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

RelA/SpoT homologue (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 Streptococcus pneumoniae, Rel(Spn), can be deleted and is the primary source of (p) ppGpp synthesis in virulent strain D39 under some conditions. A D39 Delta rel(Spn) 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 Delta rel(Spn) strains treated with mupirocin revealed rel(Spn)-independent (translation stress), rel(Spn)-dependent (stringent response) and Delta rel(Spn)-dependent changes, suggesting that rel(Spn) 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 rel(Spn)-dependent response included significant upregulation of the ply operon encoding pneumolysin toxin, whereas the Delta rel(Spn)-dependent response affected expression linked to the VicRK and CiaRH two-component systems. Finally, a D39 Delta rel(Spn) 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)(+).