Activity of the Bacillus anthracis20 kDa protective antigen component

Abstract

Background: Anthrax is caused byBacillus anthracisthat 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 (PA₈₃, 83 kDa). Likewise, the edema factor (EF) binds to the PA₈₃to 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 (PA₆₃and PA₂₀). PA₆₃forms 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 (PA₆₃and PA₂₀fragments) are found in the blood of infected animals. The 63 kDa protective antigen PA₆₃fragment has been thoroughly studied while little is known about the PA₂₀.Methods: In this study we examined the role of PA₂₀using high throughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to the PA₂₀. We constructed a PA mutant in which a Factor Xa proteolytic recognition site was genetically engineered into the protective antigen PA₈₃to obtain PA₂₀using limited digestion of this recombinant PA₈₃with 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 rPA₂₀orB. anthracisspores/vegetative cells.Conclusion: This study shows that the PA₂₀has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.