Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

Abstract

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.

Role of Serine/Threonine Phosphatase (SP-STP) in Streptococcus pyogenes Physiology and Virulence

“…The resulting plasmid pVP-SN1⌬STP was used to transform both the wild-type GAS strains, M1SF370 and M1T1, as described previously (20) to obtain M1⌬STP and M1T1⌬STP mutants, respectively. The double crossover homologous recombination event was confirmed by PCR using flanking forward primer 9 in combination with kanamycin reverse primer 10, and kanamycin forward primer 11 along with flanking reverse primer 12 along with gene-specific primer pair 13/14 (supplemental Table S2).…”

“…To assess the expression of CdhA/SibA in the GAS mutant strains devoid of STP, extracellular fractions were immunoblotted using anti-CdhA antibody (1:5000) (20), and the immunoreactive bands were visualized by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate solution.…”

“…The data presented in the mortality/survival curve were statistically evaluated using the log rank test using GraphPad Prism4 software. All animal experiments were performed in accordance with the Ohio State University Institutional Animal Care and Use Committee-approved protocol as described previously (20).…”

“…Our previous results demonstrating the correlation between long chain formation with the overexpression of CdhA (20), the STP mutants displaying long chain-forming phenotypes, loss of plane recognition, and multiple parallel septa formation led us to hypothesize that the loss of STP directly or indirectly affects CdhA in a temporal fashion. We validated this hypothesis by demonstrating an increase in the level of CdhA protein in the culture supernatants of early log phase-grown mutant strains, M1⌬STP (Fig.…”

“…Because STK has been shown previously to positively regulate cdhA (20) and its pneumococcal homolog is controlled by the WalRK regulon in S. pneumoniae (27,28), we attempted to explore the possibility of reversible phosphorylation of SP-WalR by SP-STK/STP couple. The in vitro phosphorylation assay revealed that SP-STK was able to phosphorylate SP-WalR (Fig.…”