Activity of the Bacillus anthracis 20 kDa protective antigen component

BACKGROUND: Anthrax is caused by Bacillus anthracis that produce two exotoxins, lethal toxin and edema toxin. The lethal toxin is composed of the lethal factor (LF) complexed with the cell binding protective antigen (PA(83), 83 kDa). Likewise, the edema factor (EF) binds to the PA(83 )to form the edema toxin. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the full-length, inactive protein into 63 kDa and 20 kDa antigens (PA(63 )and PA(20)). PA(63 )forms a heptamer and is internalized via receptor mediated endocytosis forming a protease-stable pore, which allows EF and LF to enter the cell and exert their toxic effects. Both proteolytically cleaved protective antigens (PA(63 )and PA(20 )fragments) are found in the blood of infected animals. The 63 kDa protective antigen PA(63 )fragment has been thoroughly studied while little is known about the PA(20). METHODS: In this study we examined the role of PA(20 )using high throughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to the PA(20). We constructed a PA mutant in which a Factor Xa proteolytic recognition site was genetically engineered into the protective antigen PA(83 )to obtain PA(20 )using limited digestion of this recombinant PA(83 )with trypsin. RESULTS: Global gene expression response studies indicated modulation of various immune functions and showed gene patterns indicative of apoptosis via the Fas pathway in a subset of the lymphoid cells. This finding was extended to include observations of increased Caspase-3 enzymatic activity and the identification of increases in the population of apoptotic, but not necrotic cells, based on differential staining methods. We identified a list of ~40 inflammatory mediators and heat-shock proteins that were altered similarly upon exposure of PBMC to either rPA(20 )or B. anthracis spores/vegetative cells. CONCLUSION: This study shows that the PA(20 )has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.