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Pathology - Molecular Interactions

Virulent strains of anthrax contain two plasmids, pX01 and pX02. Strains lacking either plasmid are avirulent in animal models. Plasmid pX02 codes for the d-glutmic acid polypeptide capsule. The capsule inhibits phagocytosis and opsonization by macrophages (12). pX01 encodes the three toxin proteins: protective antigen, PA; lethal factor, LF; and edema factor, EF.

Early on in the investigation of Anthrax, Smith and Kreppie demonstrated that sterile filtered serum derived from infected guinea pigs could induce edema if injected subcutaneously and death if injected intravenously (21). Thus the effects of anthrax were due to an exotoxin so most research has focused on understanding the role that the proteins of pX01 play.

It is important to note that although these proteins have been equated with anthrax toxins, toxins produced in vivo differ from toxins produced in vitro in that it causes death more rapidly. Thus additional factors produced during an infection may enhance the effects of the toxins (18).

Protective antigen

Of the three toxin proteins, PA is the best characterised (Gen Bank accesion number M22589). Although PA is a necessary component of the anthrax toxin, by itself it is not toxic. And in fact, the toxicity of the other proteins is dependent on the presence of PA. PA gets its name because antibodies to it seem to play a role in host immunity. However, antibodies to PA alone are not sufficient for establishing an immune state. Unfortunately, the determination of the other factors needed to achieve an immune state remain elusive. PA is an 83-kDa protein that must undergo proteolytic cleavage, resulting in the removal of a 20-kDa fragment, before it can interact with EF or LF. Cleaveage is mediated most likely by the cell membrane protein furin or similar protease. Deletions of or mutations at the site of cleavage result in an uncleaved PA that is non toxic in combination with LF and EF. The PA-63kDa fragement has also been shown to form cation-selective channels in planar phospholipid membranes. Observations with electron microscopy indicate that seven molecules associate to form each channel. Several experiments have indicated that PA mediates the entry of EF and LF via the endosomal pathway. Thus the overall idea is that PA undergoes proteolysis, binds to EF or LF and is endocytosed. In the acidic environment of the endosome, PA undergoes a conformational change an recruits other PA molecules to form a transmembrane channel that allows EF and LF access to the cytoplasm (18).

Edema Factor

Ef is a 767-amino-acid 88-kDa protein. Sequence analysis indicates that residues 1-250 at the amino-terminus share homology with the corresponding region of LF. This region has been demonstrated to mediate binding to PA. When fused to a heterologous proteins it was able to allow their translocation to the cytosol. EF has been determined to be a calcium dependent adenylate cyclase. Direct measurement of cAMP levels indicated that addition of EF and PA resulted in a 200 fold increase over non-stimulated levels. One of the consequences is the edema seen by a clinician, but the role of EF might actually be to inhibit phagocytosis since increased cAMP levels have been shown to decrease phagocytosis. Thus the role of EF might be to promote bacterial survival during the early stages of infection (18).

Lethal Factor

LF is a 776-amino-acid 90.2-kDa protein. As noted above it exhibits sequence homology at its amino terminus with EF. However, the remainder of the protein exhibited no sequence homology with any known proteins. Thus it was not until recently that some of its properties have been determined. Careful analysis indicates that LF is a Zinc-dependent metalloprotease. In 1998 Duesbery et al and later Vitale et al identified mitogen activated protein kinase kinases 1 and 2 (MAPKK1,2) as substrates for LF. MAPKK1 and 2 play complicated roles in key signal transduction pathways. LF removes residues 1-7 from the amino terminus of MAPKK1. Apparently removal is sufficient to inactivate MAPKK1 since a recombinant MAPKK1 lacking the same seven amino acids lacked activity (18).

Unfortunately, despite our knowledge of the anthrax toxin we are unable to fully explain its pathogenicity. The principle target of LF seems to be macrophages. Examination of skin lesions of cutaneous anthrax indicate that there is infiltration with leukocytes but many are necrotic. Furthermore, Friedlander demonstrated that macrophages and macrophage-like cell lines abruptly lyse when treated with LF+PA. Also Hanna et al. have found that mice depleted of macrophages by repeated injections of silica become resistent to LF+PA. Restoration of macrophages restores susceptibility. In addition, Hanna et al. have found that superoxide anion is released at about the time cell lysis begins. Administration of antioxidents prevented lysis, and resistant cell lines were found to be deficient in reactive oxygen intermediates. The suggestion of these observation is that LF leads to the production of reactive oxygen intermediates in Macrophages that in turn lyse the cell and release toxic substances. Noting the similarities between systemic shock caused by anthrax and lipopolysaccharide-mediated bacterial sepis, Hanna et al have suggested that the response may be a result of cytokine overexpression. In support of this they have demonstrated that sublytic doses of LF+PA induce expression of IL-1 and TNF-alpha in vitro. Furthermore, passive immunization with against IL-1 in mice protected them from the effects of LF+PA. Unfortunately, that is the limit of our knowledge. It is not clear how cleavage of MAPKKs are related to the pathogenisis observed in infection (18).