Roles of rel(Spn) in stringent response, global regulation and virulence of serotype 2 Streptococcus pneumoniae D39.

RelA/ SpoT homolog (RSH) proteins have (p)ppGpp synthetase and hydrolase activities that mediate major global responses to nutrient limitation and other stresses. RSH proteins are conserved in most bacteria and play diverse roles in bacterial pathogenesis. We report here that the RSH protein of S. pneumoniae, RelSpn, can be deleted and is the primary source of (p)ppGpp synthesis in virulent strain D39 under some conditions. A D39 ΔrelSpn mutant grew well in complex medium, but did not grow in chemically defined medium unless supplemented with the metals copper and manganese. Transcriptome analysis of D39 rel+Spn and ΔreSpn strains treated with mupirocin revealed relSpn-independent (translation stress), relSpn-dependent (stringent response), and ΔrelSpn-dependent changes suggesting that relSpn and (p)ppGpp amount play wide-ranging homeostatic roles in pneumococcal physiology, besides adjusting macromolecular synthesis and transport in response to nutrient availability. Notably, the relSpn-dependent response included significant up-regulation of the ply operon encoding pneumolysin toxin, whereas the ΔrelSpn-dependent response affected expression linked to the VicRK and CiaRH two component systems. Finally, a D39 ΔrelSpn mutant was severely attenuated and displayed a significantly altered course of disease progression in a mouse model of infection, which was restored to normal by an ectopic copy of rel+Spn.

RelA/SpoT同系物（RSH）蛋白具有（p）ppGpp合成酶和水解酶活性，可介导对营养限制和其他压力的主要全局反应。RSH蛋白在大多数细菌中是保守的，并在细菌致病性中发挥多种作用。我们在此报道，肺炎链球菌的RSH蛋白RelSpn可以被缺失，并且在某些条件下是有毒力的D39菌株中（p）ppGpp合成的主要来源。D39 ΔrelSpn突变体在复合培养基中生长良好，但在化学限定培养基中不能生长，除非补充金属铜和锰。用莫匹罗星处理的D39 rel⁺Spn和ΔrelSpn菌株的转录组分析揭示了不依赖relSpn的（翻译应激）、依赖relSpn的（严谨反应）以及依赖ΔrelSpn的变化，这表明relSpn和（p）ppGpp的量除了根据营养可利用性调节大分子合成和转运外，在肺炎球菌生理学中还发挥广泛的稳态作用。值得注意的是，依赖relSpn的反应包括编码肺炎溶素毒素的ply操纵子的显著上调，而依赖ΔrelSpn的反应影响与VicRK和CiaRH双组分系统相关的表达。最后，D39 ΔrelSpn突变体在感染小鼠模型中严重减毒，并显示出疾病进展过程的显著改变，通过rel⁺Spn的异位拷贝可使其恢复正常。