StkP- and PhpP-Mediated Posttranslational Modifications Modulate the S. pneumoniae Metabolism, Polysaccharide...

Pneumococcal Ser/Thr kinase (StkP) and its cognate phosphatase (PhpP) play a crucial role in bacterial cytokinesis. However, their individual and reciprocal metabolic and virulence regulation-related functions have yet to be adequately investigated in encapsulated pneumococci. Here, we demonstrate that the encapsulated pneumococcal strain D39-derived D39ΔPhpP and D39ΔStkP mutants displayed differential cell division defects and growth patterns when grown in chemically defined media supplemented with glucose or nonglucose sugars as the sole carbon source. Microscopic and biochemical analyses supported by RNA-seq-based global transcriptomic analyses of these mutants revealed significantly down- and upregulated polysaccharide capsule formation and cps2 genes in D39ΔPhpP and D39ΔStkP mutants, respectively. While StkP and PhpP individually regulated several unique genes, they also participated in sharing the regulation of the same set of differentially regulated genes. Cps2 genes were reciprocally regulated in part by the StkP/PhpP-mediated reversible phosphorylation but independent of the MapZ-regulated cell division process. StkP-mediated dose-dependent phosphorylation of CcpA proportionately inhibited CcpA-binding to Pcps2A, supporting increased cps2 gene expression and capsule formation in D39ΔStkP. While the attenuation of the D39ΔPhpP mutant in two mouse infection models corroborated with several downregulated capsules-, virulence-, and phosphotransferase systems (PTS)-related genes, the D39ΔStkP mutant with increased amounts of polysaccharide capsules displayed significantly decreased virulence in mice compared to the D39 wild-type, but more virulence compared to D39ΔPhpP. NanoString technology-based inflammation-related gene expression and Meso Scale Discovery-based multiplex chemokine analysis of human lung cells cocultured with these mutants confirmed their distinct virulence phenotypes. StkP and PhpP may, therefore, serve as critical therapeutic targets.