SPD_1495 Contributes to Capsular Polysaccharide Synthesis and Virulence in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae, a Gram-positive human pathogen, causes a series of serious diseases in humans. SPD_1495 from S. pneumoniae is annotated as a hypothetical ABC sugar-binding protein in the NCBI database, but there are few reports on detailed biological functions of SPD_1495. To fully study the influence of SPD_1495 on bacterial virulence in S. pneumoniae, we constructed a deletion mutant (D39Δspd1495) and an overexpressing strain (D39spd1495+). Comparative analysis of iTRAQ-based quantitative proteomic data of the wild-type D39 strain (D39-WT) and D39Δspd1495 showed that several differentially expressed proteins that participate in capsular polysaccharide synthesis, such as Cps2M, Cps2C, Cps2L, Cps2T, Cps2E, and Cps2D, were markedly upregulated in D39Δspd1495 Subsequent transmission electron microscopy and uronic acid detection assay confirmed that capsular polysaccharide synthesis was enhanced in D39Δspd1495 compared to that in D39-WT. Moreover, knockout of spd1495 resulted in increased capsular polysaccharide synthesis, as well as increased bacterial virulence, as confirmed by the animal study. Through a coimmunoprecipitation assay, surface plasmon resonance, and electrophoretic mobility shift assay, we found that SPD_1495 negatively regulated cps promoter expression by interacting with phosphorylated ComE, a negative transcriptional regulator for capsular polysaccharide formation. Overall, this study suggested that SPD_1495 negatively regulates capsular polysaccharide formation and thereby enhances bacterial virulence in the host. These findings also provide valuable insights into understanding the biology of this clinically important bacterium.IMPORTANCE Capsular polysaccharide is a key factor underlying the virulence of Streptococcus pneumoniae in human diseases. Thus, a deep understanding of capsular polysaccharide synthesis is essential for uncovering the pathogenesis of S. pneumoniae infection. In this study, we show that protein SPD_1495 interacts with phosphorylated ComE to negatively regulate the formation of capsular polysaccharide. Deletion of spd1495 increased capsular polysaccharide synthesis and thereby enhanced bacterial virulence. These findings further reveal the synthesis mechanism of capsular polysaccharide and provide new insight into the biology of this clinically important bacterium.

Keywords: Streptococcus pneumoniae; capsule; virulence.

FIG 1

Deletion of spd1495 affected the growth of S. pneumoniae D39 in medium containing different sugar sources. (A) Confirmation of gene deletion and overexpression of spd1495 using Western blotting. (B) SDS-PAGE results of whole-cell lysates of D39-WT, D39Δspd1495, and D39spd1495+ strains as loading controls for the Western blotting experiments of panel A. (C) Growth curves of D39-WT (red), D39Δspd1495 (blue), and D39spd1495+ (green) strains cultured in C+Y medium containing no sugar, Neu5Ac, arabinose, xylose, glucose, fructose, lactose, sucrose, and maltose. Statistical analysis was conducted using Prism 6.0.

FIG 2

Results of iTRAQ-based proteomics quantitative analysis. (A) Correlation of the fold change in differential proteins in two replicates. (B) The number of total proteins was identified in two biological replicates. (C) Distribution of biological variables in two replicates. (D) KEGG pathway enrichment analysis of differentially expressed proteins. (E) The pathways of polyketide sugar unit biosynthesis, streptomycin biosynthesis, and carbose and validamycin biosynthesis were enriched by KEGG. (F) Differentially expressed proteins involved in sugar metabolism network. (G) Differentially expressed proteins involved in the CPS synthesis network. Red represents upregulated proteins, whereas blue represents downregulated proteins.

FIG 3

SPD_1495 negatively regulated capsular synthesis. (A) SPD_1495 negative regulate the cps genes expression in D39-WT and D39Δspd1495. (B) TEM results for strains D39-WT, D39Δspd1495, and D39spd1495+. (C) Uronic acid contents in strains D39-WT, D39Δspd1495, and D39spd1495+. (D) The virulence of strains D39-WT, D39Δspd1495, and D39spd1495+. Data were analyzed by two-tailed, unpaired Student t tests, and results are expressed as means ± the SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 4

SPD_1495 interacted with ComE. (A) SDS-PAGE results of SPD_1495 expression, visualized using a PGEX-4T-1 fusion system and purified by GST affinity column. (B) Western blotting assay of SPD_1495 expression using anti-SPD_1495 antibody. (C) EMSA with SPD_1495 cannot bind to Bio-cps probe. (D) The potential proteins interacting with SPD_1495 were screened by a Co-IP assay and are indicated by arrows. (E) SPR detected the interaction of ComE^D58E with SPD_1495. (F) EMSA demonstrated that ComE^D58E, the complexes of ComE^D58E, and SPD_1495 bind to the Bio-cps probe.

FIG 5

Evolutionary analysis of SPD_1495. (A) Multiple sequence alignment of SPD_1495 with homologous proteins in various bacterial species. (B) Protein evolutionary tree of SPD_1495 and homologous proteins.

FIG 5

Evolutionary analysis of SPD_1495. (A) Multiple sequence alignment of SPD_1495 with homologous proteins in various bacterial species. (B) Protein evolutionary tree of SPD_1495 and homologous proteins.

FIG 6

Integrated model of SPD_1495 function in S. pneumoniae in this study. (A) In D39-WT, SPD_1495 interacted with ComE to negatively regulate capsular polysaccharide synthesis, resulting in normal capsule thickness. (B) In D39Δspd1495, without interaction between SPD_1495 with ComE, the bacterial capsule became thickened.