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Capsule Mediates the Cell Shape of pcsB Mutants in Serotype 2 Streptococcus pneumoniae

ABSTRACT: PcsB is a protein of unknown function that plays a critical role in cell division in Streptococcus pneumoniae and other ovococcus species of Streptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent and pcsB+ merodiploid strains indicated that pcsB is essential in serotype 2 S. pneumoniae. Quantitative Western blotting of wild-type and epitope-tagged PcsB showed that all PcsB was processed into cell-associated and secreted forms of the same molecular mass. These analyses showed that PcsB bound to cells is present in relatively low amounts of only ≈ 300 molecules per cell. Controlled expression and complementation experiments indicated that there was a causative relationship between the severity of defects in cell division and decreasing PcsB amount. These experiments also indicated that perturbations of expression of the upstream mreCD genes did not contribute to the cell division defects of pcsB mutants and that mreCD could readily be deleted in these strains. Unexpectedly, the defects in cell division and cell shape in pcsB mutants or other mutants defective in cell wall biosynthesis, such as dacA, were strongly influenced by capsule. Underexpression of PcsB did not result in changes in the amounts or composition of lactoyl-muropeptides, which were markedly different in the R6 and D39 strains, and there was no correlation between decreased PcsB amount and sensitivity to penicillin. Finally, microarray analyses indicated that underexpression of PcsB may generate a signal that increases expression of the VicRK regulon, which includes pcsB.

ORGANISM(S): Streptococcus pneumoniae

PROVIDER: GSE13107 | GEO | 2009/09/29

SECONDARY ACCESSION(S): PRJNA109941

REPOSITORIES: GEO

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Project description:PcsB is a protein of unknown function that plays a critical role in cell division in Streptococcus pneumoniae and other ovococcus species of Streptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent and pcsB+ merodiploid strains indicated that pcsB is essential in serotype 2 S. pneumoniae. Quantitative Western blotting of wild-type and epitope-tagged PcsB showed that all PcsB was processed into cell-associated and secreted forms of the same molecular mass. These analyses showed that PcsB bound to cells is present in relatively low amounts of only â 300 molecules per cell. Controlled expression and complementation experiments indicated that there was a causative relationship between the severity of defects in cell division and decreasing PcsB amount. These experiments also indicated that perturbations of expression of the upstream mreCD genes did not contribute to the cell division defects of pcsB mutants and that mreCD could readily be deleted in these strains. Unexpectedly, the defects in cell division and cell shape in pcsB mutants or other mutants defective in cell wall biosynthesis, such as dacA, were strongly influenced by capsule. Underexpression of PcsB did not result in changes in the amounts or composition of lactoyl-muropeptides, which were markedly different in the R6 and D39 strains, and there was no correlation between decreased PcsB amount and sensitivity to penicillin. Finally, microarray analyses indicated that underexpression of PcsB may generate a signal that increases expression of the VicRK regulon, which includes pcsB. Four independent hybridizations using independent RNA preparations from the strain IU1533 (reference strain) and strain IU1979 (a strain with decreased expression of PcsB protein) were performed. Dye swap was performed with the reference strain labeled with Cy3 in two hybridizations and Cy5 in the other two hybridizations.

Project description:Infection of the human host by Streptococcus pneumoniae begins with colonization of the nasopharynx, which is mediated by adherence of bacteria to respiratory epithelium. Several studies have indicated an important role for the pneumococcal capsule in this process. Here, we used microarrays to characterize the in vitro transcriptional response of human nasopharyngeal epithelial Detroit 562 cells to adherence of serotype 2-encapsulated strain D39, serotype 19F-encapsulated strain G54, serotype 4-encapsulated strain TIGR4, and their nonencapsulated derivatives (delta-cps). In total, 322 genes were found to be upregulated in response to adherent pneumococci. Twenty-two genes were commonly induced, including those encoding several cytokines (e.g., IL-1-beta, IL-6), chemokines (e.g., IL-8, CXCL1/2), and transcriptional regulators (e.g., FOS), consistent with an innate immune response mediated by Toll-like receptor signaling. Interestingly, 85% of genes was induced specifically by one or more encapsulated strains, suggestive of a capsule-dependent response. Importantly, purified capsular polysaccharides alone had no effect. Over a third of these loci encoded products predicted to be involved in transcriptional regulation and signal transduction, in particular MAPK signaling pathways. Real-time PCR of a subset of ten genes confirmed microarray data and showed a time-dependent upregulation of especially innate immunity genes. Downregulation of epithelial genes was most pronounced upon adherent D39delta-cps, as 68% of the 161 genes identified was only repressed using this nonencapsulated strain. In conclusion, we identified a subset of host genes specifically induced by encapsulated strains during in vitro adherence, and have demonstrated the complexity of interactions occurring during the initial stages of pneumococcal infection. Experiment Overall Design: We used three different pneumococcal strains and their isogenic nonencapsulated derivatives (delta-cps): serotype 2-encapsulated strain D39, serotype 19F-encapsulated strain G54, and serotype 4-encapsulated strain TIGR4. All experiments were performed in triplicate (3 independent biological replicates) and compared to uninfected control Detroit 562 cells. Bacteria were allowed to adhere to the epithelial cells for 2 hours, after which the transcriptional response of the Detroit cells was analyzed using Affymetrix Human U133 Plus GeneChips. In addition to the 6 strains mentioned above, we included transcriptional analysis of the epithelial cell response to low-dose D39delta-cps (giving adherence equivalent to wild-type D39) and purified type 2 capsular polysaccharides.

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Capsule Mediates the Cell Shape of pcsB Mutants in Serotype 2 Streptococcus pneumoniae

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