Molecular Genetics and Pathogenesis of Anthrax

National Institute Of Allergy And Infectious Diseases

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Abstract

Recently we demonstrated that the key Bacillus anthracis virulence gene regulator AtxA binds directly to the promoter region of pagA (protective antigen gene) upstream of the sigma A subunit of the RNA polymerase binding site (J Bacteriol. 2019 Nov 5;201(23): e00569-19). During this reporting period of 2021, we demonstrated the ability of the AtxA protein to bind promoter regions of other B. anthracis toxin component genes and newly identified sRNAs. These data will help understand the mechanisms by which AtxA activates gene expression in response to elevated concentrations of carbon dioxide. In separate studies during 2021, we analyzed the role of the cell surface located HtrA serine protease of Bacillus anthracis, which was suspected of acting in quality control of proteins secreted by this pathogen. This protein acts first as a chaperone to fold proteins as they transit through the cell wall peptidoglycan. However, if folding fails, then HtrA protease activity comes into play to degrade the misfolded protein. Given its key role in protein secretion, we deleted the htrA gene in our multi-protease deficient BH500 strain and found that HtrA is essential for successful secretion and production of several recombinant proteins which are key components of the tumor-targeting agents developed in this laboratory. Mutational inactivation of HtrA proteolytic activity with retention of the chaperone activity provided a host strain that has improved ability to support production of recombinant proteins. In some cases, this expression system yielded more than 25 mg of protein per liter of culture. In prior studies done collaboratively with colleagues at the CSIR-Institute of Genomics and Integrative Biology, Delhi, India, electron microscopic analysis of B. anthracis cells revealed that deletion of genes in the ClpC operon (mcsB and clpC) caused defects in septum formation leading to cell elongation and decreased spore formation (Environ. Microbiol. (2015) 17:855865). In extending morphological studies during the current reporting period of 2021, we analyzed the role of PrkC, a serine/threonine protein kinase of B. anthracis that is localized at the bacterial surface. Deletion of the prkC gene yielded a strain, BAS prkC, that grew as shorter chains throughout the bacterial growth cycle. Levels of the proteins BslO and Sap, associated with the regulation of bacterial chain length, were upregulated in the BAS prkC strain compared to the parent strain. BslO is a septal murein hydrolase that catalyzes daughter cell separation, and Sap is an S-layer structural protein required for the septal localization of BslO. PrkC disruption also had a significant effect on bacterial growth, cell wall thickness, and septum formation. The decrease in cell wall thickness and shorter bacterial chains in the prkC strain could explain the observed higher sensitivity of the prkC strain to cell wall-targeting antibiotics. Altogether, these results indicated that PrkC is required for maintaining optimum growth, cell wall homeostasis, and, most importantly, the chaining phenotype. Chaining phenotype acts as a virulence factor in B. anthracis. This is the first study that identifies a signal transduction protein with an ability to regulate the chaining phenotype in B. anthracis.