THE EFFECT OF LOW PH ON BIOLOGICALLY ACTIVE AND INACTIVE BACILLUS ANTHRACIS PROTECTIVE ANTIGEN

Abstract

Bacillus anthracis, the causative agent of anthrax, is a large gram positive bacterium that produces three toxin components: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds to cell surface receptors and is cleaved at a trypsin-sensitive site by a cell-surface protease. Conversion of 83 kDa PA to 63 kDa PA exposes a site on PA which is capable of binding either EF or LF. EF and 63 kDa PA form a complex known as edema toxin which has been shown to cause edema in experimental animals. Lethal toxin, formed when LF binds to 63 kDa PA, results in death of some experimental animals and lysis of peritoneal macrophages. EF has been shown to be an adenylate cyclase which causes elevated intracellular levels of cAMP. LF shares sequence homology with known metalloproteases, yet the intracellular target of lethal toxin remains unknown. Protease-treated Bacillus anthracis PA fragments were generated and characterized previously (Novak, J.M., et al., 1992). Native PA (N-PA) and trypsin-treated PA (T-PA) were shown to be biologically active, facilitating cell cytolysis in a macrophage lysis assay. Chymotrypsin-treatment of PA (CH-PA or CH/T-PA) resulted in PA fragment preparations that bound to cell-surface receptors and internalized LF to a pronase resistant compartment, yet were unable to facilitate cell cytolysis. B. anthracis toxins are A/B toxins (Gill, D.M., 1978) that use separate gene products to facilitate binding (PA) and toxicity (LF or EF) in vivo. Lethal toxin must pass through an acidic intracellular environment prior to exerting its toxic effect in the cytosol (Friedlander, A.M., 1986), and as such, the inability f chymotrypsin treated PA fragment preparations to facilitate cell cytolysis could result from an inability of these fragment preparations to facilitate insertion and translocation of toxin to the cytosol at low pH. Thus, the effect of low pH on both biologically active (trypsin-treated) and biologically inactive (chymotrypsin-treated) PA fragment preparations was examined to characterize the effects of low pH on active and inactive preparations of PA as well as LF. Acid treatment of PA fragment preparations bound to the cell-surface at 4°C was performed to determine if specific PA fragments were susceptible to dissociation at low pH. Interestingly, loss of PA fragments did not correlate with biological activity, but instead, correlated with typsin treatment. Pronase treatment of cell-bound fragment preparations following citric acid treatment at 4°C, removed 80% or more of both T-PA and CH/T-PA. However, the remaining cell-associated fragments suggest that after exposure to low pH, domains of PA that are sensitive to pronase removal at neutral pH, may become pronase resistant. Thus, at low pH, specific regions of PA may interact with the cell surface, either by insertion or association with cell-surface components. Increased association of PA fragments with membrane may be indicative of domains that play a role in the translocation of toxin components to the cytosol. Insertion and translocation of toxin components from an acidic intracellular environment to the cytosol necessarily requires interaction between toxin components and biological membrane components. Biochemical characterization of PA fragment preparations by Triton X-114 partitioning was performed to determine which PA fragments were able to bind detergent and thus, likely to interact with hydrophobic membrane components. The 20 kDa, 37 kDa and "17" kDa PA fragments and a carboxyl-terminal secondary fragment designated "35" kDa PA, partitioned primarily to the aqueous phase at all pH values. The 63 and 47 kDa PA fragments increasingly partitioned to the detergent phase at low pH, suggesting their possible insertion into endosomal membranes in response to low pH. Phenylarsine oxide, a potent inhibitor of endocytosis (Wiley, H.S. and D.D. Cunningham, 1982), was used to inhibit the endocytosis of '5I-LF at 37°C. Citric acid treatment of 125I-LF bound to biologically active and inactive PA fragment preparations at the cell surface at 37°C, followed by pronase treatment, demonstrated that '5I-LF bound to biologically active PA fragment preparations became protected from pronase removal at low pH while 125I-LF bound to biologically inactive PA fragment preparations remained pronase sensitive at all pH values tested. Thus, the defect associated with chymot/ypsin-treatment of PA may result from an inability of chymotrypsin-treated PA fragment preparations to facilitate the insertion or translocation of LF into or across biological membranes at low pH.