Molecular Genetics and Pathogenesis of Anthrax

National Institute Of Allergy And Infectious Diseases

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Abstract

Although it has been 25 years since we discovered AtxA, the key transcriptional regulator of toxin gene expression, much remains unknown about how it functions. We eventually showed that AtxA binds to toxin promoters, and we defined specific regions within the promoters required for AtxA binding. Two histidines in AtxA were shown to control binding and activity. However, biochemical and genetic approaches have been unable to fully explain AtxA function. We recognized that a full understanding of AtxA requires determination of a 3D structure of the transcriptionally active complex of RNA polymerase (RNAP), AtxA, and a toxin promoter. To achieve this goal, we established a collaboration with Rockefeller University researchers Seth Darst and Elizabeth Campbell, widely viewed as leaders in RNAP structural analysis. Producing recombinant B. anthracis RNAP, which consists of six subunits, is challenging. We constructed a plasmid expressing all six subunits in E. coli. The subunits assemble in the cytosol of E. coli and are purified using a His6 tag present on one subunit. While others reported success with this approach, we found by mass spectrometry that the RNAP produced in this way contained a large amount of the host E. coli RpoA. This result is not surprising (but is not widely reported) because of the strong conservation of RNAPs across species. To overcome this issue, during FY2025, we explored many alternative expression systems in both E. coli and B. anthracis. Several of these systems have yielded B. anthracis RNAP of good purity. Samples provided to Dr. Darst were transcriptionally active on anthrax toxin promoters, and importantly, the activity depended on AtxA. These protein complexes next need to be examined by cryo-EM. As part of a long-term collaboration to characterize eukaryotic-like protein kinases and phosphatases of B. anthracis, we continued analysis of PrkA, one of the two putative kinases that have been studied. Another group suggested that B. subtilis PrkA, a close homolog of B. anthracis PrkA, has both serine-protease and protein kinase activities. Because the putative protease activity was minimal, we spent considerable effort seeking better substrates. We identified several proteins that were very slowly cleaved by our “purified” PrkA protein. However, we suspected that the cleavage could be due to a trace of contaminating protease, and did then show that further purification produced a preparation having no detectable protease activity. Similarly, unlike reports about the B. subtilis homolog, we could not show that the B. anthracis PrkA could be autophosphorylated. Further work on a strain having the PrkA gene deleted established its role in producing fully native spores. While this strain could produce spores, they appeared to be altered and more sensitive to stress. Overall, our studies showed PrkA to lack demonstrable enzymatic activities but to have a significant role in spore formation and stability. The pathological effects of the anthrax edema toxin (ET) are less studied and understood than those of the lethal toxin (LT). ET is a highly active calmodulin-dependent adenylate cyclase that converts ATP to cAMP. It had been assumed that ET-induced in vivo toxicity is mediated by well-known cAMP-dependent events, specifically, the activation of protein kinase A (PKA) and EPAC (Exchange protein directly activated by cAMP). PKA activation leads to fluid loss and edema, characteristic of intoxication by cholera toxin, which also acts by raising cAMP levels. However, using inhibitors and other tools to limit PKA and EPAC action did not protect cells and mice from ET. Instead, the data showed that severe depletion of ATP concentrations is responsible for ET’s pathogenesis.