Endotoxin

Endotoxins (not to be confused with enterotoxin) are toxins associated with certain bacteria. Classically, an "endotoxin" is a toxin that, unlike an "exotoxin", is not secreted in soluble form by live bacteria, but is a structural component in the bacteria which is released mainly when bacteria are lysed.

Lipopolysaccharide and other endotoxins

Gram negative

Structure of a lipopolysaccharide

The prototypical examples of endotoxin are lipopolysaccharide (LPS) or lipo-oligo-saccharide (LOS) found in the outer membrane of various Gram-negative bacteria and is an important cause of their ability to cause disease. The term LPS is often used exchangeably with endotoxin, owing to its historical discovery. In the 1800s it became understood that bacteria could secrete toxins into their environment, which became broadly known as "exotoxin". The term endotoxin came from the discovery that portions of Gram-negative bacteria itself can cause toxicity, hence the name endotoxin. Studies of endotoxin over the next 50 years revealed that the effects of "endotoxin" were, in fact, due to lipopolysaccharide.

LPS consist of a polysaccharide (sugar) chain and a lipid moiety, known as lipid A, which is responsible for the toxic effects. The polysaccharide chain is highly variable amongst different bacteria. Endotoxins are approximately 10 kDa in size but can form large aggregates up to 1000 kDa. Humans are able to produce antibodies to endotoxins after exposure but these are generally directed at the polysaccharide chain and do not protect against a wide variety of endotoxins. Injection of a small amount of endotoxin in human volunteers produced fever, a lowering of the blood pressure, and activation of inflammation and coagulation. Endotoxins are in large part responsible for the dramatic clinical manifestations of infections with pathogenic Gram-negative bacteria, such as Neisseria meningitidis, the pathogen that causes fulminant meningitis.

Gram positive

There are, however, endotoxins other than LPS:

• The delta endotoxin of Bacillus thuringiensis makes crystal-like inclusion bodies next to the endospore inside the bacteria. It is toxic to larvae of insects feeding on plants, but is harmless to humans (as humans do not possess the enzymes and receptors necessary for its processing, followed by toxicity).

• The only gram positive bacteria that produces endotoxin is Listeria monocytogenes; however these toxins are more accurately described as "endotoxin-like" and should not be confused with that of those found in the cell wall of gram negative bacteria, coming from the paper by Wexler & Oppenheim. Another study was carried out using gas chromatography-mass spectrometry (GC-MS) in addition to Limulus amebocyte lysate (LAL) test on a number of gram-negative and gram-positive bacteria (including Listeria) by Maitra et al. (1986) Appl & Env Microbiology 52:510. The GC-MS results correlated well with LAL tests in that all the gram-negativec bacteria screened positively. Listeria, however, did not show any positive results with either GC-MS or LAL, giving evidence that it should not be considered a "true" endotoxin.

Mechanism

In humans, LPS binds to the lipid binding protein (LBP) in the serum which transfers it to CD14 on the cell membrane, which in turn transfers it to another non-anchored protein, MD2, which associates with Toll-like receptor-4 (TLR4).

CD14 and TLR4 are present in several immune system cells (including macrophages and dendritic cells), triggering the signaling cascade for macrophage/endothelial cells to secrete pro-inflammatory cytokines and Nitric oxide that lead to "endotoxic shock".

Other than TLR4, components of gram negative cell wall may also activate other pathways which may contribute to the overall endotoxic effect..

Endotoxin contamination

Endotoxins are frequent contaminants in plasmid DNA prepared from bacteria or proteins expressed from bacteria, and must be removed from the DNA or protein to avoid unwanted inflammatory responses prior to in vivo applications such as gene therapy.

Also ovalbumin is contaminated with endotoxins. Ovalbumin is one of the extensively studied proteins in animal models and also an established model allergen for airway hyper-responsiveness (AHR). Commercially available ovalbumin which is contaminated with LPS can fully activate endothelial cells in an in-vitro assay of the first step of inflammation and it falsifies research results as it does not accurately reflect the effect of sole protein antigen on animal physiology.

In pharmaceutical production, it is necessary to remove all traces of endotoxin from drug product containers as even small amounts of endotoxin will cause illness in humans. A depyrogenation oven is used for this purpose. Temperatures in excess of 300 degrees Celsius are required to break down this substance. A defined endotoxin reduction rate is a correlation between time and temperature. Based on primary packaging material as syringes or vials a glass temperature of 250°C and a holding time of 30min is typical to achieve 3log reduction on endotoxin levels.

A very sensitive assay for detecting presence of endotoxin is the Limulus Amebocyte Lysate assay, utilizing blood from the Horseshoe crab. Very low levels of LPS can cause coagulation of the limulus lysate due to a powerful amplification through an enzymatic cascade.

Endotoxemia

The presence of endotoxins in the blood is called Endotoxemia. It can lead to septic shock, if the immune response is severely pronounced.

See also

• Bioaerosol
• Exotoxin
• Depyrogenation
• Schwartzman reaction

External links

• Endotoxins at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
• Textbook of Bacteriology