The Stringent Response of Staphylococcus aureus and Its Impact on Survival after Phagocytosis through the Induction of Intracellular PSMs Expression

Abstract

The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rsh_Syn, codY and rsh_Syn, codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rsh_Syn and rsh_Syn, codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psmα1-4 and/or psmβ1,2 could complement the survival of the rsh_Syn mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis. The stringent response is a bacterial response to a multitude of different environmental stress conditions which is characterized by the synthesis of the messenger molecules (p)ppGpp. There is now growing evidence that these molecules also play a key role for pathogens to switch between specific phenotypic states within the host. This seems crucial for the adaptation to different microenvironments encountered during infection for instance after uptake by phagocytes. Killing of phagocytes as well as survival within these cells was proposed as major mechanisms for the success of the human pathogen Staphylococcus aureus to spread within the body. In the current study we demonstrate the effect of the stringent response on global gene expression in S. aureus and its impact on intracellular survival in human neutrophils. We reveal that a stringent response is induced after uptake of S. aureus in neutrophils and RSH activity is crucial for intracellular induction of psm expression, coding for cytotoxic phenol-soluble modulins (PSMs). Finally we show that this in turn mediates bacterial survival and escape after phagocytosis. These findings contribute to the understanding of how and where PSMs can act as potent cytolytic molecules and emphasise the importance of (p)ppGpp as an intracellular signalling molecule.