Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio, USA.
Infect Immun. 2023 Apr 18;91(4):e0029622. doi: 10.1128/iai.00296-22. Epub 2023 Mar 6.
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 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. C 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 P, supporting increased 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.
肺炎链球菌丝氨酸/苏氨酸激酶 (StkP) 和其同源磷酸酶 (PhpP) 在细菌胞分裂中起着至关重要的作用。然而，它们在有荚膜的肺炎链球菌中的代谢和毒力调节相关功能的个体和相互作用仍未得到充分研究。在这里，我们证明了从有荚膜的肺炎链球菌菌株 D39 衍生而来的 D39ΔPhpP 和 D39ΔStkP 突变体在含有葡萄糖或非葡萄糖糖作为唯一碳源的化学定义培养基中生长时，表现出不同的细胞分裂缺陷和生长模式。通过对这些突变体进行基于 RNA-seq 的全局转录组分析的显微镜和生化分析表明，D39ΔPhpP 和 D39ΔStkP 突变体中多糖荚膜形成和 基因显著下调和上调。虽然 StkP 和 PhpP 分别调节几个独特的基因，但它们也参与了同一组差异调节基因的调节。C 基因部分受 StkP/PhpP 介导的可逆磷酸化调节，而不依赖于 MapZ 调节的细胞分裂过程。StkP 介导的 CcpA 剂量依赖性磷酸化，使 CcpA 与 P 的结合比例增加，支持 D39ΔStkP 中 基因表达和荚膜形成的增加。虽然 D39ΔPhpP 突变体在两种小鼠感染模型中的衰减与几个下调的荚膜、毒力和磷酸转移酶系统 (PTS) 相关基因相一致，但与 D39 野生型相比，多糖荚膜数量增加的 D39ΔStkP 突变体在小鼠中的毒力显著降低，但与 D39ΔPhpP 相比，毒力增加。与人肺细胞共培养的这些突变体的基于 NanoString 技术的炎症相关基因表达和基于 Meso Scale Discovery 的多重趋化因子分析证实了它们不同的毒力表型。因此，StkP 和 PhpP 可以作为关键的治疗靶点。
