|Trypanosoma brucei brucei TREU667 (Bloodstream form, phase contrast picture. Black bar indicates 10 µm.)|
Plimmer & Bradford, 1899
T. b. brucei
Trypanosoma brucei is a species of Salivarian trypanosome. T. brucei causes African trypanosomiasis, known also as 'African sleeping sickness' in humans and 'nagana' in animals. T. brucei has traditionally been grouped into three sub-species: T. b. brucei, T. b. gambiense and T. b. rhodesiense, the latter two being human-infective.
Transmission of T. brucei between mammal hosts is usually via an insect vector, the tsetse fly. T. brucei parasites undergo complex morphological changes as they move between insect and mammal over the course of their life cycle. The mammalian bloodstream forms are notable for their variant surface glycoprotein (VSG) coats, which undergo remarkable antigenic variation, enabling persistent evasion of host adaptive immunity and chronic infection. There is an urgent need for the development of new drug therapies as current treatments can prove fatal to the patient.
Whilst not historically regarded as T. brucei subspecies due to their different means of transmission, clinical presentation, and loss of kinetoplast DNA, genetic analyses reveal that T. equiperdum and T. evansi are derivatives of T. b. brucei, and thus are members of the brucei clade.
The insect vector for T. brucei is the tsetse fly (genus Glossina). The parasite lives in the midgut of the fly (procyclic form), whereupon it migrates to the salivary glands for injection to the mammalian host on biting. The parasite lives within the bloodstream (bloodstream form) where it can reinfect the fly vector after biting. Later during a T. brucei infection the parasite may migrate to other areas of the host. A T. brucei infection may be transferred human to human via bodily fluid exchange, primarily blood transfer.
There are three different sub-species of T. brucei, which cause different variants of trypanosomiasis.
- T. brucei gambiense - Causes slow onset chronic trypanosomiasis in humans. Most common in central and western Africa, where humans are thought to be the primary reservoir.
- T. brucei rhodesiense - Causes fast onset acute trypanosomiasis in humans. Most common in southern and eastern Africa, where game animals and livestock are thought to be the primary reservoir.
- T. brucei brucei - Causes animal African trypanosomiasis, along with several other species of trypanosoma. T. b. brucei is not human infective due to its susceptibility to lysis by Trypanosome Lytic Factor-1 (TLF-1). However, as it shares many features with T. b. gambiense and T. b. rhodesiense (such as antigenic variation) it is used as a model for human infections in laboratory and animal studies.
The structure of the cell is fairly typical of eukaryotes (see eukaryotic cell). All major organelles are seen, including the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus etc. Unusual features include the single large mitochondria with the mitochondrial DNA structure known as the kinetoplast, and its association with the basal body of the flagellum. The cytoskeleton is made up primarily of microtubules. The cell surface of the bloodstream form features a dense coat of variable surface glycoproteins (VSGs) which is replaced by an equally dense coat of procyclins when the parasite differentiates into the procylic in the tsetse fly midgut.
Trypanosomatids show specific cellular forms of which two are adopted by Trypanosoma brucei at different stages of the life cycle:
- Epimastigote - Basal body anterior of nucleus, with a long flagellum attached along the cell body.
- Trypomastigote - Basal body posterior of nucleus, with a long flagellum attached along the cell body.
T. brucei is found as a trypomastigote in the slender, stumpy, procyclic and metacyclic forms. The procylic form differentiates to the proliferitive epimastigote form in the salivary glands of the insect. Unlike some other trypanosomatids, the promastigote and amastigote form do not form part of the T.brucei life cycle.
- 11 pairs of large chromosomes of 1 to 6 megabase pairs.
- 3-5 intermediate chromosomes of 200 to 500 kilobase pairs.
- Around 100 minichromosomes of around 50 to 100 kilobase pairs. These may be present in multiple copies per haploid genome.
The mitochondrial genome is found condensed into the kinetoplast, an unusual feature unique to the kinetoplastea class. The kinetoplast and the basal body of the flagellum are strongly associated via a cytoskeletal structure.
VSG surface coat
Main section: The VSG coat
The surface of the trypanosome is covered by a dense coat of Variable Surface Glycoprotein (VSG), which allows persistence of an infecting trypanosome population in the host. See below.
The cytoskeleton is predominantly made up of microtubules, forming a subpellicular corset. The microtubules lie parallel to each other along the long axis of the cell, with the number of microtubules at any point roughly proportional to the circumference of the cell at that point. As the cell grows (including for mitosis) additional microtubules grow between the existing tubules, leading to semiconservative inheritance of the cytoskeleton. The microtubules are orientated + at the posterior and - at the anterior.
The trypanosome flagellum has two main structures. It is made up of a typical flagellar axoneme which lies parallel to the paraflagellar rod, a lattice structure of proteins unique to the kinetoplastida, euglenoids and dinoflagellates.
The microtubules of the flagellar axoneme lie in the normal 9+2 arrangement, orientated with the + at the anterior end and the - in the basal body. The a cytoskeletal structure extends from the basal body to the kinetoplast. The flagellum is bound to the cytoskeleton of the main cell body by four specialised microtubules, which run parallel and in the same direction to the flagellar tubulin.
The flagellar function is twofold - locomotion via oscilations along the attached flagellum and cell body, and attachment to the fly gut during the procyclic phase.
The surface of the trypanosome is covered by a dense coat of ~5 x 106 molecules of Variable Surface Glycoprotein (VSG). This coat enables an infecting T. brucei population to persistently evade the host's immune system, allowing chronic infection. The two properties of the VSG coat that allow immune evasion are:
- Shielding - the dense nature of the VSG coat prevents the immune system of the mammalian host from accessing the plasma membrane or any other invariant surface epitopes (such as ion channels, transporters, receptors etc.) of the parasite. The coat is uniform, made up of millions of copies of the same molecule; therefore the only parts of the trypanosome the immune system can 'see' are the N-terminal loops of the VSG that make up the coat.
- Periodic antigenic variation - the VSG coat undergoes frequent stochastic genetic modification - 'switching' - allowing variants expressing a new VSG coat to escape the specific immune response raised against the previous coat.
VSG is highly immunogenic, and an immune response raised against a specific VSG coat will rapidly kill trypanosomes expressing this variant. Antibody-mediated trypanosome killing can also be observed in vitro by a complement-mediated lysis assay. However, with each cell division there is a possibility that one or both of the progeny will switch expression to change the VSG variant that is being expressed. The frequency of VSG switching has been measured to be approximately 0.1% per division. As T. brucei populations can peak at a size of 1011 within a host  this rapid rate of switching ensures that the parasite population is constantly diverse. A diverse range of coats expressed by the trypanosome population means that the immune system is always one step behind: it takes several days for an immune response against a given VSG to develop, giving trypanosomes which switched VSG time to reproduce (and undergo further switching events) unhindered. Reiteration of this process prevents extinction of the infecting trypanosome population, allowing chronic persistence of parasites in the host, enhancing opportunities for transmission. The clinical effect of this cycle is successive 'waves' of parasitaemia (trypanosomes in the blood).
VSG genes are hugely variable at the sequence level. However, for them to fulfill their shielding function, different VSGs have strongly conserved structural features. VSGs are made up of a highly variable N terminal domain of around 300 to 350 amino acids, and a more conserved C terminal domain of around 100 amino acids. N-terminal domains dimerise to form a bundle of four alpha helices, around which hang smaller structural features. This tertiary structure is well conserved between different VSGs (in spite of wide variation in amino acid sequence) allowing different VSGs to form the physical barrier required to shield the trypanosome's surface. VSG is anchored to the cell membrane via a glycophosphatidylinositol (GPI) anchor - a covalent linkage from the C terminus, to approximately four sugars, to a phosphatidylinositol phospholipid acid which lies in the cell membrane. VSGs form homodimers.
VSG archive structure
The source of VSG variability during infection is a large 'archive' of VSG genes present in the T. brucei genome. Some of these are full-length, intact genes; others are pseudogenes) typically with frameshift mutations, premature stop codons, or fragmentation. Expression of an antigenically different VSG can occur by simply switching to a different full-length VSG gene. However, only 5% of the archive is made up of such complete silent VSGs. To utilise the rest of the silent VSG archive, ‘mosaic’ VSGs can be formed by replacing part of the expressed VSG with a structurally homologous region from the archive. The combinatorial nature of mosaic formation in conjunction with the huge silent VSG archive gives the parasite a theoretically limitless VSG library, and could be a major barrier to vaccine development.
One major focus in trypanosome research is how all but one of the VSG genes are kept silent at a given time, and how these the active VSG is switched. The expressed VSG is always located in an Expression Site (ES), which are specialised expression loci found at the telomeres of some of the large and intermediate chromosomes. Each ES is a polycistronic unit, containing a number of Expression Site-Associated Genes (ESAGs) all expressed along with the active VSG. While multiple ES exist, only a single one is ever active at one time. A number of mechanisms appear to be involved in this process, but the exact nature of the silencing is still unclear.
The VSG can be switched either by changing the active expression (from the active to a previously silent site) or by changing the VSG gene in the active site. The genome contains many copies of possible VSG genes, both on minichromosomes and in repeated sections in the interior of the chromosomes. These are generally silent, typically with omitted sections or premature stop codons, but are important in the evolution of new VSG genes. It is estimated up to 10% of the T.brucei genome may be made up of VSG genes or pseudogenes. Any of these genes can be moved into the active site by recombination for expression. Again, the exact mechanisms that control this are still only partially known.
The mitotic division of T.brucei is unusual compared to most eukaryotes. The nuclear membrane remains intact and the chromosomes do not condense during mitosis. The basal body, unlike the centrosome of most eukaryotic cells, does not play a role in the organisation of the spindle and instead is involved in division of the kinetoplast.
Stages of mitosis:
- The basal body duplicates and both remain associated with the kinetoplast.
- Kinetoplast DNA undergoes synthesis then the kinetoplast divides coupled with separation of the two basal bodies.
- Nuclear DNA undergoes synthesis while a new flagellum extends from the younger, more posterior, basal body.
- The nucleus undergoes mitosis.
- Cytokinesis progresses from the anterior to posterior.
- Division completes with abscission.
- Gibson, W. C. (2007). "Resolution of the species problem in African trypanosomes.". Int J Parasitol 37: 829–838.
- Barrett MP, Burchmore RJ, Stich A, et al. (November 2003). "The trypanosomiases". Lancet 362 (9394): 1469–80. doi:10.1016/S0140-6736(03)14694-6. PMID 14602444.
- Ogbadoyi E, Ersfeld K, Robinson D, Sherwin T, Gull K (March 2000). "Architecture of the Trypanosoma brucei nucleus during interphase and mitosis". Chromosoma 108 (8): 501–13. doi:10.1007/s004120050402. PMID 10794572.
- Barry JD, McCulloch R (2001). "Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite". Adv Parasitol. Advances in Parasitology 49: 1–70. doi:10.1016/S0065-308X(01)49037-3. ISBN 978-0-12-031749-3. PMID 11461029.
- Overath P, Chaudhri M, Steverding D, Ziegelbauer K (February 1994). "Invariant surface proteins in bloodstream forms of Trypanosoma brucei". Parasitol. Today (Regul. Ed.) 10 (2): 53–8. doi:10.1016/0169-4758(94)90393-X. PMID 15275499.
- Turner CM (August 1997). "The rate of antigenic variation in fly-transmitted and syringe-passaged infections of Trypanosoma brucei". FEMS Microbiol Lett. 153 (1): 227–31. doi:10.1111/j.1574-6968.1997.tb10486.x. PMID 9252591.
- Barry, J. D.; Hall, Plenderleith (2012). "Genome hyperevolution and the success of a parasite". Ann N Y Acad Sci 1267: 11–17. doi:10.1111/j.1749-6632.2012.06654.x.
- Marcello L, Barry JD (September 2007). "Analysis of the VSG gene silent archive in Trypanosoma brucei reveals that mosaic gene expression is prominent in antigenic variation and is favored by archive substructure". Genome Res. 17 (9): 1344–52. doi:10.1101/gr.6421207. PMC 1950903. PMID 17652423.
- Barbour AG, Restrepo BI (2000). "Antigenic variation in vector-borne pathogens". Emerging Infect Dis. 6 (5): 449–57. doi:10.3201/eid0605.000502. PMC 2627965. PMID 10998374.
- Pays E (November 2005). "Regulation of antigen gene expression in Trypanosoma brucei". Trends Parasitol. 21 (11): 517–20. doi:10.1016/j.pt.2005.08.016. PMID 16126458.