Serum-Mediated Cleavage of Bacillus anthracis Protective Antigen Is a Two-Step Process That Involves a Serum Carboxypeptidase

ABSTRACT Much of our understanding of the activity of anthrax toxin is based on in vitro systems, which delineate the interaction between Bacillus anthracis toxins and the cell surface. However, these systems fail to account for the intimate association of B. anthracis with the circulatory system, including the contribution of serum proteins to the host response and processing of anthrax toxins. Using a variety of immunological techniques to inhibit serum processing of B. anthracis protective antigen (PA) along with mass spectrometry analysis, we demonstrate that serum digests PA via 2 distinct reactions. In the first reaction, serum cleaves PA 83 into 2 fragments to produce PA 63 and PA 20 fragments, similarly to that observed following furin digestion. This is followed by carboxypeptidase-mediated removal of the carboxy-terminal arginine and lysines from PA 20 . IMPORTANCE Our findings identify a serum-mediated modification of PA 20 that has not been previously described. These observations further imply that the processing of PA is more complex than currently thought. Additional study is needed to define the contribution of serum processing of PA to the host response and individual susceptibility to anthrax.