Endotoxin

The term endotoxin was coined by Richard Friedrich Johannes Pfeiffer, who distinguished between exotoxin, which he classified as a toxin that is released by bacteria into the environment, and endotoxin, which he considered to be a toxin kept "within" the bacterial cell and to be released only after destruction of the bacterial cell wall. Today, the term 'endotoxin' is used synonymously with the term lipopolysaccharide., which is a major constituent of the outer cell membrane of Gram-negative bacteria. Larger amounts of endotoxins can be mobilized if Gram-negative bacteria are killed or destroyed by detergents. The term "endotoxin" came from the discovery that portions of Gram-negative bacteria themselves can cause toxicity. Studies of endotoxin over the next 50 years revealed that the effects of "endotoxin" are, in fact, due to lipopolysaccharide.

The key effects of endotoxins on vertebrates are mediated by their interaction with specific receptors on immune cells such as monocytes, macrophages, dendritic cells, and others. Upon challenge with endotoxin, these cells form a broad spectrum of immune mediators such as cytokines, nitric oxide, and eicosanoids.

Lipopolysaccharide and other endotoxins

Gram-negative

Structure of a lipopolysaccharide

The prototypical examples of endotoxin are lipopolysaccharide (LPS) or lipooligosaccharide (LOS), found in the outer membrane of various Gram-negative bacteria, and are an important component of their ability to cause disease. The term LPS is often used interchangeably with endotoxin, owing to its historical discovery.

LPS consists 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 among different bacteria and determines their serotype. Endotoxins are approximately 10 kDa in size but can form large aggregates up to 1000 kDa. Humans are able to produce antibodies against endotoxins after exposure, but these are, in general, directed at the polysaccharide chain and can only protect against the immunoactivation from serotype-specific bacteria and/or their specific endotoxin. Injection of a small amount of endotoxin in human volunteers has been shown to produce fever, a decrease in 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 pathogens that causes meningococcal disease, including meningococcemia, Waterhouse-Friderichsen syndrome, and meningitis.

Gram-positive

Results from one 1979 study indicated that Listeria monocytogenes may produce an "endotoxin-like" substance. However, a subsequent study failed to confirm that this Gram-positive species produces an endotoxin.

Bacillus thuringiensis is known to produce an endotoxin called delta endotoxin. The expression of the toxin in plants is known to offer the plants resistance against various insects.^{[citation needed]}

Wider usage of the word

Endotoxins are toxins that are not secreted by cells, so they can be found within the cell and not in the surrounding medium. Toxins secreted are exotoxins. The delta endotoxin of Bacillus thuringiensis is a protein found in 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 and toxicity.

Mechanism of action

In humans, LPS binds to the lipopolysaccharide-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 the homodimeric 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".

Further components of the 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 frequently 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 that 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°C 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 30 minutes is typical to achieve a reduction of endotoxin levels by a factor of 1000.

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. Endotoxins cause severe diseases in human beings. In the last couple of years new assays for endotoxin detection have been developed which don't have to use the blood of horseshoe crabs by using recombinant proteins.

Endotoxemia

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

Moreover, endotoxemia of intestinal origin is considered to be an important factor in the development of alcoholic hepatitis, which is likely to develop on the basis of the small bowel bacterial overgrowth syndrome and an increased intestinal permeability.

Link to obesity

Epidemiological studies have previously shown that increased endotoxin load, which can be a result of increased populations of endotoxin producing bacteria in the intestinal tract, is associated with certain obesity-related patient groups. Other studies have shown that purified endotoxin from Escherichia coli can induce obesity and insulin-resistance phenotypes when injected into germ-free mouse models. A more recent study has uncovered a potentially contributing role for Enterobacter cloacae B29 toward obesity and insulin resistance in a human patient. The presumed mechanism for the association of endotoxin with obesity is that endotoxin induces an inflammation-mediated pathway accounting for the observed obesity and insulin resistance.

Bacterial genera associated with endotoxin-related obesity effects: Escherichia, Enterobacter

See also

• Bioaerosol
• Depyrogenation
• Exotoxin
• Lipooligosaccharide
• Lipopolysaccharide
• Schwartzman reaction

External links

• Endotoxins at the US National Library of Medicine Medical Subject Headings (MeSH)
• Textbook of Bacteriology
• Endotoxin Detection Product
• Pilot study to investigate exposure to endotoxin in farmworkers performing sheep dipping by ER Waclawski and others. Institute of Occupational Medicine Research Report TM/94/02
• The combined effects of endotoxin and dust on the lung by RT Cullen and others. Institute of Occupational Medicine Research Report TM/92/01