Anthrax protective antigen cleavage and clearance from the blood of mice and rats

Laboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892

^* To whom correspondence should be addressed. Email: sleppla{at}niaid.nih.gov.

	Abstract

Bacillus anthracis protective antigen (PA) is an 83-kDa protein that is cleaved to 63-kDa (PA63) as an essential step in binding and internalizing lethal factor (LF). To assess in vivo receptor saturating PA concentrations we injected mice with PA variants and measured the PA remaining in the blood at various times using PA83 and PA63-specific ELISAs. We found that wild type PA (WT-PA) and a receptor-binding defective mutant (Ub-PA) were cleaved to PA63 independent of their ability to bind cells. This suggested a PA-acting protease activity in the blood. The protease cleaved PA at the furin cleavage sequence because furin-site modified PA mutants were not cleaved. Cleavage measured in vitro was leupeptin-sensitive and dependent on calcium. Cell-surface cleavage was important for toxin clearance, however, as Ub-PA and uncleavable PA mutants were cleared at slower rates than WT-PA. The cell binding independent cleavage of PA was also verified by using Ub-PA (which is still cleaved) to rescue mice from LT (LF+PA) challenge by competitively binding circulating LF. This mutant was able to rescue mice even when given 12 h before toxin challenge and its therapeutic ability was comparable to that of dominant-negative PA (PA-DN), which binds cells but does not allow LF translocation, and to the protection afforded through receptor clearance by wild type PA and uncleavable receptor binding-competent mutants. The PA cleavage and clearance observed in mice did not appear to have a role in explaining the differential mouse susceptibility as it occurred similarly in LT-resistant DBA/2J and LT-sensitive mice Balb/cJ. Interestingly, PA63 was not found in LT-resistant or sensitive rats and PA83 clearance was slower than in mice. Finally, to determine the minimum amount of PA required in circulation for LT toxicity in mice, we performed time-separated injections of PA and LF and showed lethality of LF for mice after PA was no longer measurable in circulation, suggesting active PA sequestration at tissue surfaces.