Pomerantsev, A. P. | Search bioRxiv

bioRxiv2025

A novel AAA+ ATPase required for sporulation and stress response in Bacillus anthracis

AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase BAS PrkA in B. anthracis Sterne 34F2 which has 88 % protein homology to Bacillus subtilis PrkA. Conserved domain analysis confirms BAS PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on Alpha Fold3 predicted structure, we classified BAS PrkA as part of Clade III of the AAA+ superfamily. Contrary to the reported enzymatic activity in B. subtilis PrkA, we observed that BAS PrkA has negligible protease and kinase activity under in-vitro conditions. Nonetheless, BAS PrkA plays a significant role in regulating sporulation. It is temporally expressed during Stages II to VI during sporulation. A null mutant of BAS PrkA exhibits severe sporulation defects, with reduced spore viability, and down regulation of genes related to spore-coat formation. These phenotypes were restored in a complementation strain expressing BAS PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions. Analysis of the BAS PrkA interactome revealed enrichment of two proteins, ProA and EzrA, that are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BAS PrkA plays a critical role in sporulation and osmotic stress response in B. anthracis.

microbiology↗

bioRxiv2020

Bacillus anthracis chain length, a virulence determinant, is regulated by a transmembrane Ser/Thr protein kinase PrkC

Anthrax is a zoonotic disease caused by Bacillus anthracis, a spore-forming pathogen that displays a chaining phenotype. It has been reported that in a mouse infection model, systemic inoculation with longer bacterial chains caused blockade in lung capillaries. The blockade resulted in increased pathophysiological consequences viz, hypoxia and lung tissue injury. Hence, chaining acts as a virulence factor and molecules that regulate the chaining phenotype can be the potential drug targets. In this study, we have identified the serine/threonine protein kinase of B. anthracis, PrkC, localized at the bacteria-host interface, as a determinant of bacterial chain length. In vitro, prkC disruption strain (BAS {Delta}prkC) grew as shorter chains throughout the bacterial growth cycle as observed through phase-contrast and scanning electron microscopy. Since molecules such as BslO, a septal murein hydrolase, that catalyzes daughter cell separation and Sap, an S-layer structural protein required for the septal localization of BslO, are known to influence chain length, a comparative analysis to determine their levels was done through western-blot analysis. Both BslO and Sap were found to be upregulated in BAS {Delta}prkC at the majority of the time points. Additionally, PrkC disruption was observed to have a significant effect on bacterial growth and cell wall thickness. In BAS {Delta}prkC strain, a decrease in the cell wall thickness and an increase in the multi-septa formation was observed through transmission electron and confocal microscopy respectively. Altogether, we show that PrkC disruption affected chaining phenotype, cell growth and cell wall thickness and also report that the associated molecules were de-regulated. Through this work, we show for the first time that the chaining phenotype is regulated by PrkC, a transmembrane kinase with a sensor domain. During infection, PrkC may regulate the chaining phenotype through the identified signaling mechanism. Authors summaryB. anthracis, a spore-forming pathogen is the causative agent of anthrax, a zoonotic disease that primarily affects livestock and wildlife. Humans are at risk of contracting this disease through exposure to spores generated by infected animals. In the past, B. anthracis spores have been used as a bioterror agent. Hence, there has been a continuous effort to understand the biology of this pathogen to develop both therapeutic and prophylactic treatment. Various virulence factors that are essential for B. anthracis pathogenesis have been identified. The ability of B. anthracis to grow in chains acts as a virulence factor. Longer bacterial chains are reported to cause blockade of lung capillaries in the mouse infection model. In this study, we have shown that the disruption of the lone serine/threonine protein kinase, PrkC, localized at the bacteria-host interface leads to the shortening of the bacterial chains. We have seen that the depletion of PrkC results in an increase in the levels of the proteins responsible for de-chaining. Also, we have analyzed the effect of the disruption on cell growth, bacterial cell wall and septa formation. Since PrkC is a surface localized kinase with an extracellular domain that lacks homology to human proteins, it can be a target for new drugs. Disruption of PrkC activity and hence the longer chains in vivo may prevent pathophysiological consequences associated with the capillary blockade.

microbiology↗