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

Abstract

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 ΔprkC) 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 ΔprkC 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 ΔprkC 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.

Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes

“…All statistical analyses were performed in GraphPad Prism 9.2 ( 26 ). To test for significance between two groups, two-tailed paired t test was used.…”

Role of serine/threonine protein phosphatase PrpN in the life cycle of Bacillus anthracis

“…The role of protein ser/thr phosphorylation in bacterial growth and cell division is a widely investigated topic [48][49][50]. A recent report by our group in B. anthracis showed growth defects in a ser/thr kinase (PrkC) mutant strain, thus establishing a functional correlation between bacterial growth and ser/thr phosphorylation [41]. To examine the role of PrpN in B. anthracis growth machinery, we monitored the growth patterns of BAS WT, BAS ΔprpN and BAS ΔprpN::prpN strains.…”

“…We also monitored the growth profiles in the presence of salt [1 M sodium chloride (NaCl)] and oxidative [2.5 mM hydrogen peroxide (H 2 O 2 )] stresses and observed an extended lag phase in the absence of prpN (Fig 2B and 2C). Intrigued by the above growth pattern, we next checked for the temporal expression of PrpN in BAS WT by immunoblot analysis at four bacterial growth phases-(i) lag, (ii) exponential, (iii) early stationary (time points indicated in Fig 2A) and (iv) late stationary/sporulation initiation ~60 hours [41]. The bacterial cells were visualized microscopically to confirm the growth phase (Fig 2D-upper panel) prior to protein lysates preparation.…”

“…In B. anthracis, while ser/thr protein phosphorylation is implicated in various cellular pathways including bacterial pathogenesis, only a few ser/thr kinases (STK) and ser/thr phosphatases (STP) have been characterized [38][39][40][41][42][43][44][45][46]. These include the three STKs (PrkC, PrkD, and PrkG) and a single STP (PrpC).…”

The Bacillus anthracis Cell Envelope: Composition, Physiological Role, and Clinical Relevance

“…These evidences not only support the existence of a raft like entities in B. anthracis but also the role that they play in its pathology, suggesting their possible use for the development of novel drugs or vaccines against anthrax (Figure 1B). An interesting study from 2020 has identified the first sensory molecule regulating the chaining phenotype of B. anthracis, the PrkC serine/threonine protein kinase [46]. Such phenotype has been shown to contribute significantly to the virulence of this bacterium [47][48][49].…”