Molecular Genetics and Pathogenesis of Anthrax

National Institute Of Allergy And Infectious Diseases

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Abstract

We previously demonstrated that anthrax toxin activator AtxA binds directly to the promoter region of pagA (protective antigen gen) upstream of the sigma A subunit of RNA polymerase binding site (J Bacteriol. 2019 Nov 5;201(23): e00569-19). During this current reporting period, using electrophoretic mobility shift assays and in vivo analyses, we confirmed AtxA-binding sites in the promoter regions of the lef and cya genes (encoding lethal and edema factors, respectively) and of two Bacillus anthracis small RNAs (XrrA and XrrB). Activities of all four newly studied promoters were enhanced in the presence of CO2/bicarbonate and AtxA, as previously seen for the pagA promoter. Notably, the cya promoter was less activated by AtxA and CO2/bicarbonate conditions. The putative promoter of a recently described third small RNA, XrrC, showed a negligible response to AtxA and CO2/bicarbonate. RNA polymerase binding sites of the newly studied promoters show no consensus and differ from the Sigma-A-like promoter region of pagA. In silico analysis of the probable AtxA binding sites in the studied promoters revealed several palindromes. Sequence alignment of these palindromes showed some similarity between lef and cya promoters and greater similarity between the sRNA promoters, excluding XrrC. All the analyzed palindromes showed very little overlap with the palindrome of the Signa A-like pagA promoter which is responsible for AtxA binding. It remains unclear as to how AtxA and DNA-dependent RNA-polymerase identify such diverse DNA-sequences and differentially regulate promoter activation of the studied genes. Reversible protein phosphorylation at serine/threonine residues is one of the most common protein modifications, widely observed in all kingdoms of life. The catalysts controlling this modification are specific serine/threonine kinases and phosphatases that modulate various cellular pathways ranging from growth to cellular death. Genome sequencing and various omics studies have led to the identification of numerous serine/threonine kinases and cognate phosphatases, yet the physiological relevance of many of these proteins remain enigmatic. In B. anthracis, only one ser/thr phosphatase, PrpC, has been functionally characterized; it was reported to be non-essential for bacterial growth and survival. During the current fiscal year, we produced several B. anthracis phosphatases and kinases gene deletions (PrkA, PrkD, PrpY and PrpN) and characterized another ser/thr phosphatase (PrpN) of B. anthracis by various structural and functional approaches (PLoS Pathog. 2022 Aug 1;18(8): e1010729). To examine its physiological relevance in B. anthracis, a null mutant strain of prpN was generated and shown to have defects in sporulation and reduced synthesis of toxins (PA and LF) because of diminished production of toxin activator protein AtxA. We also identified CodY, a global transcriptional regulator, as a target of PrpN and the previously characterized ser/thr kinase PrkC (J Bacteriol. 2021 Mar 22;203(11): e00582-20). CodY phosphorylation strongly controlled its binding to the promoter region of atxA, as shown using phosphomimetic and phosphoablative mutants. Thus, the present study reports phosphorylation-mediated regulation of CodY activity in the context of anthrax toxin synthesis in B. anthracis, dependent on a previously uncharacterized ser/thr protein phosphatase, PrpN.