Introduction:
Secretion of toxins is s a primary way bacteria cause disease.
There are 2 types of toxins; endotoxins and exotoxins.
Endotoxins: are found only in gram-negative bacteria. ndotoxins are a specific type of toxin that is part of the outer membrane of Gram-negative bacteria and is released when the cell dies.
Endotoxins are the lipid A portion of the lipopolysaccharide of the cell membrane. Endotoxins bind to receptors on macrophages, B cells, etc and stimulate production of acute-phase cytokines, stimulate B cell growth. At a low concentration they result in fever, vasodilation and inflammation. At high concentration they can result in shock and death.
Exotoxins are produced by some gram-negative and by gram positive bacterial. Exotoxins are proteins secreted by the bacteria while they are alive.
Exotoxins are often dimeric in structure with A and B subunits (A-B toxins). B binds to a cell receptor and A is transferred into the cell. Biochemical targets include ribosomes, transport mechanisms, intracellular signals. The genes for exotoxins are encoded on a plasmid or on a lysogenic phage. One important group of exotoxins are the superantigens.
An example of an exotoxin is the Mycobacterium tuberculosis. Anthrax actually secretes 3 toxins, I. edema factor, II. protective antigen and III. lethal factor. The Factor II (protective antigen) is responsible for getting the other 2 toxins into the cell.
How Toxins are Delivered to the Cell:
Toxins are bacterial components that directly harm tissue or trigger destructive biologic activities. One common delivery mechanism of such toxins is the type III secretion system which is a pilli like apparatus that directly injects the toxin or effector molecules into the host cell which alter cell signaling. For example, enteropathogenic E coli uses a type III secretion system to deliver its own bacterially expressed receptor protein into the host cell which serves as a receptor for a bacterial adhesin which triggers a series of signalling events. Y Pestis also uses a type III secretion mechanisms to get inside its host.
Specific Types of Toxins:
Cholesterol-dependent cytolysis (CDCs): are a large family of structurally related poreforming toxins which play various roles in the pathogenesis of a wide variety of human and animals diseases caused by Gram positive bacteria. A hallmark characteristic of the CDC is the dependence on membrane cholesterol for cytolysis.
Streptococcus intermedius intermedilysin (ILY): is a member of the CDCs. ILY is created by Tereptococcus intermedius, a Gram positive pathogenic bacterium that can cause brain and liver abscesses. ILY is unusual among the CDCs in that it has been shown to specifically lyse human erythocytes; chimpanzee and cynomolgus monkey erythrocytes were 100 fold less sensitive to its cytolytic activity and all nonprimate erythrocytes examined to date are resistance its its cytolytic effects. Giddins (Natrual structural & molecular biology, November 2004) show that ILY uses humCD59 as a cellular receptor. The specificity of ILY for huCD59 is based on its recognition of the C8alpha and C9 binding domain of huCD59. Binding of ILY to human erythrocytes and ILY mediated lysis were also blocked by monoclonal antibody to huCD59.
Acceptable Endotoxin Levels in Pharmaceutical Products:
Endotoxin testing is usually perfromed according to methods nad definitions in the European Pharmacopoeia (EP) section 2.6.14, US Pharmacopeia (USP) chapter <85>, and the Japanese Pharmacopoeia (JP) chater <4.01>. Europe and the US define the threshold pyrogenic dose for parenteral products adminstiered by injection or infusion oat 5.0 endotoxin units (EU) per kilogram of patient body weight per hour (5.0 EU/kg/h) and provide instructions on how endotoxin acceptance criteria should be caclulated as K/M, where K is the threshold pyrogenic dose of endotoxin per kilogram of body weight, and M is the maximum reccomended bolus dose of product (per hour) per kilogram of body weight.
Ecipients and water used in biomanufacturing have their own acceptance criteria for endotoxin.
Tests used:
Studies comparing environmental endotoxin by the Limulus amebocyte lysate (LAL) test adn the USP rabbit pyrogen test (RPT) indcate that hte LAL method is more sensitive than the RPT.
It is well known that purified LPS from different genera/species/strains of gram negative bacteria, when experimetnally adminsitered to rabits on a uniform weight or mass basis, can product significantly different pyrogenic reactions.