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Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

ABSTRACT: Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan.

SUBMITTER: Figueiredo J

PROVIDER: S-EPMC9216600 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

Figueiredo Joana J Henriques Mafalda Xavier MX Catalão Maria João MJ Pinheiro Sara S Narciso Ana Rita AR Mesquita Francisco F Saraiva Bruno Manuel BM Carido Madalena M Cabanes Didier D Pinho Mariana Gomes MG Filipe Sérgio Raposo SR

PLoS pathogens 20220622 6

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interac ...[more]

PMID: 35731836

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Project description:Bacterial morphology is largely determined by the spatial and temporal regulation of peptidoglycan (PG) biosynthesis. Ovococci possess a unique pattern of PG synthesis different from the well studied Bacillus, and the mechanism of the coordination of PG synthesis remains poorly understood. Several regulatory proteins have been identified to be involved in the regulation of ovococcal morphogenesis, among which DivIVA is an important one to regulate PG synthesis in streptococci, while its mechanism is largely unknown. Here, the zoonotic pathogen Streptococcus suis was used to investigate the regulation of DivIVA on PG synthesis. Fluorescent d-amino acid probing and 3D-structured illumination microscopy found that DivIVA deletion caused abortive peripheral PG synthesis, resulting in a decreased aspect ratio. The phosphorylation-depleted mutant (DivIVA3A) cells displayed a longer nascent PG and became longer, whereas the phosphorylation-mimicking mutant (DivIVA3E) cells showed a shorter nascent PG and became shorter, suggesting that DivIVA phosphorylation is involved in regulating peripheral PG synthesis. Several DivIVA-interacting proteins were identified, and the interaction was confirmed between DivIVA and MltG, a cell wall hydrolase essential for cell elongation. DivIVA did not affect the PG hydrolysis activity of MltG, while the phosphorylation state of DivIVA affected its interaction with MltG. MltG was mislocalized in the ΔdivIVA and DivIVA3E cells, and both ΔmltG and DivIVA3E cells formed significantly rounder cells, indicating an important role of DivIVA phosphorylation in regulating PG synthesis through MltG. These findings highlight the regulatory mechanism of PG synthesis and morphogenesis of ovococci. IMPORTANCE The peptidoglycan (PG) biosynthesis pathway provides a rich source of novel antimicrobial drug targets. However, bacterial PG synthesis and its regulation is a very complex process involving dozens of proteins. Moreover, unlike the well studied Bacillus, ovococci undergo unusual PG synthesis with unique mechanisms of coordination. DivIVA is an important regulator of PG synthesis in ovococci, while its exact role in regulating PG synthesis remains poorly understood. In this study, we determined the role of DivIVA in regulating lateral PG synthesis of Streptococcus suis and identified a critical interacting partner, MltG, in which DivIVA influenced the subcellular localizations of MltG through its phosphorylation. Our study characterizes the detailed role of DivIVA in regulating bacterial PG synthesis, which is very helpful for understanding the process of PG synthesis in streptococci.

Project description:UnlabelledWe determined whether there is turnover of the peptidoglycan (PG) cell wall of the ovococcus bacterial pathogen Streptococcus pneumoniae (pneumococcus). Pulse-chase experiments on serotype 2 strain D39 radiolabeled with N-acetylglucosamine revealed little turnover and release of PG breakdown products during growth compared to published reports of PG turnover in Bacillus subtilis. PG dynamics were visualized directly by long-pulse-chase-new-labeling experiments using two colors of fluorescent d-amino acid (FDAA) probes to microscopically detect regions of new PG synthesis. Consistent with minimal PG turnover, hemispherical regions of stable "old" PG persisted in D39 and TIGR4 (serotype 4) cells grown in rich brain heart infusion broth, in D39 cells grown in chemically defined medium containing glucose or galactose as the carbon source, and in D39 cells grown as biofilms on a layer of fixed human epithelial cells. In contrast, B. subtilis exhibited rapid sidewall PG turnover in similar FDAA-labeling experiments. High-performance liquid chromatography (HPLC) analysis of biochemically released peptides from S. pneumoniae PG validated that FDAAs incorporated at low levels into pentamer PG peptides and did not change the overall composition of PG peptides. PG dynamics were also visualized in mutants lacking PG hydrolases that mediate PG remodeling, cell separation, or autolysis and in cells lacking the MapZ and DivIVA division regulators. In all cases, hemispheres of stable old PG were maintained. In PG hydrolase mutants exhibiting aberrant division plane placement, FDAA labeling revealed patches of inert PG at turns and bulge points. We conclude that growing S. pneumoniae cells exhibit minimal PG turnover compared to the PG turnover in rod-shaped cells.ImportancePG cell walls are unique to eubacteria, and many bacterial species turn over and recycle their PG during growth, stress, colonization, and virulence. Consequently, PG breakdown products serve as signals for bacteria to induce antibiotic resistance and as activators of innate immune responses. S. pneumoniae is a commensal bacterium that colonizes the human nasopharynx and opportunistically causes serious respiratory and invasive diseases. The results presented here demonstrate a distinct demarcation between regions of old PG and regions of new PG synthesis and minimal turnover of PG in S. pneumoniae cells growing in culture or in host-relevant biofilms. These findings suggest that S. pneumoniae minimizes the release of PG breakdown products by turnover, which may contribute to evasion of the innate immune system.

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Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

Publications

Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

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