Anthrax: Biomedical Mechanics
Bacillus anthracis possesses three known virulence factors: an antiphagocytic capsule and two protein exotoxins called the lethal and edema toxins. The role of the capsule in pathogenesis was demonstrated in the early 1900s when anthrax strains lacking a capsule were shown to be avirulent. In more recent years, the genes encoding synthesis of the capsule were found to be encoded on a 110-kilobase (kb) plasmid. Molecular analysis revealed that strains cured of this plasmid no longer produced the capsule and were attenuated, thus confirming the critical role of the capsule in virulence. The capsule is composed of a polymer of poly-D-glutamic acid, which confers resistance to phagocytosis and may contribute to the resistance of anthrax to lysis by serum cationic proteins.
In his initial studies on anthrax, Robert Koch suggested the importance of anthrax toxins. In 1954, Smith and Keppie demonstrated a toxic factor in the serum of infected animals that was lethal when injected into other animals. The role of toxins in virulence and immunity was firmly established by many scientists following Smith and Keppie.
The genes encoding the synthesis of the two protein exotoxins are located on a 60-kb plasmid, distinct from that encoding for the capsule. In an environment of increased bicarbonate and carbon dioxide and increased temperature, such as is found in the infected host, there is increased transcription of the genes for synthesis of the two toxins, as well as for the capsule.
Anthrax toxins, like many bacterial and plant toxins, possess two components: a cell-bind, or B, domain; and an active, or A, domain that has the toxic and, usually, the enzymatic activity. The B and A anthrax toxin components are synthesized from different genes and are secreted as noncovalently linked proteins. The two toxins are unusual in that the B protein called protective antigen, is shared by both toxins. Thus the lethal toxin is composed of the protective antigen combined with a second protein, which is known as the lethal factor. The lethal toxin is lethal for experimental animals and the lethal factor has been shown to possess homology to metalloproteases, although no direct enzymatic activity has yet been discovered.
The endemic toxin, consisting of the same protective antigen together with a third protein, edema factor, causes edema when injected into the skin of experimental animals. The edema factor is a calmodulin-dependent adenylate cyclase, which elevates intracellular cyclic adenosine monophosphate, and which is likely to be responsible for the marked edema often present at the site of bacterial replications.
Each of the three toxin proteins- the B protein and both A proteins- individually is without biological activity. The critical role of the toxins in pathogenesis was established when it was shown that deletion of the toxin encoding plasmid or the protective antigen gene alone attenuates the organism. The lethal toxin also appears to be more important for virulence in a mouse model tan the edema toxin.
Recent studies in cell culture models have given a clearer understanding of the molecular interactions of the toxin proteins. Protective antigen first binds, most likely by a domain at its carboxy-terminus to a specific cell receptor. Once bound, it is cleaved by a protease located on the cell surface, resulting in retention on the cell surface of a 63-kilodalton fragment of protective antigen. This cleavage creates a binding site on the protective antigen to which either the lethal factor or the edema factor can bind with high affinity. The complex is then internalized and passes through an acidic vesicle and is translocated to the cell cytosol, where it expresses its toxic activity.
Route of Infection
Infection begins when the spores are inoculated through the skin or mucosa. It is thought that spores are ingested at the local site by macrophages, in which they germinate to the vegetative bacillus with production of capsule and toxins. At these sites, the bacteria proliferate and produce the edema and lethal toxins that impair host leukocyte function and lead to the distinctive pathological findings: edema, hemorrhage, tissue necrosis, and a relative lack of leukocytes. In inhalational anthrax, the spores are ingested by alveolar macrophages, which transport them to the regional tracheobronchial lymph nodes, where germination occurs.
Once in the tracheobronchial lymph nodes, the local production of toxins by extracellular bacilli gives rise to the characteristic pathological pictures: massive hemorrhagic, edematous, and necrotizing lymphadenitis; mediastinitis. The bacilli can then spread to the blood, leading to septicemia with seeding of other organs and frequently causing hemorrhagic meningitis. Terminally, toxin is present in high concentrations in the blood, but both the site of toxin action and the molecular mechanism of death remain unknown. Death is the result of respiratory failure associated with pulmonary edema, overwhelming bacteremia, and often, meningitis.
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