Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release

Abstract

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.

Disentangling competence for genetic transformation and virulence in Streptococcus pneumoniae

“…Interestingly, these aforementioned findings by Benton et al (1995) and Greene et al (2015) are in conflict with our findings that pneumolysin is responsible for CbpD, CibAB and p1-LytA-related virulence, because we did not observe the cross-feeding effect of pneumolysin in our competitive assays (Fig. 2b, CI of cbpD, cibAB and p1 in D39 background should be ≥1 if pneumolysin displays a cross-feeding effect).…”

“…Two studies have shown that some pneumolysin molecules are localized to pneumococcal cell wall (Greene et al 2015;Price and Camilli 2009), and its release involves both complete autolysis as well as cell wall remodeling of intact pneumococcus by choline binding proteins, including CbpD and LytA (Greene et al 2015). Similar cell lysis independent release of cytoplasmic proteins also has been observed in Staphylococcus (Ebner et al 2015).…”

“…However, during a competitive infection, pneumolysin released by co-infecting wild-type bacteria can cross-feed ΔcbpD and therefore, compensate the attenuated inability of ΔcbpD to release pneumolysin. Given the evidence that pneumolysin-deficient mutant (Δply) shows reduced virulence in single infection model (Benton et al 1995), and its virulence reduction can be compensated by co-infecting wild-type strain during both intravenous (Benton et al 1995) and intranasal (Greene et al 2015) infections, it is reasonable to argue that cross-feeding of pneumolysin by wild-type occurs within close proximity, as well as at a distance, and is accessible to both wild-type and Δply at similar concentrations.…”

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

“…The MurMN enzymes catalyze cross-bridge formation in D39 and other pneumococcal strains (60)(61)(62)(63). We showed previously that laboratory strain R6 contains substantially more cross-bridges than its D39 progenitor strain (40).…”

Staying in Shape: the Impact of Cell Shape on Bacterial Survival in Diverse Environments

“…Gram-positive organisms can elaborate a thicker wall with altered chemical properties as a barrier to noxious chemicals and conditions, while the thin and relatively simpler cell wall of Gram-negative organisms may necessitate minimizing the surface area as a primary strategy to avoid interactions with a hostile environment. Early experiments suggested the Gram-positive cell wall as a barrier to release of secreted ␣-amylase in Bacillus amyloliquefaciens (144), and the production of branched PG stem peptides in pneumococcus PG was recently found to limit pneumolysin release (145).…”