and the bulb. A number of rhoptry neck proteins are conserved between apicomplexan species and are involved in host cell invasion. Bulb proteins in contrast are less well conserved between the apicomplexa and most likely evolved for a particular lifestyle. In the majority of species studied to date, rhoptry content is involved in formation and maintenance of the parasitophorous vacuole.

Rhoptery proteins

The rhoptery neck proteins (RONs) along with the micronemal AMA1 protein are important in the penetration of the erythrocyte. The invasion mechanism while common to all Apicomplexa is unique and involves a tight interaction between the host cell and the parasite surfaces called the moving junction. The moving junction, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (proteins RON-2, -4, -5, -8), derived from the rhoptries, into the host cell membrane.

The mechanisms involved in this process are still being elucidated. The protein RON8 appears to be central to the binding of parasite to the erythrocyte surface. Apical Sushi Protein and Rhoptry Neck protein 2 are released early following the formation of the tight junction between the merozoite and the erythrocyte. The rhoptry protein PFF0645c is released only after invasion is complete.

The P. falciparum apical sushi protein is the homolog of the P. vivax RON1 protein.

The rhoptry protein 2 of Plasmodium vivax has been cloned. The 1,369 amino acid protein is encoded PVX_099930 gene. The gene has nine introns and the protein contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. It is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to its homolog in P. falciparum.

RON2 is inserted as an integral membrane protein in the host cell.

The residues of the RON2 protein that bind to the AMA-1 protein have been identified. It also appears that the formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Positive diversifying selection appears to have acted in the RON2 protein of Plasmodium vivax.

In Plasmodium berghei RON4 is required for sporozoite invasion of hepatocytes.

RhopH2 is transcribed and localised to the endoplasmic reticulum of the trophozoites by 28 hours post invasion. This pathway is Brefeldin A-sensitive. By 32 hours the protein is distributed in the schizonts' cytoplasm but not in the parasitophorous vacuole.

A protein - Armadillo Repeats-Only - has been localised to the cytosolic face of the rhoptries. A putative signal sequence in the first 20 amino acids has also been identified.

A number of heparin binding proteins are present in the rhopteries. Heparin like molecules bound to the surface of the erythrocyte appear to be important in the adhesion process which also involves the merozoite surface protein 1.

A protein S-acyl transferase with a DHHC-cysteine-rich domain is present in the rhoptry. This enzyme may play a role in the apical positioning of the rhoptry organelles.

Micronemal proteins

Apical membrane antigen-1 (AMA-1) - the product of the Pfama1 gene - is a surface exposed protein that plays a role in erythrocyte invasion. It is shed from the parasite surface predominantly via the action of the protease Sub2 Using gene deletion mutants AMA-1 has been shown to be a non essential protein. While attached to the surface of the parasite AMA-1 can bind to RON2 - a protein that is inserted by the parasite into the erythrocyte membrane. This binding seems to be involved in the invasion process.

The receptor binding site of AMA-1 comprises the hydrophobic groove and a region that becomes exposed by displacement of the flexible domain II loop.

Sub2 is released from the micronemes and can also act on the MSP1/6/7 complex and PTRAMP - another micronemal protein. Sub2 appears to be an essential gene.

Proteins whose location is unclear

Several proteins are involved in the binding of the sporozoite to the various tissues it attaches to. TRAP, S6 and TLP have been implicated in these processes.

Maurer's clefts

In 1902 the German physician Georg Maurer discovered an unusual staining pattern in the cytoplasm of erythrocytes infected with P. falciparum. These structures were subsequently named Maurer's clefts. These consist of a convoluted set of membranes that lie within the erythrocyte's cytoplasm and appear to be involved in secrection from the erythrocyte. They are known to have proteins of parasite origin within them including the Maurer's cleft two transmembrane proteins (PfMC-2TM) The clefts appear to originate from vacuoles budding off them the parasitophorous vacuole membrane which then diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.

Another protein associated with these structure is skeleton-binding protein 1 (SBP1). This protein is involved in transport of the var gene protein, PfEMP1 (erythrocyte membrane protein 1) to the erythrocyte surface.

Other proteins associated with these structures include membrane associated histidine rich protein 1 and ring exported protein 1 and 2.

Mutations in the ring exported protein 1 (Rex 1), a protein normally found in Maurer's clefts, reduces transport of the var gene products to the erythrocyte surface.

The erythrocyte protein ankyrin is found in these structures.

The parasite generates a host derived actin cytoskeleton within the cytoplasm of the erythrocytes that connects the Maurer's clefts with the host cell membrane and to which transport vesicles are attached. Hemoglobin oxidation products which are enriched in hemoglobin S and C containing erythrocytes inhibit actin polymerization. This may account for their protective role in malaria.

The protein trophozoite exported protein 1 (PFF0165c) is located within the clefts. The protein's N-terminal region is intrinsically unstructured but it also has a coiled coil domain. It appears to lack export motifs such as PEXEL, signal sequence/anchor or a transmembrane domain. Transport of this protein to the clefts is sensitive to inhibition by Brefeldin A. This is normally associated with proteins that are have co-translation translocation into the endoplasmic reticulum or posttranslational insertion into the endoplasmic reticulum followed by vesicular transport from the endoplasmic reticulum via Golgi apperatus to the cell surface.

Mutations in REX1 and Pf332 proteins result in distortion of Maurer's clefts morphology suggesting that they play a role in its structure.

The actin network exerts skeletal functions by anchoring the Maurer's clefts within the erythrocyte cytoplasm and restrains the Brownian motion of this organelle. Haemoglobin S and C appear to interfere with this organisation.

The protein Surfin 4.1 is found in the clefts.

The membrane associated histidine rich protein 2 is attached to Maurer's clefts.

There are three multigene families organized into 9 highly conserved clusters with the Pfmc-2tm genes in the subtelomeric regions of the chromosomes. These genes are expressed at early trophozoite stages. Like the PfMC-2TM proteins, the PfEPF1, 3 and 4 proteins encoded by these families are exported to the Maurer's clefts, as peripheral or integral proteins of the Maurer's cleft membrane and largely exposed to the red cell cytosolic face of this membrane.

Within the PfMC-2TM proteins there is a conserved domain MC-TYR.

Dense granules

These are small vacuoles that can be seen on electron microscopy. They bind osmium and appear dark on the images. Little is known about these organelles but they are thought to play a role in the maintenance of the parasitophorous vacuole. One protein - p377 - has been localised to these organelles. Disruption of this protein reduces osmiophilic body formation, and leads to a marked decrease in female fitness and impaired infectivity to mosquitoes.

A protein - the ring membrane antigen (RIMA) - has been localised to the dense granules. The protein's molecular weight is 14 kiloDaltons and it is synthesized late in schizogony. At the late schizont stage this protein is distributed diffusely throughout the intracellular schizont. During the segmenter stage it is then localized to the dense granules.

Parasitophorous vacuole

Within a red blood cell, P. falciparum resides inside the parasitophorous vacuole. This is formed during erythrocyte invasion.

The proteins originating in the parasite pass through the membrane of the parasitophorous vacuole and are transported to the cytoplasm or membrane of the erythrocyte. Although this transport mechanism is largely unknown some details have been elucidated. Ingestion of the erythrocyte cytoplasm begins in mid-ring-stage parasites. Host cytoplasm is internalised via cytostome-derived invaginations and then concentrated into several acidified peripheral structures. Haemoglobin digestion and haemozoin formation occur within these vesicles. The ring-stage parasites can adopt a deeply invaginated cup shape, but they do not take up haemoglobin via macropinocytosis. As the parasite matures the haemozoin containing compartments coalesce to form a single acidic digestive vacuole (pH 4.5 - 5.5) that is then fed by haemoglobin containing vesicles. Some haemoglobin degradation also occurs in compartments outside the digestive vacuole.

The enzyme phosphatidyl-inositol-3-kinase (PI3K) has been implicated in this process. PI3K is located in vesicular compartments near the membrane and in the digestive vacuole and is involved in endocytosis from the host and trafficking of hemoglobin in the parasite. Its inhibition with wortmannin or LY294002 results in entrapment of hemoglobin in vesicles within the parasite cytoplasm preventing its transport to the digestive vacuole.

The pH of the digestive vacuole is maintained by a V-type H(+)-ATPase.

A signal sequence at the N terminal of proteins targeted to the arasitophorous vacuole has been identified. The signal appears to reside in the 55 amino acids of the N terminal of the protein. There may be a retention signal at the C terminal.

The micronemal protease ROM1 appears to be essential for proper parasitophorous vacuole modification to allow parasite development. This protease is able to cleave the proteins AMA1 and MAEBL.

Uric acid precipitates are present in the cytoplasm of the parasitophorous vacuole. These are released when the merozoites are released. Uric acid is highly inflammatory and can cause maturation of dendritic cells.

The protein PFA0210c is a member of the START domain family which is involved in the transport of phospholipids, ceramide or fatty acids between membranes. It associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. It is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite.

Apicoplast

Plasmodium falciparum, and most other members of the phylum Apicomplexa, contain an organelle termed an apicoplast. It was first discovered in 1996. The apicoplast is an essential plastid, homologous to a chloroplast, although the apicoplast itself lacks any photosynthetic function. Evolutionarily it is thought to have been derived through secondary endosymbiosis. As humans do not harbor apicoplasts, this organelle and its constituents are seen as a possible target for antimalarial drugs.

The apicoplast varies in size during the life cycle from ~0.5 µm × 0.15 µm in the merozoite to 1.6 µm × 0.35 µm in the trophozoites. Only one copy is present until it replicates in late schizonts. The apicoplast always adheres to the mitochondrion, along its whole length in merozoites and early rings but only at one end in later stages. Regions of the apicoplast are also closely related to the pigment vacuole, nuclear membrane and endoplasmic reticulum. In merozoites the plastid is anchored to a band of 2-3 subpellicular microtubules.

The apicoplast has four membranes is normally located between the nucleus and the rhoptries. Its matrix contains ribosome sized particles and membranous whorls. The gap between the second and third membranes is frequently larger than between the other membranes. The interior matrix of the apicoplast contains ribosome-like granules and a network of fine branched filaments.

It contains a 35-kb genome, which encodes for 30 proteins. The genome of this organelle has now been sequenced for several species. It appears to be conserved and to encode ~30 genes in all species examined.

The plastid genome replicates at the late trophozoite stage of the parasite intraerythocytic cycle. It proceeds predominantly via a D-loop/bi-directional ori mechanism with replication ori localized within the inverted repeat region. The process of replication involves a nuclear-encoded DnaJ homolog that binds to the ori site.

The DNA polymerase involved in the replication of its genome is Pfprex (Klenow-like polymerase). This enzyme has been cloned, expressed and purified. The enzymes is relatively error prone and shows a bias toward T->C mutations.

Other nuclear encoded proteins are transported into the apicoplast. Transport into the apicoplast are not well understood. These proteins has a signal in the N terminal but unlike many other organisms this appears to be a disordered chain rather than a conserved sequence. It was thought that a specific signal peptide was responsible for this targeting and it was estimated that 551, or roughly 10%, of the predicted nuclear-encoded proteins are targeted to the apicoplast. This hypothesis now appears to be incorrect. It appears that a relative enrichment within the protein of positively charged amino acid residues (Arginine, Histidine, Lysine) particularly at the N terminal of the protein may be sufficient to target the protein to the apicomplast.

The biosynthesis of this organelle is not well understood. Phosphatidylinositol 3-monophosphate has been shown to be involved in its biosynthesis in the apicomplexian Toxoplasma gondii. It seems likely that this enzyme is involved in the formation of this organelle in the Plasmodium species also.

This organelle appears to be essential in the liver stages.

The functions of this organelle remains to be fully determined but it appears to be involved in the metabolism of fatty acids, isoprenoids and heme. There are two pathways for protein lipoylation in Plasmodium - one in the mitochondrion and the other in the apicoplast. The apicoplast pathway is not found in the vertebrate host and relies on de novo lipoic acid synthesis.

The role of the apicoplast in the blood stages has been clarified. Inhibition of isoprenoid precursor biosynthesis with the antibiotic fosmidomycin (an inhibitor of the enzyme DOXP reductoisomerase) causes delayed death in this parasite. This effect can be overcome with the addition of isopentenyl pyrophosphate (IPP) to the culture medium. Continued culture in the presence of this agent leads to the loss of the apicoplast genome and these mutants fail to process or localize organelle proteins. These auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival.

Iron-sulphur prosthetic groups are assembled in this organelle. One component (SufB) is encoded in the apicoplast genome and a second (SufC) is encoded in the nucleus. SufB also exhibits ATPase activity. Other pathways that have been linked to this organelle include biosynthesis of isoprenoid precursors, fatty acids, heme and lipoic acid.

Iron-sulfur (Fe-S) cluster cluster synthesis pathways are found in the apicoplast. In the Suf pathway, SufS - a cysteine desulfurase - and its partner SufE are found exclusively in the apicoplast. In the Isc pathway IscS and its effector Isd11 were solely mitochondrial. Interruption of the Suf pathway results in a phenotpe that is dependent on external isopentenyl pyrophosphate. This phenotype is also associated with the loss of the apicoplast organelle and its organellar genome suggesting a role for this pathway in the maintenance of the apicoplast itself.

The [Fe-S] cluster protein NFUapi - a protein with a nitrogen fixation factor U (NifU)-like domain - is localised to the apicomplast. This protein may have a role in merosome formation and appears to be non essential for the blood stages.

A gene Plasmodium-specific Apicoplast protein for Liver Merozoite formation (PALM) has been shown to be important for merozoite formation. Knock out mutants are unable to release merozoites into the blood from the liver stages. Mutants lacking this gene appear to be able to elicit at least temporary immunity.

Falcilysin a zinc metalloprotease is found in the apicoplast. It is a member of the M16 protease group and has maximal activity at neutral pH. It appears to be an essential gene. Its function in this organelle is not quite clear but it appears to be involved in the degradation of transit peptides.

The enzyme thioredoxin peroxidase is found in the apicoplast, the mitochondrion and the cytosol.

Autophagy is membrane-mediated degradation process that involves a series of proteins known as Atg proteins. Atg8 is expressed during development and localises to the apicoplast.

Two C3 sugar phosphate transporter are present in the membrane of the apicoplast of Plasmodium berghei - triose phosphate transporter and phosphoenolpyruvate transporter. Knock out mutants of the triose phosphate transporter fail to survive. Phosphoenolpyruvate transporter knock out mutants survive in the blood stages but suffer defects during the liver stages development.

A pyruvate dehydrogenase complex with four subunits (E1alpha, E1beta, E2, E3) is present in the apicoplast. Unlike plants there is only a single copy of these genes in the genome. pyruvate dehydrogenase deficient parasites have no apparent blood stage growth defect, they are unable to progress beyond the oocyst phase of the parasite's mosquito stage.

An ATP dependent caseinolytic protease (ClpP) is present in the apicomplast. Its function is currently unknown.

The lipid composition of the apicoplast has been analysed. some apicoplast lipids are generated de novo by the organelle itself. Phosphatidylinositol and other phospholipids in the organelle are enriched in saturated fatty acids. Lipids atypical for plastids - sphingomyelins, ceramides and cholesterol - are present. Galactoglycerolipids, dominant in plant and algal plastids, are not present suggesting that these glycolipids are a hallmark of photosynthetic plastids and were lost when these organisms assumed a parasitic lifestyle.

The endoplasmic reticulum associated degradation (ERAD) machinery is a quality control mechanism that retro-translocates misfolded secretory proteins across the endoplasmic reticulum membrane. Several components of this system including the membrane protein Der1, the AAA ATPase Cdc48 and its cofactor Ufd1 appear to be present in the apicoplast and are involved in transmembrane transport.

ClpB is a molecular chaperone and a member of the AAA+ superfamily of ATPases. There are 2 isoforms of ClpB (PfClpB1 and PfClpB2) found in the apicoplast. PfClpB1 contains all characteristic AAA+sequence motifs but there is a 52-residue long non-conserved insert middle domain. ATP induces self association of PfClpB1 into hexamers like in most AAA+ ATPases. It catalyzes the hydrolysis of ATP and its ATPase activity is activated in the presence of casein and polylysine.

A glutamyl-tRNA synthetase (GluRS) is present in the apicoplast. It is it is non-discriminating glutamylating both apicoplast tRNA-Glu and tRNA-Gln. It appears to be an essential enzyme.

An elongation factor G which is involved in translation is present in the apicoplast. It has GTPase activity that can be inhibited by fusidic acid.

Mitochondrion

Plasmodium lacks mitochondrial pyruvate dehydrogenase and the hydrogen ion translocating NADH dehydrogenase (Complex I, NDH1). The mitochondrion contains a minimal DNA genome (~6 kilobases) and carries out oxidative phosphorylation in the insect vector stages by using 2-oxoglutarate as an alternative means of entry into the tricarboxylic acid cycle and a single-subunit flavoprotein as an alternative NADH dehydrogenase (NDH2). In the blood stages mitochondrial enzymes are down regulated and parasite energy metabolism relies mainly on glycolysis. The enzyme malate quinone oxidoreductase was acquired from an epsilon proteobacteria via lateral gene transfer. This transfer occurred in an ancestor of the Apicomplexa.

The ATP synthase is localised to the mitochondrion, is assembled as a large dimeric complex and appears to be essential for in the blood stages of the life cycle. Its function in these stages is not yet clear.

The sulfhydryl:cytochrome c oxidoreductase Erv1/ALR/GFER/HSS (Essential for Respiration and Vegatative growth/Augmenter of Liver Regeneration/Growth Factor Erv1-like/Hepatic regenerative Stimulation Substance/hepatopoietin) is an essential sulfhydryl oxidase for required oxidative protein import into the mitochondrial intermembrane space. It is one of several enzymes involved in electron transferase activity. It is encoded by all eukaryotes and cytoplasmic DNA viruses sequenced to date. The enzyme from P. falciparum differs significantly from that found in yeast and humans with altered cysteine motifs and intermolecular disulfide bonds. Despite successful cloning and expression in yeast, the parasite enzyme fails to function in yeast. A second related enzyme - Mia40 - does not appear to be present in P. falciparum.

Deletion of the gene in the rodent parasite Plasmodium berghei for the flavoprotein subunit of succinate dehydrogenase - part of the complex II - showed impairment of ookinete function and oocyst formation.

The gene for the flavoprotein subunit of succinate dehydrogenase can be disrupted in the parasite. Its disruption causes growth retardation of the intraerythrocytic forms. It appears that complex II functions as a quinol-fumarate reductase to form succinate from fumarate in the intraerythrocytic parasite.

The dicarboxylate-tricarboxylate carrier homolog has been cloned from P. falciparum. This protein may mediate the oxoglutarate-malate exchange across the inner mitochondrial membrane required for the branched pathway of tricarboxylic acid metabolism.

The ClpQ protease and ClpY ATPase have been cloned. ClpQY function disruption caused hindrance in the parasite growth and maturation of asexual stages of parasites. Features of apoptosis like cell death are also found.

The mitochondrial pathway of protein lipoylation relies on scavenging from the host and can be inhibited with the lipoic acid analog 8-bromo-octanoic acid. Use of this agent inhibits growth and significantly reduces merosome formation. Schizogony is the phase most affected by this inhibition.

Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation site of the mitochondrial cytochrome bc1 complex and is used as an antimalaria agent. Inhibition of this enzyme leads to the collapse of the mitochondrial membrane potential and disruption of pyrimidine biosynthesis. These effects are lethal to the parasite.

The mitochondrial RNA polymerase appears to be an essential gene for the erythrocytic stages.

Over half the genome of the mitochondrion encodes the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes 34 RNA genes of which 27 have been assigned to ribosomal RNA (12 to the small subunit and 15 to the large subunit). These genes are fragmented and are encoded on both strands.

The mitochondrial thioredoxin peroxidase-2 does not appear to be essential.

The ATP dependent ClpQY system is a prokaryotic proteasome like multisubunit machinery localized in the mitochondrion. It has two components - a ClpQ threonine protease and ClpY ATPase. The ClpQ threonine protease appears to be an essential gene.

The bc 1 complex of the mitochondrial respiratory chain is essential for the parasite's life cycle. The drug atovaquone inhibits its activity. Atovaquone binds to the Q₀ site on this complex.

An AAA+/FtsH protease homolog (PfFtsH1) that exhibits ATP- and Zn²⁺-dependent protease activity is present in the inner mitochondrial membrane. It seems likely that this is a regulatory protein for organelle biogenesis.

An elongation factor G involved in protein translation is present in the mitochondrion. It has GTPase activity that can be inhibited by fusidic acid.

Digestive vacuole

During growth of the parasite and as part of its digestion of the erythrocyte's haemoglobin, fusion of digestive vesicles occurs and gives rise to a large digestive vacuole. This vacuole the interior of which is maintained at a low pH (pH 4.5 - 5.5), processes 60-80% of the ingested hemoglobin and provides a pool of amino acids that is crucial for parasite growth and development. The membrane contains ion pumps and transporters that maintain its low pH. During haemoglobin digestion the heme is released from hemoglobin. Haem is toxic to the parasite and is detoxified by biocrystallization to hemozoin within the vacuole. Quinoline drugs, including chloroquine, act by binding to heme and thus prevent its sequestration into hemozoin.

It has been shown that micromolar concentrations of chloroquine partially permeabilized the parasite's digestive vacuole membrane and that this event appears to precede mitochondrial dysfunction.

Quinine has been shown localise to a non acidic compartment within the digestive vacuole. It may colocate with haemozoin. It's localisation within the parasite is not altered by the presence or absence of a functional multidrug resistance gene.

The digestive vacuole is able to activate both the alternative complement and the intrinsic clotting pathway. The digestive vacuole membrane has the capacity to assemble prothrombinase, a key enzyme of the intrinsic clotting pathway. The capacity of this membrane to activate both complement and coagulation can be suppressed by low molecular weight dextran sulfate. Phagocytosis of these membranes drives the polymorphonucleocytes into a state of functional exhaustion.

Two multi-spanning digestive vacuole membrane proteins are known: the multidrug resistance protein 1 and Chloroquine Resistance Transporter (CRT). The CRT protein moves from the endoplasmic reticulum to the Golgi apparatus before becoming associated with the digestive vacuole. The digestive vacuole forms in the ring stages of the parasites life cycle. Chloroquine sensitivity is not influenced by the absence of CRT from the digestive vacuole bringing into question its relationship (if any) to chloroquine resistance. Mutations in the CRT gene have been associated with sensitivity and resistance to quinolines. In particular a lysine to isoleucine at codon 76 (Adenosine -> Thymine at base 227) mutation and a valine to phenylalanine (Guanine -> Thymine at base 1108) mutation have been associated with changes in drug sensitivity. The mutant CRT protein increases the transport of glutathione into the digestive vacuole. The role - if any - that this has on chloroquine resistance is presently unknown.

The membrane of the digestive vacuole is four nanometers in thickness with patches that may be up to 12 nanometers in thickness.

The digestion of haemoglobin produces large quantities of ferriprotoporphyrin IX which it unable to digest and is potentially toxic to the parasite. To avoid the toxicity the ferriprotoporphyrin is converted to haemozoin. Chloroquine inhibits this process. The mechanisms behind this process are still unclear. In vitro conversion of ferriprotoporphyrin to haemazoin is enhanced at a temperature of 41C when compared to its conversion at 37C. It is possible that the rise in temperature that occurs in malaria may be part of a strategy to enhance this reaction at the later stages of growth when the ferriprotoporphyrin concentration is likely to be high.

Inhibition of the falcipains involved in the digestion of haemoglobin results in enlargement of the digestive vacuole.

Nucleus

Unlike most other eukaryotes the chromosomes never become condensed even during mitosis and remain difficult to visualise by llight microscopy.

Studies with antibodies to the nuclear pore protein subunit PfNup116 show that the nuclear pores are highly polarized during the ring and schizont stages whereas in trophozoite stage the nuclear pores redistributed over the entire nuclear surface.

PfSec13 is a nucleoporin. It appears to be a fusion between Sec13 and Nup145C and it associates with the nuclear pore complexes and microtubules. It appears to be an essential gene.

Nucleolus

A hat-like structure polarized towards one side of the nucleus that stains with nucleolar markers has been described It seems likely that this unusual structure is the nucleolus.

The histone deacetylase Sir2a is found in the nucleolus in addition to the telomeres. It functions there to control the transcription of the ribosomal RNA levels.

The ribosomal RNA genes are found localised to this organelle.

Endoplasmic reticulum

This forms a set of reticular structures adjacent to the nuclear regions during the trophozoite and schizont stages. In the late schizont stage it forms globular structures surrounding each budding merozoite.

A Ca²⁺ ATPase 6 has been associated with resistance to artemisinin resistance. The gene has a single copy in the genome. Reistance has been associated with four mutations: codon 263 Lysine->Glutamic acid, codon 431 Glutamic acid->Lysine, codon 623 Alanine->Glutamic acid and codon 769 Serine->Asparagine. This gene is a SERCA pump and appears to be essential in the asexual stages.

Many proteins in the genome carry a host targeting signal. This signal sequence is recognised by phosphatidylinositol-3-phosphate in the endoplasmic reticulum.

Ribosomes

Unlike other eukaryotes studied to date Plasmodium species have two or three distinct SSU rRNA (18S rRNA) molecules encoded within the genome. These have been divided into types A, S and O. Type A is expressed in the asexual stages; type S in the sexual and type O only in the oocyte. Type O is only known to occur in Plasmodium vivax at present. The reason for this gene duplication is not known but presumably reflects an adaption to the different environments the parasite lives within.

The Asian simian Plasmodium species - Plasmodium coatneyi, Plasmodium cynomolgi, Plasmodium fragile, Plasmodium inui, Plasmodium fieldi, Plasmodium hylobati and Plasmodium simiovale - have a single single S-type-like gene and several A-type-like genes. Phylogenetic analyses has shown that gene duplication events giving rise to A- and S-type-like sequences took place independently at least three times in the Plasmodium evolution.

The phosphoprotein P0 occurs as a complex with two other small acidic ribosomal proteins (P1 and P2). A pentameric complex [(P1–P2) P0 (P1–P2)] form the stalk of the large ribosomal subunit, which seems to play a role in the GTPase elongation centre of the ribosome.

The P2 protein is exported to the infected erythrocyte surface at 30 hrs post merozoite invasion, concomitant with extensive oligomerization. It is largely largely composed of alpha helical and random coil domains.

Acidocalcisomes

Acidocalcisomes are acidic calcium stores and are present in many organisms including bacteria, Plasmodium and humans. The organelles possess an acidic matrix that contains several cations bound to phosphates. These are mainly present in the form of short and long polyphosphate chains. The matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca²⁺/H⁺ countertransporting ATPase located in the membrane of the organelle.

Motility

Thrombospondin Related Anonymous Protein (TRAP) is a type I transmembrane proteins which has several extracellular adhesive domains and a cytoplasmic domain that recruits the glycolytic enzyme aldolase. Normally only small amount of TRAP found on the sporozoite surface. TRAP is involved in cell motility.

Its tandem von Willebrand factor A and thrombospondin type I repeat domains connect through the proline rich stalk, transmembrane and cytoplasmic domains to the parasite's actin dependent motility apparatus. Binding is dependent on the presence of a metal ion. The protein is capable of considerable conformational changes. There is also a potential heparan sulphate binding site in the von Willebrand factor A domain.

The cytoplasmic domain binds to F-actin which connects to myosin A. Within the transmembrane domain it has a canonical rhomboid cleavage site (Ala-Gly-Gly-Ile-Ile-Gly-Gly). Rhomboid proteases are a family of serine proteases that require helical instability in the transmembrane domain and have specific residue requirements in their P1, P4 and P2′ positions. These proteases are responsible for intramembraneous cleavage.

TRAP binds to receptors on the host and is translocated posteriorly by the actomyosin motor. It is then normally cleaved by a calcium independent serine protease. Removal of the cytoplasmic domain abolish the motility of the parasite. Mutations in the rhomboid cleavage site are defective in TRAP shedding and display slow, staccato motility and reduced infectivity. The reduction in infectivity is particularly marked if the sporozoites are inoculated intradermally rather than intravascularly. Prevention of cleavage of the TRAP protein entirely renders the sporozoites uninfectious and immobile. The rhomboid protease normally involved in TRAP cleavage appears to be the ROM4 protease. This protease is found across the entire sporozoite surface suggesting it has functions in addition to TRAP cleavage.

The circumsporozoite- and thrombospondin-related adhesive protein (CTRP) is a modular multidomain protein containing six tandem von Willebrand factor A like domains and seven tandem thrombospondin type I repeat-like domains. The A domains of CTRP are critical for ookinete gliding motility and oocyst formation. The thrombospondin domains are fully redundant.

The cell-traversal protein for ookinetes and sporozoites (CelTOS) is a protein involved in the invasion of both vertebrate and insect host cells.

The C-terminal tail of myosin A (MyoA) and its light chain, myosin A tail domain interacting protein (MTIP) are essential parts of the gliding motility apperatus.

Dynein light chain 8 is present in P. falciparum as a homodimer. The dimer is formed by the interaction of the β₀ chains on one molecule with the β₂ chains of the second.

A kinesin with the ability to depolymerise microtubules has been cloned.

Autophagy

A number of proteins involved in autophagy are known to be present in the genome. These include Atg8 and Atg3. The functions of these proteins in the parasite are still being elucidated.

Autophagosomes fuse with the endosomes before being routed to the digestive vacuole. The digestive vacuole is probably used for this purpose because the parasite lacks lysosomes.

Crystaloids

These are transient structures whose presence is restricted to the mosquito specific ookinete and young oocyst stages of the parasite. They are cytoplasmic aggregations of closely packed spherical particles 25–35 nanometers in diameter and disappear after ookinete-to-oocyst transition. Although first described in the 1962 their function is unknown. They contain a scavenger receptor like protein which is formed in the macrogametocytes. They also contain a number of LCCL proteins.

Effect of radiation

Gamma rays may be used to produce attenuated parasites. Despite this the effects on the parasite have rarely been studied. Gamma irradation acts in a dose dependent fashion: morphologically it induces defective mitosis, sparse cytoplasm, fewer ribosomes, disorganized and clumped organelles and large vacuoles. The transcription of a number of genes is altered.

Molecular biology and biochemistry

Redundancy of binding/invasion proteins

Proteins involved in this process are subject to three levels of host cell selection:

• selection between host species (species specific tropism)
• selection among individuals within a host species (erythrocyte receptor diversity)
• selection of subpopulations within an individual (age dependent invasion)

These proteins are necessarily surface exposed and are also subject to selection by the host's immune system

Because of the multiple levels of selection it is to be expected that a number of proteins are involved in this process. This also in part explains the apparent redundancy of these proteins.

Surface exposed proteins

Merozoite surface protein (MSP) family

The cleavage of MSP 1 appears to involve a purinergic signalling pathway.

The MSP1 protein binds the pro inflammatory protein S100P. This binding appears to prevent the usual NFκB activation in monocytes and chemotaxis in neutrophils. S100P appears to be able to bind to at least 2 alleles of MSP1 which are separated by at least 27 million years of evolution suggesting that this inhibition mechanism may also be of considerable age.

Merozoite surface protein 2 is one of the most abundant proteins on the surface of merozoites, is intrinsically unstructured and forms amyloid-like fibrils in solution.

Merozoite surface protein 7 appears to enhance the virulence of the parasite at least in the rodent.

A protein PfMSPDBL1 (encoded by PF10_0348 gene) that is a member of the MSP3 family and has both Duffy binding-like (DBL) domain and secreted polymorphic antigen associated with merozoites (SPAM) domain appears to be critical for erythrocyte invasion. The merozoite surface proteins DBL1 and -2 (PfMSPDBL1 and PfMSPDBL2) (PF10_0348 and PF10_0355) are extrinsically associated with the merozoite. MSPDBL2 appears to have a role in resistance to halofantrine, mefloquine and lumefantrine.

Circumsporozoite protein (CSP)

The circumsporozoite protein (CSP) forms a dense coat on the sporozoite's surface. It consists of approximately 400 amino acids organized into three domains: an N-terminal domain containing a conserved pentapeptide (region I), a highly repetitive species specific central domain and a C-terminal domain containing a second conserved sequence (region II). It is involved in invasion of the mosquito's salivary glands and the binding sporozoites to liver cells.

The circumsporozoite protein has been shown to be an inhibitor of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Its nuclear localization signal alone is sufficient to block NF-κB activation.

CSP binds to salivary glands and is involved in its invasionIn by the sporozoites. An Anopheles salivary gland protein - CSP-binding protein - to which CSP binds during this process has been identified.

Early transcribed membrane protein (ETRAMP) family

The ETRAMP family is characterized by a predicted signal peptide, a short lysine rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. The highly charged terminal region appears to be involved in protein-protein interactions. They are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and to vesicle like structures exported to the host erythrocyte cytosol. The gene ETRAMP 10.3 has been shown to be expressed in the liver, sporozoites and blood stages. Within the liver and blood stages it is localized to the parasitophorous vacuole membrane. It is also exported to the erythrocyte during the blood stages. It appears to be an essential gene in the blood stages. In Plasmodium berghei two members of the ETRAMP family (uis3 and uis4) localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. Another member of this family - SEP2 - is expressed in the gametocytes, in the mosquito and in the liver stages. In the liver stage SEP2 is routed to the parasitophorous vacuole membrane. In the ookinete and sporozoite stages, it instead localizes to the parasite surface. It is also released during gliding motility of salivary gland sporozoites.

6-cys domain proteins

A group of proteins known as the 6-cys domain proteins - so called because they contain modules with six characteristic cysteines forming three intra-molecular disulphide bonds between C1 and C2, C3 and C6, and C4 and C5 - are surface exposed proteins. The first P12 - named after the clone it was isolated from - was described in 1990. There are at least nine members of the 6-cys family. Most family members contain two 6-cys modules, but up to seven modules can be found in a single protein, in addition to incomplete modules containing fewer cysteine residues. About half of the 6-cys family members characterised to date possess glycosylphosphatidylinositol (GPI) moieties that anchor them to the outer leaflet of the plasma membrane, while those that lack GPI-anchors presumably remain associated with the parasite surface via interactions with other membrane proteins. Of this family P12, P38 and P41 are blood stage antigens. P230 and P48/45 - another two members of this family - are expressed on the surface of gametes.

P12 has only two s48/45 domains while other members have up to fourteen. Pf12 is highly conserved and under purifying selection. It forms a heterodimeric complex with Pf41.

LCCL/lectin adhesive-like protein family

There are a family of LCCL/lectin adhesive-like protein (LAP) proteins encoded in the genome. The six members are expressed in gametocytes and form a multi-protein complex. There are normally six of these proteins in the genome. They are essential for parasite transmission to the mosquito.

Pf332 protein

The 700 kiloDalton protein Pf332 is the largest known exported asexual malaria protein. The protein has three parts: an N-terminal Duffy binding like domain followed by a putative transmembrane region and a large number of negatively charged repeats that are not identical but have the consensus (X)₃-EE-(X)₂-EE-(X)_2–3 where E is glutamic acid and X is a hydrophobic amino acid. The repeat portion of the protein consititue more than 90% of the protein. The protein has a predicted isoelectric point (pI) of 3.8. It is known to associate with the erythrocyte plasma membrane.

The Pf332 protein can first be detected within the parasite at 20–24 hours post invasion, after which it translocates across the parasitotopherous vacuole membrane into the host cell cytosol. It is initially synthesised in the endoplasmic reticulum and eported to the host cytosol. From there it is trafficked as part of a multimeric protein complex to Maurer's clefts. It may interact with two chaperone proteins - PF14_0700 (a hypothetical protein with a J domain) and PFB0595w (a heat shock protein 40). It is associated with the cytoplasmic side of Maurer's clefts in a peripheral manner throughout trophozoite maturation and schizogony. In the clefts both the N and C-termini are localised to the erythocyte cytosol. Export of Pf332 is sensitive to treatment with Brefeldin A The export signal appears to be encoded in the N terminal domain

It interacts with the erythrocyte cytoskeleton and binds actin.

PHIST family

The Plasmodium helical intersperse sub-telomeric family is a collection of 72 small exported proteins.

Other proteins

The merozoite specific thrombospondin related anonymous protein (MTRAP) is thought to be released from the micronemes during merozoite invasion and mediates motility and host cell invasion through an interaction with aldolase. MTRAP is a highly extended bifunctional protein that binds to an erythrocyte receptor and the merozoite motor. MTRAP specific antibodies fail to inhibit parasite development in vitro.

Thrombospondin related apical membrane protein (PTRAMP) is a surface exposed protein whose function is currently unknown.

The phosphoprotein P0 is surface exposed during the asexual erythrocytic stages and antibodies to this protein appear to be protective. It is also present on the surface of the merozoites.

The 60S stalk ribosomal acidic protein P2 (gene PFC0400w) as well as forming part of the ribosome complex is surface exposed where it forms homo-tetramers. This protein is exported to the erythrocyte surface 26-28 post invasion and persists there for 6–8 hours. Treatment with antiP2 antibodies causes mitotic arrest at the first nuclear division and disruption of the tubovesicular network which is set up during the trophozoite stages. Removal of the antibodies al lows the reformation of the tubovesicular network and mitotic division to continue.

The receptor for the attachment protein PfRh4 has been identified as complement receptor 1.

The mature parasite-infected erythrocyte surface antigen (MESA) is exported to the erythrocyte cytoplasm where it binds to the N-terminal 30 kiloDalton domain of the erythrocyte protein 4.1R via a 19-residue sequence. This sequence is also found in a number of other proteins in the parasite. Their role in remodeling of the erythrocyte are still under investigation.

The Ring-Infected Erythrocyte Surface Antigen (RESA/Pf155) protein appears to affect the mobility of the erythrocyte membrane.

The proteins Pf12, Pf34, Pf92 and Pf38 are associated with detergent resistant membrane microdomains through glycosylphosphatidylinositol anchor sequences. These microdomains are considered organizing centers for the assembly of molecules implicated in cell signaling.

The erythrocyte binding like 175 protein (EBL) is normally found in the micronemes. It has a signal sequence and a cysteine rich domain both of which are required for localisation to the micronene. In addition to these two ssequences for correct trafficking it also requires a sequence in its region 5.

Enolase is bound to the surface of P. falciparum and several other pathogens. In this location it binds plasminogen which is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion.

The gene PFE0565w is transcribed in both the erythrocytic and sporozoite stages. The protein is only expressed in the salivary gland sporozoite stage.

Surfin 4.1 - a type I transmembrane protein located on the merozoite surface - is responsible for reversible adherence to the erythrocyte before invasion. The gene is highly polymorphic, particularly at the C-terminal side of the variable region located just before a predicted transmembrane region. Positive diversifying selection is detectable in this region and in the conserved N-terminally located cysteine-rich domain.

Heparin has been shown to bind to infected erythrocytes. What role this binding has in the pathology of this infection - if any - is not yet clear.

Erythrocyte proteins taken up

A small number of erythrocytic proteins are taken up by the parasite during the course of its life cycle. The role these play is not clear. Among these proteins is dematin which interacts with the parasite's 14-3-3 protein.

The parasite is capable of making use of the erythrocyte's own enzymes. The enzymes PAK1 and MEK1 neither of which are encoded in the Plasmodium genome have been shown to be phosphorylated and activated during the course of infection' In vitro work has shown that inhibition of these enzymes is fatal to the parasite.

Plasmodium ingests kininogen from which its proteases generate vasoactive peptides. The role this may play in the pathophysiology of malaria is not yet understood.

Another enzyme that is imported by the parasite is the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1). This imported protein accounts for ~50% of the total thioredoxin peroxidase activity in parasite extracts. In the presence of chloroquine the parasite increases its imports of this protein. This protein is found both in the cytosol and in Maurer's clefts.

Another enzyme that is taken up is delta-aminolevulinate dehydrase - an enzyme in the haem biosynthesis pathway.

Transport/secretion

The uninfected erythrocyte lacks a regulated transport system. Vesicular transport within both the parasite and the infected erythrocyte cytoplasm must be provided by the parasite itself.

Both the cytoplasmic pH (7.3) and the inside negative plasma membrane potential (-95 mV) are kept fairly constant during the intra erythrocytic cycle. This is due to the action of a V-type H(+)-ATPase which is also responsible for the pH of the digestive vacuole. There is also a Na⁺ ATPase in the plasma membrane.

Transport

The intracellular concentration of chloride ions has been estimated to be 48 milliMolar. It appears to actively import using ATP both hydrogen ions and chloride ions in a linked fashion via a DIDS sensitive transporter in the cytoplasmic membrane.

One difficulty the parasite has in acquiring nutrients from the cytoplasm is the presence of phosphate groups on these molecules. It appears to have overcome this by secreting an acid phosphatase (glideosome-associated protein 50 - GAP50 ) into the cytoplasm that is then taken up into the digestive vacuole.

The parasite has an absolute requirement for isoleucine - an amino acid absent from human haemoglobin. A saturable neutral amino acid (methionine, leucine, isoleucine) transporter appears to be encoded by the parasite and this protein functions in the infected erythrocyte membrane.

The P. falciparum Na+/H+ exchanger (PfNHE1) is located on chromosome 13 (gene PF13_0019). This gene may be involved in resistance to quinine.

Two folate transporters (PfFT1 and PfTF2) have been cloned. Substrates include folic acid, folinic acid, the folate precursor pABA and the human folate catabolite pABAG(n). 5-methyl tetrahydofolate is not transported by PfFT1 and only poorly by PfFT2. The activity of both transporters may be inhibited by probenecid or methotrexate. Folate transport appears to be an ATP requiring activity and dependent on a proton gradient.

The parasite possesses its own equilibrative nucleoside transporter 1. All members of this protein family have 11 transmembrane segments. The gene product is located in the parasite's plasma membrane and knock out mutants have shown that this is an essential gene at least at physiological concentrations. In the 11th transmembrane segment two mutations have been shown to affect its activity: a phenylalanine (Phe) to leucine (Leu) at residue 394 (F394L) via cytosine (C) or uracil (U) to adenosine (A) or guanine (G) at the third codon position and a cysteine (Cys) to glycine (Gly) mutation at either glycine in a conserved glycine-X-X-glycine motif (where X is any amino acid) via a cytosine to uracil at the second codon position. Additional work suggests that the 11th transmembrane segment is largely alpha helical. It has been suggested that this transmembrane segment may be the actual purine transport channel.

The parasite is unable to synthesize purines (including adenosine, hypoxanthine and adenine) and must take these up from the host. Purines are transported across the parasite plasma membrane entry into the infected erythrocyte P. falciparum nucleoside transporter 1 (PfNT1). This transport system carries hypoxanthine, inosine and adenosine into the parasite. At least some of the hypoxanthine is converted into uric acid by the parasite.

Three purine transporters have been studied: the human equilibrative nucleoside transporter (hENT1), the human facilitative nucleobase transporter (hFNT1) and the parasite-induced new permeation pathway (NPP). The bulk of transport is facilitated by host's own transporters rather than through the NPP. Hypoxanthine and adenine were transported mainly through the hFNT1 pathway whereas adenosine entered predominantly through the hENT1 system. The rate of purine uptake in infected cells was approximately twice that of uninfected erythrocytes. The rate of adenosine uptake was greater than the rate of hypoxanthine uptake in infected human red blood cells. Furosemide inhibits the transport of purine bases through the hFNT1.

An intracellular purine permease (PfNT2) has been shown to be localised to the endoplasmic reticulum. This protein is a member of the equilibrative nucleoside transporter family.

Within the genome there are encoded four equilibrative nucleoside transporters (ENTs). ENT 1 is the major route of purine nucleoside/nucleobase transport in the erythrocytic stages. Knock out mutants have been generated that can survive. ENT4 has been cloned and expressed. It does not appear to transport either hypoxanthine or adenine monophosphate but does transport adenine and 2'-deoxyadenosine. It is inhibited by dipyridamole.

The parasite can uptake polyamines from the host. Two of these - putrescine and spermidine - are taken up in a temperature, pH and membrane potential dependent mechanism.

The clag3 genes on chromosome 3 appear to be involved in anion transport rather than in cell adherence as originally thought.

At least two of the clag3 genes appear to be involved in the surface anion channel which functions in nutrient uptake.

The clag3 gene family encode a parasite ion channel known as the plasmodial surface anion channel. Its activation appears to involve an intracellular domain. The anion channel is formed by 5 clag genes and forms a complex with RhopH and other proteins. The expression of the anion channel can be suppressed by reversible histone modification.

Two paralogous clag3 genes - clag3.1 and clag3.2 - show mutually exclusive expression and can be silenced by epigenetic mechanisms. They appear to offer different transport efficiency for some solutes. The expression of at least one of these genes is essential for the function of the anion channel.

Positive diversifying selection has acted upon clag2, clag8 and clag9 but not in clag3.1 and clag3.2. These proteins appear to be involved in anion transport.

The plasma membrane protein aquaglyceroporin mediates the transport of both glycerol and water.

A copper transport protein (PF14_0369) has been identified This protein is expressed in early ring stage and translocating from the erythrocyte plasma membrane to a parasite membrane as the parasites developed to schizonts. Inhibition of copper uptake with neocuproine inhibits the ring to trophozoite transition.

The parasite is dependent on the acquisition of pantothenate from the host. The transporter - PfPAT - has been cloned. The transporter is located in the parasite's plasma membrane and plays an essential role in its intraerythrocytic development. It can be inhibited by the drug fenpropimorph.

Secretion

Protein export into the infected erythrocyte is critical for malaria parasite survival and the majority of effector proteins are thought to export via a proteinaceous translocon. This is found in the parasitophorous vacuole membrane surrounding the parasite. Exported protein 2 is a critical component of this system.

A translocon (PTEX) of several proteins is located in the vacuole membrane. These include heat shock protein 101 (HSP101) a ClpA/B-like ATPase from the AAA+ superfamily, of a type commonly associated with protein translocons, a novel protein termed PTEX150 and a known parasite protein - exported protein 2 (Exp2). Exp2 is the potential channel as it is the membrane-associated component of the core PTEX complex. Two other proteins - a new protein PTEX88 and thioredoxin 2 (Trx2) - are also PTEX components. These latter two proteins do not appear to be essential. Deletion of either of these last two proteins is associated with a reduction in the replication rate in the blood.

PTEX88 is diffusely located within the blood stage parasites. In trophozoites, PTEX88 is also localized to previously unrecognized extensions reaching from the parasite surface into the erythrocyte cytoplasm.

The thioredoxin 2 protein is part of the multi-protein complex embedded within the parasitophorous vacuolar membrane and is thought to be involved in protein secretion. This protein is located in distinct punctate organelles of unknown identity.

Within the genome are encoded 11 Rab GTPases. These proteins are typically involved in vesicle transport. Casein kinase-1 has been shown to interact with RAB5B and the catalytic subunit of cAMP-dependent protein kinase A interacts with RAB5A and RAB7.

Several proteins are transported across its plasma membrane, the surrounding parasitophorous vacuole membrane and into its host erythrocyte. Most of these exported proteins contain a host targeting motif. Cleavage is of this motif by the protease plasmepsin V is normally part of this process. This process is not linked to the next component in the export pathway. The fifth reside in the host motif is important for the action of plasmepsin V. Mutation of the fourth and fifth positions of this motif, as well as amino acids further downstream, block or affect the efficiency of protein export.

Surfin 4.1 is a type 1 transmembrane protein expressed on the surface of infected erythrocytes. It is exported to the surface via the endoplasmic reticulum/Golgi apperatus. Its structure varies between strains: in 3D7 there are 19 amino acids after the transmembrane region while FCR3 there are two tryptophan rich domains after the transmembrane domain. The transmembrane region is required for the initial movement of the protein to the endoplasmic reticulum. The subsequent sorting step to the parasitophorous vacuole is determined by two independent signals located within the N terminual 50 amino acids. It may form a homodimer during transport.

Sodium is actively extruded from the parasite: at least one of the mechanisms involved in this process is PfATP4.

A mutation in the mu-chain of the AP2 adaptor complex - a component of the endocytic machinery - has been associated with artemisinin resistance in Plasmodium chabaudi. This protein interacts with a cargo recognition protein.

An essential protein - Cleft-like Protein 1 - has been cloned in P. berghei. This protein has two predicted transmembrane domains in the C-terminal end. It is found in discrete convoluted, vesico-tubular membranous structures in the erythrocyte cytoplasm.

Band 4.1 is clover leaf shaped protein which interacts with multiple erythrocytic proteins via its three arms known as the N, C and alpha lobes. This protein is involved in maintaining the biconcave shape, elasticity, and mechanical stability of human erythrocytes, and defects in 4.1R are one cause of hereditary erythrocyte elliptocytosis. The mature parasite-infected erythrocyte surface antigen (MESA) interacts with the C lobe. A secreted protein - PF3D7_0402000 - is localised to the parasitophorous vacuole membrane and interacts with Band 4.1.

Several of the exported proteins - PfEMP1, PfEMP3, ring associated erythrocyte surface antigen (RESA) and knob associated histidine rich protein (KAHRP) - interact with the preponderant erythrocyte skeleton protein spectrin. KAHRP also binds to ankyrin R. KAHRP binds to ankyrin via a 79 residue segment: the reciprocal binding site for KAHRP is a subdomain (D3) of the ankyrin R membrane binding domain. KAHRP is normally associated with the host cell membrane. Blocking this interaction with ankyrin R prevents KAHRP movement to the host membrane: instead KAHRP remains diffusely distributed throughout the erythrocyte cytosol.

Kinases

Although several kinases are known in P. falciparum (~90 in total) very little is known about them.

Cyclin dependent kinases

A subgroup of cyclin-dependent kinases (CDK) including crk-5 have an activation loop that contains a novel Proline-Threonine-x-Cytosine motif which is absent from all known CDKs outside the Apicomplexa.

The protein PFD0975w appears to be homologous with the right open reading frame 2 kinase RIO-2, a kinase involved in ribosome biogenesis and other cell cycle events. This enzyme is unique among the kinases in the genome because along with the kinase domain, it also has a highly conserved N-terminal winged helix domain.

The right open reading frame 2 protein kinase may be a potential drug target.

Cyclin dependent like kinases

Several are cyclin dependent like kinases (CLK): of these two - the Lammer kinase homologue PfCLK-1 and PfCLK-2 have been cloned. CLKs in other eukaryotes are involved in the regulation of mRNA splicing through phosphorylation of serine/arginine-rich proteins. Both are transcribed throughout the asexual blood stages and in gametocytes. PfCLK-1/Lammer possesses two nuclear localization signal sites while PfCLK-2 possesses one of these signal sites upstream of the C-terminal catalytic domains. The two PfCLKs form complexes with proteins with predicted nuclease, phosphatase or helicase functions.

Although the kinases are primarily localized in the parasite nucleus, PfCLK-2 is also present in the cytoplasm. They are important for completion of the asexual replication cycle. Substrates phosphorylated by the PfCLKs include the Sky1p substrate, splicing factor Npl3p, and the plasmodial alternative splicing factor PfASF-1.

NIMA kinases

Within the genome is a family of four protein kinases (Pfnek-1 to -4) that are related to the NIMA (never-in-mitosis/Aspergillus) family of kinases. The members of this latter family play important roles in mitosis and meiosis. Pfnek-1 (PFL1370w) is expressed in asexual parasites and male gametocytes. It is an essential gene for completion of the asexual cycle. The other three - Pfnek-2 (PFE1290w), -3 (PFL0080c) and -4 (MAL7P1.100) - are expressed predominantly in gametocytes.

Pfnek-2 is predominantly expressed in gametocytes and is required for DNA replication during meiosis and ookinete development.

The plasmodial mitogen-activated protein kinase kinase Pfnek-3 has both serine/threonine and tyrosine kinase activities.

Pfnek-4 is expressed in stage II to V gametocytes and in a subset of asexual stage parasites undergoing schizogony. It is also required for the completion of meiosis in the ookinete.

Adenylate kinases

There are at least three adenylate kinases (AK) encoded in the genome - PfAK1, PfAK2 and a GTP:AMP phosphotransferase (PfGAK). There are two additional adenylate kinase-like proteins - PfAKLP1 (which is homologous to human AK6) and PfAKLP2. PfAK1, PfAKLP1, and PfAKLP2 are found in the cytosol. PfGAK is located in the mitochondrion. PfAK2 is located at the parasitophorous vacuole membrane and this localization is driven by N-myristoylation.

Adenylate kinases are phosphotransferases that catalyze the interconversion of adenine nucleotides. There are at least three adenylate kinases (PfAK1, PfAK2 and GTP:AMP phosphotransferase) encoded in the genome. PfAK1 and PfAK2 both catalyse the conversion of ATP and AMP to two molecules of ADP. PfGAK instead has a preference for GTP and AMP and does not accept ATP as a substrate.

Calcium dependent kinases

The calcium dependent protein kinases (CDPK) are part of a superfamily found in plants, ciliates and some apicomplexa. They are not present in fungi or animals. They have three domains: a variable N-terminal region involved in substrate recognition and protein interaction, a kinase catalytic domain and a regulatory domain. The regulatory domain has two subdomains - an autoinhibitory junction domain and a calmodulin like domain. The calmodulin domain has four EF hands. These hands, upon binding calcium, undergo a structural change that moves the junction domain from its autoinhibitory interaction with the substrate binding site of the kinase domain which in turn activates kinase domain catalytic activity.

Seven CDPKs are present in the genome. The first of these cloned was PfCDPK2 in 1997.

Calcium dependent protein kinase 1 is expressed in parasite asexual blood and mosquito stages. This protein has a specific auto inhibitory junction region (J). It localises to the parasite plasma membrane of very young intracellular parasites, replicating and invasive forms. It does not appear to be exported into the erythrocyte. Inhibition of this protein results in the arrest of parasite development late in the cell cycle during early schizogony. This protein also appears to have a role in microneme secretion during the process of merozoite invasion of the erythrocyte. It also phosphorylates members of the actin-myosin complex.

Calcium dependent protein kinase 1 autophosphorylates at several site and has an ATP binding site in its N terminal. The protein is myristoylated at its N terminus and is localised to the parasitophorous vacuole and the tubovesicular system of the parasite. It is also palmitoylated at its N terminus and has a basic motif located there.

The homolog of calcium dependent protein kinase 1 (CDPK1) in Toxoplasma gondii is calcium dependent protein kinase 3 (TgCDPK3). This protein in Toxoplasma is localised to the inner membrane and is not an essential gene. It is involved in Ca(2+) ionophore control and host cell egress. The role of this protein in Plasmodium is not currently known. It is however expressed and localises with proteins at the periphery of the schizonts and merozoites involved in gliding motility and can can phosphorylate these proteins. Inhibition of CDPK 1 is associated with a block in development at the schizont level. In P bergei CDPK1 regulates transcription of stored mRNA during ookinete development in the mosquito midgut.

In P. falciparum CDPK5 controls parasite egress from host cells. In P. bergei CDPK3 is essential for the ookinete to traverse the mosquito midgut epithelium and CDPK4 is involved in development of the male gametocyte. In P. falciparum CDPK4 appears to be involved in the exflagelation of the male gametes.

Mitogen activated kinases

A mitogen activated protein kinase (MAP kinase) gene is located on is located on chromosome 14. It is predominantly expressed in gametocytes and gametes/zygotes. The protein has 882 amino acid residues and possesses a TDY dual phosphorylation site upstream of the highly conserved VATRWYRAPE sequence within subdomain VIII. Within the carboxyl-terminal segment the protein contains an unusually large and highly charged domain. This region includes two repetitive sequences of either a tetrapeptide or octapeptide motif.

Two mitogen activated protein kinases are present in the genome. Both genes appear to be transcribed during the liver stages.

An atypical mitogen activated protein kinase (MAPK) - Pfmap-2 - is known. It posses the usual properties of a MAPK - including (i) the ability to undergo autophosphorylation, (ii) the ability to phosphorylate myelin basic protein, a classical MAPK substrate, (iii) the regulation of kinase activity by a MAPK-specific phosphatase and (iv) the ability to be activated by component(s) present in cell extracts. It is expressed in gametocytes. It lacks the conserved threonine-X-tyrosine activation motif usually found in enzymes of this family and instead has a threonine-serine-histidine at the same location.

FIKK kinases

A groups of 20 kinases with a Phe-Ile-Lys-Lys sequence motif appear to be unique to P. falciparum. One of these kinases (PfFk4.1, PFD1165w) has been cloned. It autophosphoralates and phosphorlyates dematin, a cytoskeletal protein found at the erythrocyte spectrin-actin junction.

Other kinases

The protein kinase CK2, a serine/threonine protein kinase, has one catalytic subunit (PfCK2) and two regulatory ones (PfCK2beta1 and PfCK2beta2). This enzyme is found both in the cytoplasm and the nucleus. Substrates include the nucleosome assembly proteins (Naps), histones and two members of the Alba family. Both of the two regulatory subunits are required for completion of the asexual erythrocytic cycle.

The cyclic guanine monophosphate dependent protein kinase is essential for the initiation of gametogenesis and for blood stage schizont rupture and may also be involved in ookinete differentiation and motility and liver stage schizont development.

Pantothenate kinase, the first enzyme involved in converting pantothenate to coenzyme A is present in the genome. It appears to be an essential enzyme.

A putative O-phosphoseryl-tRNA(Sec) kinase - an enzyme involved in the formation of selenocysteine tRNA - has been identified in the genome. A protein kinase C has been identified. Although predominantly cytosolic it is also present in the membrane faction. Its activation requires Ca²⁺, phosphatidyl serine and either diacylglycerol or phorbol myristate acetate. Its activity is 9 time greater in the trophozoites than in the ring forms. On activiation in the trophozoites the activity in the membrane farction increase significantly. Ita actvity is inhibited in a dose dependent fashion by chloroquine. The inhibition appears to be non compeditive. Chloroquine resistant strains do not show this inhibition of activity.

A tyrosine kinase like kinase (PfTKL2) of the IRAK/RLK/Pelle protein family has been identified in the genome. The gene is expressed in asexual blood stages and in gametocytes. It is also secreted into the culture media. Its function is not presently known.

Another kinase - PfPK7 - is involved in the rate of asexual growth in erythrocytes, the production merozoites in the schizonts and in the production of oocysts in the mosquito vector.

Phosphatases

There are 27 putative protein phosphatases in the genome. These can be classed into groups: phosphoprotein phosphatases, metallo-dependent protein phosphatases, protein tyrosine phosphatases and NLI interacting factor-like phosphatases.

A Shewanella-like protein phosphatase that is expressed at all stages of the parasite life cycle but is particularly bundant in asexual blood stages and expressed at all stages of the parasite life cycle is essential for ookinete (zygote) development and microneme formation.

The open reading frame PF3D7_1305500 encodes an atypical MAPK phosphatase. This gene appears to be involved in the regulation of the transition from the pre-S phase to the M phases of asexual intraerythrocytic development.

Protein phosphatase 1 is a key enzyme that plays diverse and essential roles in cell biology. Its dephosphorylation activity/specificity is governed by the interaction of its catalytic subunit (PP1c) with regulatory proteins. A homolog of the inhibitor of protein phosphatase 1 has been cloned. This gene is essential for survival and appears to be localised to the nucleus. A conserved 41- Lysine-Valine-Valine-Arginine-Tryptophan-45 motif is essential for its inhibition activity. Another inhibitor - inhibitor 2 - has also been cloned. Within the protein are two motifs - 12-Lysine-Threonine-Isoleucine-Serine-Tryptophan-16 and 102-Histadine-Tyrosine-Asparagine-Glutamine-105 - that are critical for its activity. It appears to be an essential gene and seems likely to act at the G2/M point of the cell cycle.

Cysteine proteases

A number of cysteine proteases have been identified this organism including four falcipains, serine repeat antigens (SERA), dipeptidyl aminopeptidase 1, dipeptidyl aminopeptidase 3 and a calpain homolog.

Falcipains

The falcipains belong to the papain family of enzymes (clan CA).

Falcipain-1 appears to be important in the development of the oocysts in the mosquito. The ortholog of this protein in Plasmodium chabaudi - chabaupain-1 - is localised preferentially to the apical portion of the ookinete.

Falcipain-2 is involved in the hydrolysis of haemoglobin and appears to be a non essential gene. It also promotes host cell rupture by cleaving the skeletal proteins of the erythrocyte membrane.

Falcipain-3 appears to be an essential gene but its function has yet to be firmly established.

Dipeptidyl aminopeptidases

Dipeptidyl aminopeptidase 1 is found in the digestive vacuole and is also an essential gene.

Dipeptidyl aminopeptidase 2 is specific to the gametocyte. It does not appear to be an essential gene.

Dipeptidyl aminopeptidase 3 appears to be involved in the release of the merozoites from the erythrocyte.

Serine repeat antigens

The serine repeat antigens are a set of nine proteins in P. falciparum (two in P. gallinaceum, five in P. bergei, 12 in P. vivax) with a central domain homologous to the papain-like (clan CA, family C1) protease family. They are named after the presence of tandem repeats of serine residues in the protein.

The only known homolog of the SERA proteases is found in the parasite Theileria. In a study of 18 species the majority of the SERA genes were found to be clustered between between two conserved genes: a conserved hypothetical protein (HP) gene and the iron-sulfur assembly protein gene (hesB). There is a signal peptide sequence at the N-terminus. The family can be divided into four groups: Groups I to III have a cysteine at the active site and Group IV has a serine residue. Group II and III are sister clades and are more closely related to Group I than to Group IV. With a few exceptions the SERA gene family has a common structure of four exons and three introns. SERA genes of Group I in general have a six exon and five intron structure. In the mammalian malaria parasites, gene duplication occurred only in Group IV SERA genes and was particularly frequent in primate species. The reasons are unknown presently.

Although most SERAs are cysteine proteases the majority in P. falciparum (6 of the 9) have a serine residue at the active site. The SERAs possessing a canonical Cys residue at the position of the active site nucleophile in their papain-like central domains are with SERA6, 7 and 8.

SERA5 and SERA6 are indispensable in blood-stage parasites. SERA 5 (molecular weight 120 kiloDaltons) is abundantly produced at the late trophozoite to schizont stages of parasite development. SERA 6 is produced in much smaller quantities than SERA 5 and appears to be involved with egress from the erythrocyte. SERA 6 is activated by SUB1 - a seine protease that is stored in exonomes.

SERA8 in P. falciparum (SERA5 in P. berghei) is expressed exclusively during mosquito stages of the parasite life cycle, where it has been shown to be required for egress of midgut sporozoites from oocysts.

Other proteases

A calpain homolog has been cloned. Although shorter than other calpains it possesses a typical catalytic triad (Cysteine-Histamine-Asparagine).

A cysteine protease inhibitor - falstatin - is involved in regulating proteolysis during erythrocyte infection. It is found in vesicles within the asexual blood stage parasite cytoplasm, the parasitophorous vacuole and is exported to dynamic exomembrane structures in the infected erythrocyte. In sporozoites, its is found in the rhoptries and in intracellular vesicles distinct from the micronemes. In the final stages of liver infection it is released into the infected hepatocyte during parasitophorous vacuole membrane breakdown.

Homologs of these proteins have been identified and cloned in Plasmodium knowlesi. These three knowpains are found in the digestive vacuole, are active at acidic pH and are capable of degrading haemoglobin. Two of them (KP2 and KP3) cleave only if the P2 position is occupied by an leucine residue. KP4 shows a moderate preference for leucine at the P2 position but is more active if this position is occupied by an arginine residue. Although found in the digestive vacuole KP4 is also found in the parasite periphery and may play a role in parasite egress from the erythrocyte.

Metallopeptidases

There is at least one M1 family aminopeptidase in the genome (PfA-M1). This is a zinc binding metalopeptidase with optimal activity at pH 7.4, and remains at least 40% active between pH 5.8-8.6. It is an alanyl aminopeptidase. Immunofluorescence studies have shown that in trophozoites that it diffusely found in the parasite cytoplasm with accumulations outside the digestive vacuole while in schizonts it is progressively located to a vesicle like pattern ending as a single location in released merozoites. It exists as two major isoforms, a nuclear 120 kDa species and a processed species consisting of a complex of 68 and 35 kDa fragments.

There are at least 2 essential metallopeptidases encoded in the genome - PfA-M1 and Pf-LAP. Specific inhibition of PfA-M1 causes swelling of the parasite digestive vacuole and prevented proteolysis of haemoglobin derived oligopeptides. This inhibition is lethal to the parasite probably by starvation.

Leucyl aminopeptidase (LAP) is a member of the M17 family. It has a predicted molecular weight of 67.831 kiloDaltons and is localized in the cytosol. It is inhibited by bestatin. Its inhibition is lethal to parasites early in the life cycle, prior to the onset of haemoglobin degradation suggesting a different role for this enzyme.

Falcilysin a zinc metalloprotease found in the apicoplast. It is a member of the M16 protease group and has maximal activity at neutral pH. It appears to be an essential gene. Its function in this organelle is not quite clear but it appears to be involved in the degradation of transit peptides.

M18 AAP is a metallo-aminopeptidase that has a highly restricted specificity for peptides with an N-terminal glutamine or asparagine residue.

The gene PFI1625c appears to be a metaloprotease.

Aspartyl proteases

At least four aspartyl proteases known as plasmepsins are involved in the degradation of haemoglobin by Plasmodium falciparum.

The histo-aspartic protease (HAP) has been crystallised. This protein has high sequence similarity to pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced in this enzyme by a histidine residue. The propeptide interacts with the C-terminal domain of the enzyme, forcing the N- and C- terminal domains apart. This mechanically separates His32 and Asp215 and prevents formation of the mature active site. This mechanism is similar to those of other proplasmepsins. The enzyme has a number of unique features and may be a useful drug target.

There are at least 10 aspartic proteases encoded within the genome. Plasmepsins I, II, IV and histo-aspartic protease are known to be involved in the digestion of haemoglobin. These four enzymes share 50-79% amino acid sequence identity and are located on chromosome 14 (gene identifiers PF14_0076, PF14_0077, PF14_0078, and PF14_0075 respectively). Plasmepsins I and II are present in the food vacuole and make the initial cleavages in the hemoglobin molecule. The proplasmepsins I and II are both type II integral membrane proteins that are transported through the secretory pathway before cleavage to the soluble form. This reaction occurs within the food vacuole and the cleavage occurs immediately after a conserved Leucine-Glycine dipeptidyl motif. This reaction may be blocked calpain inhibitors. It appears that plasmepsin II and IV are capable of autoactivation as well as activation each other's inactive form. These two proteins are not glycosylated. Plasmepsin I is synthesized and processed to the mature form soon after the parasite invades the red blood cell, while plasmepsin II synthesis is delayed until later in development.

Intramembrane proteases

Plasmepsin V, an integral membrane protein, is located within the endoplasmic reticulum but not in the Golgi apperatus. The gene is expressed over the course of asexual intraerythrocytic development. The amount of the protein in the parasite is lowest in the ring stage and increases steadily through schizogony. It appears to be involved in the export of proteins to the erythrocyte. Plasmepsin V cleaves N-terminal sequences from RIFIN, STEVOR and RESA multigene families but does not cleave the N-terminal sequence of erythrocyte membrane protein 1, skeleton binding protein (SBP-1) or REX-2.

A presenilin-like signal peptide peptidase is known to be present in the endoplasmic reticulum.

Serine proteases

A SUMO specific protease PfSENP1 (PFL1635w) has been identified in the genome but its importance if any is not known

There are at least 3 subtilisin like proteases encoded in the genome. These are serine proteases.

SUB1 has multiple roles in the parasite's life cycle: in the blood stages it is involved in the egress of merozoites from the infected erythrocytes and also in priming merozoites for subsequent erythrocyte invasion. It also appears to be involved in escaping from the parasitophorous vacuole membrane in the hepatocytes. Within the merozoites SUB1 is stored in exonemes and released in response to a rise in calcium. Blocking this release results in failure to process serine repeat antigen 5 (PfSERA5) and parasitophorous vacuolar membrane rupture and merozoite egress from the hepatocytes. SUB1 also acts on merozoite surface protein 1 (MSP1), MSP6 and MSP7 on the merozoite surface. Blocking the action of SUB1 on these proteins reduces the ability of the merozoite to invade erythrocytes. SUB1 also acts on serine repeat antigen 6 (SERA 6) - a cysteine protease found in the intraerythrocytic parasitophorous vacuole. The action of SUB1 activates the proteolytic function of SERA 6.

In the merozoites SUB2 has been implicated in shedding of adhesins at a juxtamembrane position. PfSUB2 is located on the surface of the parasite. Posttranslationally it is transported first to the micronemes and then to the surface. Its usual promoter is required for its final location. Its transmembrane domain is required for transport to the microneme and the cytoplasmic domain is required for its surface localization.

Another protease - PfSUB3 - is expressed at late asexual blood stages. This enzyme is known to act on profilin (PfPRF).

Intramembrane proteases

At least two rhomboid proteases (ROM1 and ROM4) are present in the genome. They are able to act on a number of substrates including TRAP, CTRP, MTRAP, PFF0800c, EBA-175, BAEBL, JESEBL, MAEBL, AMA1, Rh1, Rh2a, Rh2b and Rh4 but not PTRAMP.

The protease rhomboid-1 (ROM1: PF11_0150) is located within a single, thread-like structure on one side of the merozoites that appears to be in close proximity to the subpellicular microtubules. This organelle has been named the mononeme. The mononeme first appears in the schizonts. On the release of merozoites from schizonts, ROM1 moves from a lateral asymmetric localization to the merozoite apical pole and the posterior pole. It can cleave the apical membrane antigen 1.

ROM4 (PFE0340c) appears to be involved in the gliding motility of the sporozoite.

Actin

Within the genome are encoded two forms of the protein actin - I and II. The first form (I) is present in significantly greater quantities. Actin II appears to be essential for the process of exflagellation. Deletion of this gene results in viable asexual stages. During the formation of the male gametes actin I is found initially in both the nucleus and the cytoplasm. After activation it is found only in the cytoplasm. In actin II deletion mutants actin I remains in both the nucleus and the cytoplasm after activation. Morphologically in the actin II mutants male gametocyte DNA was replicates normally and axonemes are assembled but egress from the host cell is inhibited and axoneme motility is abolished.

Two proteins P. falciparum actin-depolymerizing factor 1 (PfADF1) and P. falciparum actin-depolymerizing factor 2 (PfADF2) are involved in the polymerisation of actin. PfADF1 has ben crystallised and despite having significant differences from other proteins with similar function it is capable of severing actin filaments. PfADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing the filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of PfADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. PfADF2 structurally resembles the canonical members of the ADF/cofilin family.

The actins found In Plasmodium and in Toxoplasma are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. This inherent instability of parasite's actin filaments is a critical adaptation for their gliding motility.

Actin is involved in the expression of the var genes. The var introns interact with an 18 base pair nuclear protein binding element which recruits actin and repositions the var DNA from a transcriptionally repressive to a transcriptionally active perinuclear compartment.

The presence of actin microfilaments has been demonstrated in the ookinete in the pellicle and in the apices.

There are two formin genes encoded in the genome. These associate with and nucleate both mammalian and Plasmodium actin filaments. Another gene profilin - also encoded in the genome but only as a single copy - sequesters actin monomers preventing their polymerisation.

Aldolase, an actin binding protein, is involved in the moving junction that forms during the invasion of the erythrocyte.

Several hemoglobinopathies that protect carriers from severe malaria may do so by interfering with host actin reorganization.

The cyclase associated proteins are among the most highly conserved regulators of actin dynamics. They catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from ADF/cofilin for new rounds of filament assembly. The Plasmodium falciparum cyclase associated protein is entirely composed of β-sheet domains and efficiently promotes nucleotide exchange on actin monomers.

Ubiquitin

The addition of the small protein ubiquitin to other proteins as part of post translational processing is widespread in most eukaryotes. This is also the case with P. falciparum where this process occurs at all stages of the asexual life cycle. Ubiquitylation involves the covalent attachment of a ubiquitin moiety to lysine residues of protein substrates via the hierarchical intervention of an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase that is usually involved in specific substrate recognition. This process is also known as sumoylation (small ubiquitin related modifiers).

Ubiquitylation is involved in removing misfolded proteins from the endoplasmic reticulum - a process known as Endoplasmic-reticulum-associated protein degradation. This process is a prerequisite for subsequent retro-translocation to the cytosol and destruction by the 26S proteasome. Aberrant proteins are recognized by endoplasmic reticulum luminal chaperone proteins and protein disulfide isomerases to help discriminate properly folded proteins from misfolded proteins. Misfolded proteins are shuttled to the DER1 translocon complex which forms a hydrophobic pore to allow the retro-translocation of proteins through the endoplasmic reticulum membrane. Several components of the system are known to be present in the parasite: HRD1 (E3 ubiquitin ligase), UBC (E2 ubiquitin conjugating enzyme) and UBA1 (E1 ubiquitin activating enzyme). HRD1 localizes to the endoplasmic reticulum membranes, while UBC and UBA1 localize to the cytosol. HRD1 interacts with membrane bound proteins needed for retro-translocation and helps form the hydrophobic pore complex. Another member of this pathway is the signal peptide peptidase.

The enzymatic mechanism is reasonably well understood. The E1 enzyme adenylates ubiquitin at its C-terminus, creating a mixed anhydride: this process requires ATP. The sulfhydryl group of the E1 active site cysteine then attacks the anhydride: thus results in the formation of a high energy thio-ester linking ubiquitin to E1. Ubiquitin is then passed to the active site cysteine of the E2 enzyme. Finally, with the aid of an E3 ligase, ubiquitin is transferred from E2 and covalently attached to the ε-amino group of a lysine in the target protein.

The genes PFL1245w is the E1 ubiquitin activating enzyme, PFL0190w is the E2 ubiquitin conjugating enzyme and PF14_0215 is the E3 ubiquitin ligase. PFL1245w (E1) contains a ubiquitin activating enzyme active site, two ubiquitin like activating enzyme catalytic domains, two ThiF repeats and a catalytic cysteine at the N-terminal end. PFL0190w (E2) is 147 amino acid residues in length and contains an ubiquitin conjugating enzyme domain takes up almost its whole length. PF14_0215 (E3) has multiple transmembrane domains, an E3 RING zinc finger (zf-C3HC4) domain on its C-terminal half and a predicted signal peptide consistent with endoplasmic reticulum targeting. The presence of four transmembrane domains is compatible with a pore forming ability and to be able to participate in the recognition and translocation of misfolded proteins across the endoplasmic reticulum membrane.

The addition of ubiquitin to a protein frequently precceds its digestion in the proteasome. The proteasome mediates the nonlysosomal degradation of cytosolic proteins in eukaryotic cells. It is a large complex consisting of two multisubunit structures, the 20S and 19S (PA700) or P28 complexes which combine to form the 26S particles. The proteasome subunit 4 ATPase has been cloned from P. falciparum. The protein is similar to those found in eukaryotes. It lacks introns.

The E1 and E2 enzymes interact to recognize and modify RanGAP1. Sumoylation actvity peaks during mid stages of the intra-erythrocyte developmental cycle.

A mutation in a deubiquitinating enzyme in Plasmodium chabaudi has been reported to cause both artesunate and chloroquine resistance.

It appears that ubiquitylation may be a mechanistic requirement of apicoplast protein import independent of the proteasomal degradation pathway.

Heat shock proteins

A number of heat shock proteins 40 (hsp40) have been predicted from the sequenced genome. Only one is predicted to be a cytosolic canonical Hsp40 capable of interacting with the major cytosolic Hsp70 an interaction that has been confirmed experimentally.

Heat shock protein 20 has been shown to have a critical role in sporozoite motility. This role appears to be via substrate adhesion.

PfGECO is a type IV heat shock protein 40 expressed in gametocyte stages I to IV and is exported to the erythrocyte cytoplasm. This gene appears to be non essential.

A Hsp40 class of chaperone (PFB0090c; PF3D7_0201800; KAHsp40) is located in a chromosomal cluster together with knob components KAHRP and PfEMP3. This protein has a PEXEL motif required for transport to the erythrocyte compartment. It occurs in punctuate spots in the erythrocyte periphery, distinctly from Maurer's clefts. These structures may be knobs particularly since it is found in a complex the known knob proteins KAHRP, PfEMP3 and Hsp101.

There are 6 HSP70 proteins in the genome. Five of these proteins are found in other species: the exception being PfHsp70-x.

Heat shock proteins Hsp70 and Hsp90 are both expressed in P. falciparum. They are linked by an essential adaptor protein known as the Hsp70-Hsp90 organising protein (Hop). This protein co-localises with PfHsp70 and PfHsp90 at the trophozoite stage and forms a complex with them.

A heat shock protein PfHsp70-x with endoplasmic reticulum signal peptide has been identified. It is maximally expressed at the schizont stage of intra erythrocytic life cycle. Although the majority of the protein localizes to the parasitophorous vacuole, some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. It lacks an endoplasmic reticumlum signal and interacts with at least some of the HSP40 proteins.

The protein Aha1 interacts with HSP90.

The heat shock protein 110 (Hsp110) is an essential protein in P. falciparum. Although its usual function is still under investigation it is known to prevent the aggregation of asparagine repeat rich proteins within the parasite. These repeat rich proteins tend to form aggregates particularly at the elevated temperatures that occur with malaria.

A 20 kiloDalton protein (cpn20) that acts as a heat shock protein and is localised in the apicoplast has been cloned.

Redox balance

The infected host cell is under considerable oxidative stress. Normal erythrocytes have a ratio of reduced (GSH) and oxidized glutathione (GSSG) of 321.6 while the GSH/GSSG ratio in infected cells is 26.7. The ratio in the parasite is 284.5. Efflux of GSSG from the intact infected cell is more than 60-fold higher than the rate observed in normal erythrocytes. This export process is mediated by permeability pathways that the parasite induces in the erythrocyte's membrane. Exogenous gamma-glutamylcysteine is not converted into GSH in the infected erythrocyte suggesting that the erythrocytes' own GSH synthetase may not be functional. This may be due to the lower levels of magnesium (Mg²⁺) in the infected erythrocyte (0.5 milliMolar) compared to the normal erythrocytes (1.5-3 mM). The lower level of results in cessation of gamma-glutamylcysteine synthesis and of GSH synthesis in the infected erythrocyte. The parasite maintains a level of 4 mM magnesium. The parasite membrane is impermeable to both gamma-glutamylcysteine and GSH.

The proteins gamma-glutamylcysteine synthetase and glutathione synthetase which are involved in the synthesis of glutathione appear to be essential genes in P. falciparum. Inhibition of the glutathione biosynthesis by the parasite is lethal. Its levels appear to be tightly regulated. The enzyme glutathione reductase is highly specific for its substrate glutathione disulfide.

Glutathione export from parasitized cells is inhibited partially by both the compound MK571 and by furosemide. These agents are inhibitors of the 'new permeability pathways' induced by the parasite in the host erythrocyte membrane.

A possible connextion between the redox system and chloroquine resistance has been described. Strains carrying the mutant chloroquine resistant transport gene have lower levels of glutathione and are more sensitive to inhibition of glutathione synthesis. The mutant chloroquine resistance transport gene selectively transports glutathione into the digestive vacuole. Also in the mutant strains the haemozoin levels are lower and there is reduced chloroquine binding by haemozoin. It seems likely that the increased transport of glutathione into the digestive vacuole may alter both the synthesis of haemozoin within the digestive vacuole and the binding of chloroquine to haemozoin within the digestive vacuole.

The thioredoxin system like the glutathione system is responsible for maintaining the redox balance in the cell. The thioredoxin reductase reduces thioredoxin and a number of other low molecular weight compounds. The other members of this system include five peroxiredoxins differentially located in the cytosol, apicoplast, mitochondria and nucleus with partially overlapping substrate preferences. It also includes members of the thioredoxin superfamily with three thioredoxins, two thioredoxin-like proteins, a dithiol and three monocysteine glutaredoxins and a redox-active plasmoredoxin being encoded in the genome.

Within the cytoplasm two peroxiredoxins - T peroxiredoxin-1 (TPx-1) and 1-Cys peroxiredoxin (1-Cys Prx) - are produced at differing points in the life cycle. Disruption of the T peroxiredoxin-1 enzymes renders the parasite hypersensitive to heat stress. This does not occur with knock out mutants of 1-Cys peroxiredoxin suggesting that these enzymes have different roles in the life cycle.

Investigation of the liver stages of these enzymes in Plasmodium berghei has shown that both TPx-1 and 1-Cys Prx are present in the cytosol but differ in their expression patterns. TPx-1 is transcribed shortly after infection of the hepatocyte and expression continues until the schizont stage. Transcription of 1-Cys Prx starts after the parasite has developed into the schizont stage.

The 1-Cys peroxiredoxin enzyme appears to be located in the cytoplasm. The peroxiredoxin requires both glutaredoxin and glutathione for its activity.

The crystal structure of thioredoxin reductase has been solved. There are significant differences between the human enzyme and the plasmodial which are most apparent in the Plasmodium-specific insertion and the conformation of the flexible C-terminal arm.

Lysis of the erythrocyte releases methaemoglobin. Exposure of uninfected erythrocytes to methaemoglobin renders them susceptible to osmotic stress and haemolysis. This mechanism may form part of a chain reaction resulting in the haemolysis that is found in severe malaria. Both N-acetyl cysteine and mannitol can reduce this oxidative stress. Clotrimazole irreversibly inactivates methaemoglobin and abolishes its peroxidase activity.

Ferriprotoporphyrin IX is released inside the digestive vacuole of the malaria parasite during the digestion of host cell hemoglobin. Undegraded ferriprotoporphyrin IX accumulates in the membrane fraction and is degraded by reduced glutathione in a radical mediated mechanism.

Three phosducin-like proteins have been identified in Plasmodium berghei. Their role in the parasite's metabolism has yet to be clearly established.

The type II NADH:ubiquinone oxidoreductase has been shown to be redundant in the blood forms but to be essential in the mosquito midgut.

At least one function of B₆ in this parasite is as an antioxidant.

The thioredoxin like protein TRX2 is not essential but its deletion reduces growth rate.

The glyoxalase system is the main catabolic route for methylglyoxal, a non-enzymatic glycolytic byproduct with toxic and mutagenic effects. In Plasmodium falciparum the glyoxalase pathway is glutathione dependent. Its glyoxalase I is an atypical monomeric enzyme with two active sites.

Calcium fluctuation

In recently invaded erythrocytes the Ca²⁺ concentration increases about 10 fold. The Ca²⁺ content increases as the parasite matures. In infected erythrocytes, Ca²⁺ is almost exclusively localized in the parasite compartment and changes but little in the cytosol of the host cell.

Cytosolic calcium²⁺ increases evoked by extracellular stimuli are may be observed in the form of oscillating Ca²⁺ spikes in eukaryotic cells. Spontaneous spikes in the calcium levels have been observed in Plasmodium falciparum. The frequency of Ca2+ oscillations are higher in early ring forms than that in early trophozoites. Blockage of this oscillation results in the cessation of intraerythrocytic maturation and death of the parasite. This effect is maximal in the trophozoites.

An inositol phosphate kinase with a role in calcium metabolism has been cloned.

Expression of the calmodulin gene is developmentally regulated throughout the blood-stage cycle.

Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, usually by liberation of calcium from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate. These genes are found in P. falciparum and appear to be essential. The genes are twice as long as their mammalian counterparts and belong to the delta class of phospholipase C proteins.

Nucleotide metabolism

P. falciparum is unable to biosynthesize purines. Instead, the parasite is able to transport and interconvert host purines. The enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase converts hypoxanthine to inosine monophosphate and is essential for purine salvage.

Conversely, the parasite can produce pyrimidines de novo using glutamine, bicarbonate and aspartate.

A gene encoding S-adenosyl-L-homocysteine hydrolase is present in the genome.

P. falciparum contains both cytosolic and mitochondrial serine hydroxymethyltransferase isoforms. This is a pyridoxal phosphate dependent enzyme which plays a vital role in the de novo pyrimidine biosynthesis pathway. Both genes are expressed throughout the erythrocytic stages. Both enzymes appear to be essential.

A conserved trytophan residue is involved in the activity of the purine nucleoside phosphorylase enzyme.

Orotate phosphoribosyltransferase catalyzes the magnesium dependent condensation of orotic acid with 5-α-D-phosphorylribose 1-diphosphate to yield diphosphate and the nucleotide orotidine 5'-monophosphate. This enzyme has been crystallised.

Molecular biology

The long adenosine/thymidine tracts that are scattered through the genome may play a role in gene duplication.

The centromeres occupy a 4-4.5 kilobase region in each chromosome. The centromeres cluster to a single nuclear location prior to and during mitosis and cytokinesis but dissociate soon after invasion.

The DNA polymerase is unusual. They share a large amino-terminal domains with putative helicase/primase elements features that are known only in the thermophilic viruses and Aquificae. A horizont transfer seems the most likely explanation for these findings.

The telomerase (tert) is a large protein (2518 codons) and has a predicted molecular weight of ~280 kiloDaltons. It has the usual telomerase specific motifs within the N-terminal half of the protein (GQ/N, CP, QFP and T) and reverse transcriptase (RT) specific motifs in the C-terminal half. The N-terminal half is required for efficient binding of the RNA template, defining the 5′ RNA template boundary, multimerization and interactions with associated proteins. The RT domain is essential for the catalytic activity. The protein contains several nuclear localization signals and is found in the nucleolus.

A putative tyrosine site specific recombinase has been isolated. The N-terminus has the typical alpha helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. The C-terminal domain has the putative tyrosine recombinase conserved active site residues Lysine-Histadine-Lysine-(Histadine/Tryptophan)-Tyrosine. The gene is expressed differentially during the erythrocytic stages being maximal in the schizont stage. The open reading frame encodes a ∼57 kiloDalton protein. Knockout mutants are viable and appear normal. DNA binding studies suggest a number of targets include the subtelomeric regions.

A number of mini chromosome maintenance proteins are present in the genome. These are large proteins and members of the AAA ATPase family with a conserved region of ~200 amino acids responsible for nucleotide binding. They are responsible for unwinding DNA at the replication forks and are involved in other chromosome transactions such as transcription, chromatin remodeling and genome stability.

A SIP2 gene, a member of the ApiAP2 family of putative transcription factors, has been cloned. It appears to be involved in maintenance of the chromosome ends rather than in the regulation of particular genes.

A novel DNA/RNA binding protein PfAlba has been described. This protein is related to the archaeal protein Alba (Acetylation lowers binding affinity). There are at least four paralogs of the PfAlba gene and these proteins form a complex with the P. falciparum specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric regions. Also associated with the TARE6 regions are PfSir2 a histone deacetylase. In the early blood stages the PfAlba proteins are enriched at the nuclear periphery and associate with the PfSir2 proteins. When the parasite switches from trophozoite to the schizont stage the PfAlba proteins move to the cytoplasm. These proteins will also bind single stranded RNA but the reason for this binding is not known.

The single stranded DNA binding protein (SSB) plays an important role in all known organisms. A SSB protein is encoded in the genome and localises to the apicoplast. It forms a homo-tetramer alone and when bound to single stranded DNA. The protein binds 52-65 nucleotides/tetramer. While similar in its overall structure to that of the SSB of E. coli it differs at the carboxy terminal region. Although it binds single stranded DNA in a similar fashion to the SSB of E. coli it does so with the opposite polarity. There are a number of other functional differences between this protein and that of E. coli. The basis for these differences has yet to be determined.

A protein - RPA1L - is the homologue of the bacterial single stranded binding protein (SSB) and acts in initiating homologous pairing and strand exchange activity. It is negatively regulated in a dose dependent manner by RPA1S.

The eukaryotic homologue of the bacterial RecA protein is Rad51. The Plasmodium falciparum Rad51 protein exhibits ATPase activity and promotes DNA strand exchange. This protein interacts with Rad54 and replication protein A.

SET is a conserved nuclear protein involved in chromatin dynamics. In P falciparum it is expressed in both asexual and sexual blood stages but strongly accumulates in male gametocytes. In P falciparum there are two distinct promoters upstream. One is active in all blood stages while the other is active only in gametocytes and in a fraction of schizonts possibly committed to sexual differentiation. In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte specific promoter is silent and the reporter gene is only transcribed from the constitutive promoter.

Plasmodium appears to lack both DNA methylation and the RNA interference machinery.

DDX6/DOZI (development of zygote inhibited) is a member of the DEAD box family and is involved in the sexual development of the protozoan parasite. The gene is known as PfDZ50 in P. falciparum. It binds DNA and RNA and has nucleic acid dependent ATPase and RNA unwinding activities. It interacts with eIF4E mainly through domain 1 and inhibits translation. It is localized mainly in the granular bodies found throughout the cytoplasm during the asexual intraerythrocytic developmental stages.

A novel transcription factor - PREBP - has been described. This protein has 4 KH homology domains which are found in RNA-binding or single-stranded DNA-binding proteins. PREBP is well conserved in Plasmodium species and partially conserved in phylum Apicomplexa. It acts on the pf1-cys-prx, a gene expressed in the trophozoite/schizont stages.

DNA repair proteins

Several nucleic acid repair pathways are known. These include the nucleotide excision repair, the mismatch repair, the base excision repair, the double strand break repair and the cross link repair pathways. DNA replication errors - base substitution mismatches and insertion-deletion loops - are primarily corrected by the mismatch repair system.

The MutL homolog (MLH) - part of the DNA mismatch repair system - has been cloned. MLH possess ATPase and endonuclease activities. Its expression is maximal in the schizont stage.

Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to replace missing nucleotides and rejoin broken strands. A P. falciparum gene encoding a protein with 24% homology to human PNKP has been cloned. This enzyme dephosphorylates single-stranded substrates or double-stranded substrates with a short 3'-single-stranded overhang, but not double-stranded substrates that mimicked single-strand breaks.

RNA binding proteins

The messenger RNA capping system appears to be more similar to that of fungi than to vertebrates. It encodes RNA guanylyltransferase (Pgt1) and RNA triphosphatase (Prt1) enzymes. The triphosphatase enzyme is a member of the fungal/viral family of metal dependent phosphohydrolases. These are structurally and mechanistically unrelated to the cysteine phosphatase type RNA triphosphatases found in metazoans and plants.

A protein (PfSR1) involved in alternative splicing has been described. It appears that regulation of this gene is essential for the parasite's normal physiology.

A number of the DExD/DExH-box containing pre-mRNA processing proteins (Prps) - PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p - are present in the genome. PfPrp16p a helicase and a member of DEAH-box protein family with nine collinear sequence motifs has been cloned. It binds to RNA, hydrolyses ATP and appears to be involved in splicing.

The RNA polymerase II has an unual expansion in the C terminal domain. The primate infecting species posses a larger number of repeats that are found in either the bird or rodent species.

A cyclin (Pfcyc-1) has been cloned and its structure has been solved. The protein has a typical cyclin box which consists of two tandemly repeating five-helix bundles separated by a linker hinge peptide.

The origin recognition complex subunit 1 (Orc1) associates with fSir2 in the sliencing complex.

At least seven proteins are known to be involved in the splicing process. They are expressed at asexual blood stages of the parasite, show nucleo-cytoplasmic localization and form a ring like heptameric complex like other eukaryotes. The interaction of PfSMN (survival of motor neuron, Tudor domain containing protein) or PfTu-TSN (tudor domain of Tudor Staphylococcal nuclease) with the PfSmD1 protein is methylation dependent. The arginine methylase PfPRMT5 appear to interact with PfSmD1 suggesting a role for arginine methylation in the assembly of the spliceosome complex.

Helicases

An unusual helicase - a homologue of Dbp5 and DDX19 from yeast and human respectively - has been cloned. It possesses DNA and RNA unwinding, nucleic acid dependent ATPase and RNA binding activities. A Q motif is required for its activity.

Another DEAD box helicase - a homolog of Has1p from yeast which has DNA and RNA unwinding, nucleic acid dependent ATPase and RNA binding activities - has also been cloned.

The DNA helicase II (uvrD) is a superfamily 1A helicase which plays an essential role in the mismatch repair pathway. Homologs of UvrD include the proteins PcrA and Rep. These proteins have a two domain (1 and 2) structure with each domain made of two sub-domains (1A, 1B, 2A and 2B) and a C-terminal extension. They are DNA-dependent ATPases with 3′ to 5′ helicase activity. The helicase activity is located in the N terminal domain.

The UvrD protein of P. falciparum has been cloned. This gene (PFE0705c) is located on chromosome 5 and contains no introns. It is 4326 bases in length, encodes a protein of 1441 amino acids and has a predicted molecular weight of ~170 kiloDaltons. The two domains and their subdomains are present: The 1A domain is from amino acid 1–722; the 1B domain is from amino acid 150–464; the 2A domain is from amino acid 723–1441; and the 2B domain is from amino acid 896–1359. There is no C-terminal extension. The ATPase and helicase activity are confined to domain 1A and 1B (the N-terminal and first half of the C terminal). It is expressed in the schizont stages of intraerythrocytic development and it colocalizes with PfMLH, a protein involved in mismatch repair. Both PfDH60 - another helicase - and PfMLH are also expressed in schizont stages.

A helicase - PfH45 - of 398 amino acid residues (molecular weight 45 kiloDaltons) is a unique bipolar helicase with both the 3' to 5' and 5' to 3' directional helicase activities. It is expressed in all the intraerythrocytic developmental stages and has a role in translation.

A 3'-5' DNA helicase has been identified in the parasite. The apparent molecular weight is 90 kiloDaltons. Its activity is dependent on the presence of magnesium and ATP.

A homolog of UAP56 (U2AF65 associated protein) - a member of the DEAD box helicase family - has been cloned. This homolog - PfU52 - contains the RNA dependent ATPase, RNA helicase and RNA binding activities. This protein is expressed in all the intraerythrocytic developmental stages of the parasite. Residues glycine 181, isoleucine 182 and arginine 206 are involved in RNA binding and this binding activity is required for its enzymatic activities.

The RuvB proteins belong to AAA+ family of enzymes which are involved in diverse cellular activities. There are at least 3 copies of this protein in the genome. The PfRuvB1 protein has considerable homology with human as well as yeast RuvB1 and contains both Walker motif A and Walker motif B. It is an ATPase and this activity increased significantly in the presence of single stranded DNA. It also has DNA helicase activity and translocates preferentially in 5' to 3' direction. It is constitutively expressed during all the stages of intraerythrocytic cycle and localizes mainly to the nucleus.

PfRuvB2 similarly has both ATPase and weak DNA helicase activities. It is expressed in all the asexual intraerythrocytic developmental stages and localizes mainly in the nucleus during merozoite, ring and trophozoite stages while during schizont stage it relocalizes partially in the nucleus and partially towards cytoplasm. It interacts with PfRuvB3 and not with PfRuvB1.

RuvB3 possesses the Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. It has single stranded DNA dependent ATPase activity. The protein is mainly expressed during intraerythrocytic schizont stages and localizes to the nuclear region. In the merozoite the protein relocalizes to the sub nuclear region. The PfRuvB2/PfRuvB3 complex preferentially translocates and unwinds DNA in the 5'-3' direction.

Histones and their modifying enzymes

Histones are essential for the correct packing and function of DNA in eukaryotes. Variants of these proteins are known to occur throughout eukaryotes and these are thought to play a role in epigenetic control. Although variants of H2A and H3 are common, variants of H2B and H4 are much less so. H2B.Z is an apicomplexan specific H2B variant. H2B.Z localises to euchromatic intergenic regions throughout intraerythrocytic development and with H2A.Z may form nucleosomes. These H2A.Z and H2B.Z nucleosomes are more common in promoters over 3' intergenic regions and their occurrence is related to the promotor's strength. The presence of H2B.Z is correlated with the base composition of the underlying DNA.

Other variants of histones known to occur in this genome are H2Bv, H3.3 and CenH3. Covalent modification of the histones is well known with at least 44 different types having been described. The deacetylation and subsequent tri-methylation of lysine 9 on histone H3 (H3K9me3) as well as the recruitment of heterochromatin protein 1 are found in heterochromatic islands and are involved in the regulation of the var genes. Other modifications that have been described include H3K4me3, H3K9ac, H3K14ac and H4ac. H2A.Z is consistently found at both 5′ and 3′ ends of the euchromatic genes. The histone acetyl transferases (Gcn5 and Esa1) that can act on H2A.Z are present in the genome.

Posttranslational covalent modifications of the histones (acetylation, methylation and phosphorylation) are known to occur. How these influence gene transcription is poorly understood. The 14-3-3 protein appears to act as a phosphorlyated histone 3 reader.

A histone deacetylase (HDAC1) has been cloned. The protein has 449 amino acid residues and localises to the nucleus. Its molecular weight is 50 kiloDaltons and it is predominantly expressed in mature asexual blood stages and in gametocytes.

Sir2A is a member of the sirtuin family of nicotinamide adenine dinucleotide dependent deacetylases. In P. falciparum it has been has been shown to regulate the expression of surface antigens to evade the detection by host immune surveillance. While it is a poor deacetylator of histones it also catalyzes the hydrolysis of medium and long chain fatty acyl groups from lysine residues. Proteins are present in P. falciparum with these modifications and these can be removed by can be removed by PfSir2A in vitro. This suggests that this may be its role rather than the deacetylation of histones.

During the life cycle the telomeres and telomere associated repeat elements are transcribed as long non coding RNAs. They are transcribed by RNA polymerase II as single-stranded molecules. In the ring stage, these transcripts are located in a single perinuclear compartment that does not co-localize with any known nuclear subcompartment. During the schizont stage they are found at several nuclear foci. At least some of these can form stable and repetitive hairpin structure that is able to bind histones. Their function requires further elucidation.

Reversible histone modifications can cause changes in gene expression and alter the phenotype.

Gene regulation

A number of novel DNA binding sites have been identified along the genome. Their function - if any - remains to be determined.

Apetala 2 (AP2) family proteins are transcription factors that have DNA-binding domains of ~60 amino acids called AP2 domains. 27 AP2-family genes have been identified in the Plasmodium falciparum genome. One of these proteins appears to play a critical role in the liver stage development of the parasite.

The transcription factor NF-YB is localised in the nucleus during the erythrocytic stages of the life cycle. Melatonin and cyclic adenosine monophosphate modulate the expression of NF-YB. NF-YB is also more ubiquitinated in the presence of melatonin.

The protein PfMyb1 is a transcription factor belonging to the tryptophan cluster family. Inhibition of this gene reduces growth by ~40% with the mortality being concentrated at the trophozoite-schizont interface.

SAP1 has been shown to be involved in the post transcriptional control of liver stage genes.

CCR4-associated factor 1 is involved in the regulation more than 1000 genes during malaria parasite's intraerythrocytic stages. Mutations in this gene result in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress and invasion of new host cells. This leads to the premature release of predominantly half-finished merozoites in turn drastically reducing the intraerythrocytic growth rate of the parasite.

Mutation rates

The overall mutation rate of the genome is 1.0-9.7 x 10^-9 mutations per base pair per generation. The rate in genes involved in immune evasion is higher - 9.5 x 10^-6 structural variants per base pair per generation.

Protein metabolism

It has been hypothesized that the parasite obtains all, or nearly all, of its amino acids by salvaging from the host or through the degradation of hemoglobin. This is supported by the fact that genomic analysis has found no enzymes necessary for amino acid biosynthesis, except for glycine-serine, cysteine-alanine, aspartate-asparagine, proline-ornithine, and glutamine-glutamate interconversions.

Deoxyhypusine hydroxylase catalyses the final stage of the synthesis of the amino acid hypusine. In eukaryotes this amino acid is only found in eukaryotic initiation factor 5A. This gene has been identified in the genome of P. falciparum, P. knowlesi, P. vivax and P. yoelii.

Deoxyhypusine synthase catalyzes the first step in hypusine biosynthesis of eukaryotic initiation factor 5A (EIF5A). Inhibitors of this enzyme may be of use therapeutically.

Much of the digestion of haemoglobin occurs within the digestive vacuole. Multiple enzymes are involved in this process including four distinct plasmepsins (aspartic acid proteases).

Protein translation

There are two translation elongation factor G proteins encoded in the genome. One is located in the mitochondrion and the second in the plastid. Both appear to be inhibitable with fusidic acid

Two Ribosome Recycling Factors (RRF1 and RR2) are present in the genome. Both proteins are targeted to both the apicoplast and the mitochondrion. RRF2 is also present in the cytoplasm. Unusually it forms dimers. RRF1 has a 108 amino acid insert compared with that of other organisms. The function of this insert - if any - is currently unknown.

The eukaryotic translation initiation factor 2α has a regulatory serine at position 51. This can be phosphorylated by several kinases. Three are known in P falciparum: IK1, IK2 and PK4. IK1 regulates stress response to amino acid starvation; IK2 inhibits development of malaria sporozoites present in the mosquito salivary glands; and PK4 is essential for the completion of the parasite's erythrocytic cycle.

Unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number.

Aminoacyl tRNA synthetases are required for protein synthesis. Alanine tRNA synthetase, glycine tRNA synthetase and threonine tRNA synthetase are dually localised to the cytosol and the apicoplast. These enzymes do not appear to be present in the mitochondrion.

Tyrosyl tRNA synthetase is secreted by the parasite into the cytoplasm of the infected erythrocyte. On lysis of the erythrocyte it is released into the blood stream where it is pro inflammatory. It is specifically bound by and taken up by host macrophages and leads to enhanced secretion of the cytokines tumor necrosis factor-alpha and interleukin 6. This interaction also increases the adherence linked host endothelial receptors ICAM-1 and VCAM-1.

The cytoplasmic lysyl-tRNA synthetase is dimeric unlike the human version which may be dimeric or tetrameric. It is capable of synthesizing the signalling molecule diadenosine tetraphosphate using ATP as a substrate.

The tryptophanyl-tRNA synthetase appears to be localised to the cytoplasm. This enzyme has an unusual N-terminal extension that appears to be essential for its activity.

Post translational modifications

Palmitoylation - the reversible addition of a lipid moiety to a cysteine residue - appears to be common in this parasite. Its role in its biology is not yet understood.

An N-myristoyltransferase is present in the genome. It is involved in protein trafficking.

Lysine acetylation appears to be common in this organism. Lysine-acetylated proteins are present in the nucleus, cytoplasm, mitochondrion and apicoplast. The acetyltransferase PfMYST appears to be involved in this process. The effects of these modifications are not yet understood.

Hemoglobin metabolism

During the erythrocytic stage of the parasite's life cycle, it uses intracellular hemoglobin as a food source. The protein is broken down into peptides, and the heme group is released and detoxified by biocrystallization in the form of hemozoin.

Heme biosynthesis by the parasite has been reported. Mutations have been induced in both the first and last enzymes (δ-aminolevulinate synthase and ferrochelatase) of this pathway. Although these mutations do not appear to have an effect on the growth in blood cells, growth in both the mosquito and in the liver requires an intact pathway. The muatated strains form decreased numbers of oocyts and fail to form sporozoites. These strains fail to be infective to hosts. The parasites incorporate both host haemoglobin heme and parasite synthesized heme into haemozoin and into the mitochondrial cytochromes.

Haemozoin

Haemozoin (malarial pigment) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. It is an insoluble phase of iron (III) protoporphyrin-IX. The structure of haemozoin was solved in 2000 by powder X-ray diffraction. It is a crystalline structure composed of heme units interlinked to form cyclic dimers via reciprocal iron-oxygen (propionate) bonds. Its nucleation occurs at the inner membrane of the digestive vacuole, with crystallization occurring in the aqueous rather than lipid phase. Acylglycerol lipids are involved in the nucleation process.

Ferriprotoporphyrin IX (haematin) competes with NADH for the active site of the enzyme lactate dehydrogenase. This competition may be fatal to the parasite. To detoxify the ferriprotoporphyrin IX the parasite polymerizes haematin to haemozoin.

The conversion of haemoglobin to haemozoin is mediated by a ~200 kiloDalton protein complex within the digestive vacuole. This complex contains a number of proteins including falcipain 2/2', plasmepsin II, plasmepsin IV, histo-aspartic protease and heme detoxification protein. The proteins spontaneously associate with each other and can convert haemoglobin to haemozoin. The heme detoxification protein has two heme-binding sites. The drugs chloroquine and artemisinin both act during the heme polymerization step. Chloroquine also acts at the haemoglobin degradation step.

Although the parasite has a heme oxygenase like protein which binds bound heme and protoporphyrin IX with modest affinity, this protein does not catalyze heme degradation in vivo. It lacks a critical heme-coordinating histadine residue and this probably accounts for its lack of heme oxygenase activity. The function of this protein remains unknown.

Drugs interfering with haemozoin formation

Artemisinin an anti malarial agent appears to require haemoglobin digestion for its activity. Inhibition of hemoglobinase activity with cysteine protease inhibitors, knockout of falcipain 2 or deprivation of host cell lysate reduce the activity of the drug against the parasite.

Chloroquine causes a dose dependent decrease in haemozoin by disrupting hemozoin crystal growth. This results in mosaic boundaries in the crystals formed in the parasite. This disruption causes a redistribution of heme from the parasite digestive vacuole to the its cytoplasm.

Other drugs that inhibit haemozoin formation include amodiaquine, artesunate, lumefantrine, mefloquine and quinine. Neither pyrimethamine nor sulfadoxine affect haemazoin formation.

Effects on the host

Haemozoin has multiple effects on the host's immune system including increasing the level of matrix metalloproteinase-9. Its presence within the lung is correlated with lung inflammation and it may be important in the pathogensis of acute respiratory distress syndrome in malaria.

Haemozoin has been shown to promote inflammation mediated lysozyme release from human monocytes through p38 mitogen activated protein kinase and nuclear factor κB dependent mechanisms. This process may also involve 15-hydroxyeicosatetraenoic acid.

Both matrix metalloproteinase 9 and its endogenous inhibitor - tissue inhibitor of metalloproteinase 1 - are induced in macrophages by haemozoin. Both p38 MAPK and NF-κB appear to be involved in this induction pathway.

Haemazoin is recognised by a number of immune receptors including the Toll-like receptors. Phagolysosomal formation within the macrophages after haemozoin ingestion is normal but the haemozoin remains stored inside these cells for months or even longer without any detectable degradation.

Haemozoin inhibts erythropoetin induced proliferation of erythroid precursors. The mechanism is unknown but this probably contributes to the anemia associated with malaria.

Carbohydrate metabolism

During erythrocytic stages, the parasite produces its energy mainly through anaerobic glycolysis, with pyruvate being converted into lactate.

Genes encoding for the TCA cycle enzymes are present in the genome, but it is unclear whether the TCA cycle is used for oxidation of glycolytic products to be used for energy production, or for metabolite intermediate biosynthesis. It has been hypothesized that the main function of the TCA cycle in P. falciparum is for production of succinyl-CoA, to be used in heme biosynthesis.

Genes for nearly all of the pentose phosphate pathway enzymes have been identified from the genome sequence.

Glycerol is a major glucose metabolite.

Pools of several sugars (UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose and GDP-fucose) are present in the cytoplasm of the blood stages. The enzymes GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase are present in the genome.

Both the asexual and gametocyte stages of the life cycle utilise the Kreb's cycle to generate adenosine triphosphate. Glutamine is the preferred substrate over glucose in the asexual stages while in the gametocytes, glucose is preferred. Inhibition of this cycle in the asexual stages has little effect while inhibition in the gametocytes is lethal.

The enzyme hexokinase limits the rate of entry of glucose into glycolysis.

Mutations in the glycerol kinase gene limit the production of glycerol-3-phosphate which is used in phosphlipid biosynthesis. Exogenous glycerol may be used as an alternative source.

Lipid metabolism

Phosphatidylcholine is a major and essential membrane phospholipid in the parasite. Its synthesis occurs via the CDP-choline and the serine decarboxylase phosphoethanolamine methylation pathways. The substrates of these pathways are the host's choline, serine and fatty acids. Both pathways share the final two steps catalyzed by two essential enzymes CTP:phosphocholine cytidylyltransferase and choline-phosphate transferase. Mutations in phosphoethanolamine N-methyltransferase are associated with severe alterations in gametocyte development, are incapable of producing mature-stage gametocytes and are not transmittable to mosquitoes.

The major source of phosphatidylcholine is the CDP-choline Kennedy pathway. Both phosphoethanolamine-Nmethyltransferase and phosphatidylethanolamine-N-methyltransferase are important in its synthesis. Most of the serine derived phosphatidylethanolamine is formed via serine decarboxylation, whereas the majority of phosphatidylserine is formed by base exchange reactions.

The other major component of the parasite's membranes is the phospholipid phosphatidylethanolamine. This phophlipid is not found in the erythrocyte. Phosphatidylethanolamine is synthesised de novo by the CDP-ethanolamine dependent Kennedy pathway. The rate limiting step in this pathway is the step involving CTP:phosphoethanolamine cytidylyltransferase. This enzyme is composed of two cytidylyltransferase domains separated by a linker region. Only the N-terminal domain appears to be enzymatically active. The enzyme appears to function as a dimer and to obey Michaelis-Menten kinetics.

A phosphatidylserine decarboxylase has been cloned from the parasite Plasmodium knowlesi. It seems highly probably this enzyme is also found in P. falciparum.

β-hydroxyacyl-acyl carrier protein dehydratase catalyzes the third and important reaction of the fatty acid elongation cycle. The P. falciparum gene has been cloned and the crystal structure of the enzyme solved.

α-lipoic acid (6,8-thioctic acid: LA) is a vital co-factor of α-ketoacid dehydrogenase complexes and the glycine cleavage system. It is essential for the erythrocytic and liver stages of Plasmodium and is the co-factor of the acetyltransferase subunit of pyruvate dehydrogenase located in the apicoplast.

LA biosynthesis, comprising octanoyl-acyl carrier protein (ACP): protein N-octanoyltransferase and lipoate synthase is exclusively found in the apicoplast where it generates LA de novo from octanoyl-ACP, provided by the type II fatty acid biosynthesis pathway which is also present in this organelle.

Other members of the fatty acid synthesis type II pathway present in the genome are PfFabI, PfFabG and PfFabZ.

Two lipoic acid protein ligases (LplA1 and LplA2) are present in the genome. LplA1 is confined to the mitochondrion while LplA2 is found in both the mitochondrion and the apicoplast. LplA1 exclusively uses salvaged LA and lipoylates α-ketoglutarate dehydrogenase, branched chain α-keto acid dehydrogenase and the H-protein of the glycine cleavage system. LplA2 cannot compensate for the loss of LplA1 function during blood stage development.

The parasite syntheses 2-C-methyl-d-erythritol-4-phosphate. Other enzymes in the this pathway include 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase and 1-deoxy-D-xylulose 5-phosphate synthase (the first enzyme in the pathway). This pathway is responsible for the production of the essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate.

The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase can be inhibited by the antibiotic fosmidomycin which has been shown to potentially useful as an antimalarial.

Isoprenoid biosynthesis may be inhibited by fosmidomycin which in turn reduces protein prenylation. One of the proteins that is normally prenylated is Rab5 - a protein involved in vesicle transport. On inhibition by fosmidomycin the Rab5 proteins mislocalize and cause marked defects in food vacuolar morphology and integrity.

HMB-PP synthase (IspG), an iron-sulphur (4Fe4S) protein involved in isoprenoid biosynthesis, has two domains - a TIM barrel and a 4Fe4S domain - in bacteria. In plants and malaria parasites, there is an additional large insert domain. This is a second TIM barrel that interacts with the other TIM barrel.

Rather than using sphingomyelin as the primary complex sphingolipid, yeast, plants and some protozoa utilise an evolutionarily related inositol phosphorylceramide synthase to synthesize inositol phosphorylceramide. In the P. falciparum genome there is a single copy of a putative sphingomyelin synthase. This gene's homolog in Toxoplasma gondii is the functional orthologue of the yeast's inositol phosphorylceramide synthase.

The parasite's requirement for acetyl-CoA are supplied by several pathways including acetyl-CoA synthetase and a pyruvate dehydrogenase like enzyme. The nature of the pyruvate dehydrogenase like enzyme is not quite clear but it seems likely that the branched chain ketoacid dehydrogenase complex is performs this function. This latter pathway contributes glucose derived acetyl-CoA to the tricarboxylic acid cycle in a stage independent process whereas anapleurotic carbon enters the this cycle via a stage dependent phosphoenolpyruvate carboxylase / phosphoenolpyruvate carboxy-kinase process that decreases as the parasite matures.

Membrane biogenesis

Membrane biogenesis in this organism involves the enzyme phosphoethanolamine methyltransferase which catalyses the methylation of phosphoethanolamine to phosphocholine. This pathway is found in plants and nematodes but not in humans. The enzymes in P. falciparum is a multi-functional unlike that of plants and nematodes. The enzyme from P. falciparum has been cloned and its structure solved.

Four putative autophagy genes - those involved in ATG8 lipidation - are present in the genome. Atg8 lipidation requires Atg7 (an E1-type ligase), Atg3 (an E2-type ligase) and Atg4 (a cysteine protease). PfAtg7 is the activating enzyme of PfAtg8. PfATg7 appears to be essential for normal growth.

Trace metals

Zinc is essential to the parasite and it actively accumulates it during growth. During the life cycle zinc pools are formed in both the cytosol and the mitochondria.

A copper channel has been identified in the genome. Removal of copper from the growth medium is associated with early developmental arrest.

Vitamin A

The parasite takes up vitamin A from its host and uses it in own metabolism. Children with malaria have lower vitamin A levels than normal.

Miscellaneous proteins

Polyamine biosynthesis in these parasites is controlled by a unique bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC). On the secondary structure level PfAdoMetDC is similar to that of the human protein. This bifunctional enzyme ensure coordination decarboxylated AdoMet and putrescine for the subsequent synthesis of spermidine.

The first two reactions of the pentose phosphate pathway in P. falciparum are catalysed by a single bifunctional enzyme - glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase. This is distinct from the case in humans where the enzymes are separate. In animals this pathway is usually found in the cytosol while in plants it is found in the plastids. The location of this reaction is not currently known in P. falciparum.

Fusions between these two enzymes (glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase) have also been reported in chordates. The chordate fusion differs in its orientation to that in Plasmodium (in Plasmodium the 6-phosphogluconolactone is found at the N-terminus of the glucose 6-phosphate dehydrogenase protein) indicating that at least two separate fusion events have occurred. The metazoan fusion appears to have occurred near the bases of the metazoan and apicomplexan lineages. This fusion event was not found in any of the three sequenced Cryptosporidium genomes. It was not found in Perkinsus marinus or in either of the ciliate (Paramecium tetraurelia and Tetrahymena thermophila) genomes. More data will be needed to estimate the timing of this fusion event.

Only one of the two metazoan paralogs of glucose 6-phosphate dehydrogenase is fused, indicating that the fusion occurred after a duplication event. This duplication event occurred in an ancestor of the choanoflagellates and metazoans. Another fusion event between these enzymes occurred in an ancestor of the protozoan parasites Trichomonas and Giardia lamblia. In Giardia, the proteins are fused in opposite orientations. A third fusion event occurred between glucose 6-phosphate dehydrogenase with phosphogluconate dehydrogenase in a diatom species (Phaeodactylum tricornutum).

The mechanism of action of the triose phosphate isomerase enzyme has been investigated in some detail. The conserved glutamic acid residue at position 97 is involved in the catalytic proton transfer. Modification of this residue may reduce the rate of catalysis by 9000 fold.

The shikimate pathways is functional in P. falciparum and vitamin E biosynthesis also occurs.

The parasite actively synthesises pyridoxal-phosphate (vitamin B₆). The 2-C-methyl-d-erythritol-4-phosphate pathway is involved in its synthesis. This process involves two sets of reactions: condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate and ammonia produced from glutamine. These actions are carried out by separate subunits. The synthase domain is known as Pdx1 and the glutaminase domain as Pdx2. In P. falciparum the core Pdx1 is a dodecamer and forms the core of the enzyme. There are up to 12 Pdx2 subunits surrounding the Pdx1 subunit. The majority of the synthesis is carried out by Pdx1. The pentose substrate is covalently attached through its C1 and forms a Schiff base with the Lysine 84 residue. The ammonia transfer between Pdx2 glutaminase and Pdx1 active sites is regulated by a transient tunnel.

Chorismate synthase (CS) catalyses the seventh and final step of the shikimate pathway. P. falciparum chorismate synthase (PfCS) is unique in terms of enzymatic behavior, cellular localization and in having two additional amino acid inserts compared to any other CS.

There are several versions of the enzyme glutamate dehydrogenase (GDH) encoded in the genome. Of these, GDH1 and 3 appear to localise in the cytoplasm and GDH2 to the apicoplast.

Within the genome, there are two adenylyl cyclases - ACα and ACβ. ACα contains six predicted transmembrane domains and a single carboxy-terminal catalytic domain homologous to sAC-like ACs. It is a predicted bifunctional protein comprising both a potassium channel and an AC that is conserved in the alveolates. It is expressed in the gametocytes. ACβ has no predicted transmembrane regions and possesses two AC catalytic domains. It has a marked pH dependence and is required for the erythrocytic stages.

Asymmetrical diadenosine 5',5″-P¹,P⁴-tetraphosphate hydrolase (EC 3.6.1.17) catalyses the conversion of diadenosine 5',5″-P¹,P⁴-tetraphosphate (Ap₄A) to ATP and AMP and diadenosine 5',5″-P¹,P⁵-pentaphosphate (Ap₅A) to ATP and ADP. This enzyme from the parasite has been cloned and expressed.

A glycerophosphodiesterase has been cloned. This enzyme is found in the parasitophorous vacuole, digestive vacuole and cytosol. It appears to be an essential gene but its specific function is currently unclear.

The translationally controlled tumor protein appears to bind artemisinin. This may contribute to its anti malarial action.

A bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase has been cloned. It is encoded by the gene PF3D7_1128400.

Thiamine appears to be essential to the parasite's metabolism. Thiamine is converted to its active form - thiamine pyrophosphate - by thiamine pyrophosphokinase. The enzymes oxoglutarate dehydrogenase and pyruvate dehydrogenase are both dependent on thiamine pyrophosphate.

A thymidylate kinase - an enzyme that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate - is present in the genome.

Human immune system evasion

var family

The var genes encode for the P. falciparum erythrocyte membrane protein 1 (PfEMP1) proteins. The genes are found in the subtelomeric regions of the chromosomes. There exist an estimated 59 var genes within the genome. The proteins encoded by the var genes are ultimately transported to the erythrocyte membrane and cause the infected erythrocytes to adhere to host endothelial receptors. Due to transcriptional switching between var genes, antigenic variation occurs which enables immune evasion by the parasite.

The hypervariable var gene repertoire is to a large extent generated by frequent meiotic ectopic recombination in the mosquito gut. Mitotic recombination may also occur.

Gene structure

The var genes are classified into a number of subfamilies (A, B and C) that possess distinctive upstream and downstream flanking regions. A number of intermediate types - B/A and B/C - have also been described. The classification system is based primarily on the orientation of the genes: subtelomeric A and B family genes are oriented tail to tail (3′ to 3′) while central C family genes are oriented head to tail in a tandem repeat manner. The two intermediate groups - B/A and B/C- have chromosomal positions or domain composition that differ from those that would be expected from their orientation.

From N- to C-terminal the genes are organised in the following fashion: an N-terminal segment (NTS), Duffy binding-like (DBL) domains, Cysteine rich inter-domain regions (CIDR), C2 domains, one transmembrane region (TM) and the acidic terminal segment (ATS).

The extracellular region of PfEMP1 comprises multiple adhesion domains called Duffy Binding Like (DBL) and Cysteine Rich Interdomain Region (CIDR). The number of DBL domains in each var gene varies between 2 and 9: the usual number is ~6. The DBL domains have been classified into different major classes - α, β, γ, δ, ζ and ε - and a number of sub-classes based on sequence criteria. Within the DBL are seven regions of considerable variability known as variable blocks (VB1-6). In all 147 subtypes have been recognised. In addition to these 21 conserved tandem runs of specific domains (domain cassettes) have also been identified. The DBL domains have been also characterized by definition of 10 semi-conserved homology blocks (HBa-j) interspersed by hypervariable regions and by the definition of three structural subdomains (S1–3). Some biological correlations with the DBL domains have been described: DBLα has been associated with binding to heparin sulfate, blood group A antigen and complement receptor 1; DBLβ domains have affinity for ICAM-1; and DBLδ adheres to platelet-endothelial cell adhesion molecule 1 (PECAM-1).

The CIDR domains have been divided into three classes - α, β, and γ - and have three regions: the minimal CD36 binding region (M2) which is flanked flanked by the less conserved M1 and M3 regions. Several CIDRα class domains mediate binding to the human CD36 receptor but this binding only occurs in the B and C var families. The CIDRα domains also bind immunoglobulin M and PECAM-1.

An association between the length of the gene and its sequence variation has been reported. Shorter genes tend to be more variable and long genes to be more conserved. This probably is the result of a trade off between optimizing within host fitness and minimizing between host immune selection pressure.

Evolution

These genes appear to have evolved before the separation Plasmodium falciparum and Plasmodium reichenowi.

Gene expression

The var genes undergo antigenic switching - only a single gene is expressed at a given time and the expression varies during the course of infection. This variation begins when the merozoites leave the liver and the mechanisms driving this process are not presently understood. A method of erasing the epigenetic memory which appears to be involved in this process has been described. An analysis of the results showed that a subtype of var genes - the upsA vars - which are rarely expressed in culture systems but appear to be important in clinical infections, are activated early in the switching process. The switching rate appears to be a function of the gene structure rather than its chromosomal position or promoter.

The protein PfSir2 associates with promoter regions of silenced genes involved in antigenic variation.

A histone 3 lysine 4 methyltransferase - PfSET10 - which localizes exclusively to the perinuclear active var gene expression site and is required to maintain the active var gene in a poised state during division for reactivation in daughter parasites.

The variant silencing SET gene is an analog of the Drosophila melanogaster ASH1. It controls histone H3 lysine 36 trimethylation (H3K36me3) on the var genes. Knocking this gene out results in the transcription of virtually all the var genes in the genome and their subsequent translation and localization to the cell membrane. This protein is present along the entire gene body including the intronic promoter and the transcription start site.

The origin recognition complex 1 protein binds the telomeres via its N terminal and appears to play some role in gene regulation.

Within the regulatory region of the var genes there are a number of insulator like elements. These along with the strict pairing of the 5' promoter with the second promoter within the intron are essential for the normal regulation of the var genes.

There is a small upstream (5') open reading frame associated with the var2csa gene. This gene is only expressed in placental malaria. The product of the open reading frame interacts with the sequence surrounding the var2csa gene to control its transcription.

Expression of gene appears to be non random with a global activation hierarchy favouring short and highly diverse genes in central chromosomal location. Longer and more conserved genes are rarely activated.

Molecular biology

The intracellular portion of the EMP1 protein binds to PFI1780w - a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family.

A low level solution of the ~300 kiloDalton ectodomain of PfEMP1 has been reported. It is rigid, elongated and monomeric and interacts with intercellular adhesion molecule 1 through the Duffy binding Lβ domain alone, forming a 1:1 complex.

The strength of adhesion between the EMP1 and chondroitin sulfate A is decreased at 40C compared with its binding at 37C.

The subdomain 3 which connects the h6 and h7 α-helices of PfEMP1-DBL1α appears to be important in the process of parasitized erythrocytes forming rosettes with uninfected erythrocytes.

Clinical notes

It is thought that associations may exist between different var types and the clinical syndromes seen in malaria. The VAR2CSA variants bind to glycosaminoglycan chondroitin-sulfate A and have been associated with placental malaria. Group A and specifically Domanin Casette type 8 have been associated with cerebral malaria in Benin. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes 8 and 13. These subtypes bind the endothelial protein C receptor (EPRC) with their amino terminal cysteine rich interdomain region. This binding interferes with protein C binding to EPCR.

A difference between homology blocks has been noted in the phenotypes of erythrocyte rosetting and impaired consciousness. The clinical significance - if any - of this finding is not presently clear.

rif family

The rif genes encode for repetitive interspersed family (rifin) proteins. The genes are found in the subtelomeric regions of the chromosomes. There exist an estimated 149 rif genes within the genome.

The rifins are divided into 2 types (A and B) depending on the presence/absence of a 25 amino acid domain in the semiconserved domain. One of the rifins (PF13_0006) is transcribed both in the sexual and asexual stages. It is present on both gametocytes and merozoites.

Rifin proteins are ultimately transported to the erythrocyte membrane. The function of these proteins is currently unknown.

stevor family

The stevor genes encode for the sub-telomeric variable open reading frame (stevor) proteins. The genes are found in the subtelomeric regions of the chromosomes. There exist an estimated 28 stevor genes within the genome.

These proteins appear to affect membrane deformability.

Additional material

• Jewett , Sibley (2003). "Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites". Mol Cell. 11 (4): 885–94. doi:10.1016/S1097-2765(03)00113-8. PMID 12718875. {{cite journal}}: |first2= missing |last2= (help)
• Bergman (2003). "Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites". J Cell Sci. 116 (1): 39–49. doi:10.1242/jcs.00194. {{cite journal}}: Invalid |display-authors=1 (help)
• Baum (2005). "Invasion by P. falciparum merozoites suggests a hierarchy of molecular interactions". PLoS Pathog. 1 (4): e37. doi:10.1371/journal.ppat.0010037. {{cite journal}}: |first2= missing |last2= (help); |first3= missing |last3= (help); |first4= missing |last4= (help); |first5= missing |last5= (help)CS1 maint: unflagged free DOI (link)
• Bosch (2007). "The closed MTIP-myosin A-tail complex from the malaria parasite invasion machinery". J Mol Biol. 372 (1): 77–88. doi:10.1016/j.jmb.2007.06.016. PMC 2702245. PMID 17628590. {{cite journal}}: |first2= missing |last2= (help); |first3= missing |last3= (help); |first4= missing |last4= (help); |first5= missing |last5= (help); |first6= missing |last6= (help); Invalid |display-authors=1 (help)
• Bosch (2007). "Aldolase provides an unusual binding site for thrombospondin-related anonymous protein in the invasion machinery of the malaria parasite". Proc Natl Acad Sci USA. 104 (17): 7015–20. doi:10.1073/pnas.0605301104. PMC 1855406. PMID 17426153. {{cite journal}}: |first2= missing |last2= (help); |first3= missing |last3= (help); |first4= missing |last4= (help); |first5= missing |last5= (help); |first6= missing |last6= (help); |first7= missing |last7= (help); |first8= missing |last8= (help); |first9= missing |last9= (help)
• Daher , Soldati-Favre (2009). "Mechanisms controlling glideosome function in apicomplexans". Cur Opin Micro. 12 (4): 408–414. doi:10.1016/j.mib.2009.06.008. {{cite journal}}: |first2= missing |last2= (help)
• Sibley (2010). "How apicomplexan parasites move in and out of cells". Cur Opin Biotech. 21 (5): 592–598. doi:10.1016/j.copbio.2010.05.009.
• Hain and Bosch (2013). "Autophagy in Plasmodium, a multifunctional pathway?". CSBJ. 8 (11). doi:10.5936/csbj.201308002. {{cite journal}}: |first2= missing |last2= (help)
• Boucher and Bosch (2013). "Development of a multifunctional tool for drug screening against plasmodial protein-protein interactions via surface plasmon resonance. |". J. Mol. Recognit. 26 (10): 496–500. doi:10.1002/jmr.2292. PMID 23996492. {{cite journal}}: |first2= missing |last2= (help)

External links

• MR4, The NIAID funded Malaria Research and Reference Reagent Resource Center
• PlasmoDB
• GeneDB
• Malaria IDC Strain Comparison Database
• Malaria IDC Transcriptome Database
• Malaria Parasite Metabolic Pathways
• ApiCyc
• Library of Apicomplexan metabolic pathways