Structure and Function of Virulence Factors of Bacillus anthracis

National Institute Of Allergy And Infectious Diseases

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Abstract

Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and LF and EF translocate to the cytosol. EF is a calcium and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). [unreadable] Because the tissue distribution and relative importance of the two alternative toxin receptors, CMG2 and TEM8, are not known, we have sought to develop reagents to distinguish the two. Polyclonal and monoclonal antibodies were developed, but these have not yet proven highly effective in detecting the receptors in cell lysates or on intact cells. A previously described system for bacteriophage display of domain 4 of PA has been used in additional selections, and mutations have been identified that discriminate between the two receptors. Variants of PA have been obtained that retain high affinity for CMG2 while retaining very little affinity for TEM8. Evidence from several areas confirms the view that TEM8 expression is more restricted than that of CMG2, and that the latter is probably the more important receptor. [unreadable] Production of many batches of the toxin proteins for our own use and that of collaborators, and comparison of their potencies, made us aware that the specific activity of LF was variable. Close scrutiny related differences in potency to the identity of the N-terminal residue. By creation of variants having defined N-termini produced by removal of an extension containing a Factor Xa cleavage site, we demonstrated that LF potency appears to be controlled by the N-end rule, which states that the identity of the terminal residue determines the rate of ubiquitinylation and proteosomal degradation. The differences in potency were seen both in the macrophage cytotoxicity assay and in mouse and rat challenges with lethal toxin, the combination of PA and LF. This finding has significance because the macrophage cytotoxicity assay is widely used in characterization of drugs, vaccines, and therapeutics targeting the toxin. [unreadable] Work also continued to understand the role of diphthamide, the unique post-translational modification on elongation factor 2 that is the target of ADP-ribosylation by Pseudomonas exotoxin A and diphtheria toxin. Cells lacking diphthamide are viable, making it difficult to understand why a target for a toxin would be maintained. We have now found that cells lacking diphthamide are 3-fold more susceptible to a ribosome inactivating toxin (RIP), ricin. Although it is speculative, we suggest that one role of diphthamide and a possible reason for its selection during evolution may be the added protection it confers to RIPs. The diphthamide modification is located in the region of the ribosome to which both RIPS and elongation factor 2 bind, and so it may sterically block access by ricin to its target in the ribosome. RIPs may exert a considerable selective pressure because they are produced by many plants, including ones widely eaten as foods.