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
2015
ABSTRACT We determined whether there is
turnoverof the peptidoglycan (PG) cell wall of the ovococcus bacterial pathogen Streptococcus pneumoniae (pneumococcus). Pulse-chase experiments on serotype 2 strain D39 radiolabeled with
N-
acetylglucosaminerevealed little
turnoverand release of PG breakdown products during growth compared to published reports of PG
turnoverin 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 infusionbroth, in D39 cells grown in
chemically defined mediumcontaining 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
turnoverin 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
pentamerPG 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
turnovercompared to the PG
turnoverin rod-shaped cells. IMPORTANCE PG 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
turnoverof 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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