The Stringent Response of Staphylococcus aureus and Its Impact on Survival after Phagocytosis through the Induction of Intracellular PSMs Expression Tobias Geiger 1 , Patrice Francois 2 , Manuel Liebeke 3¤ , Martin Fraunholz 4 , Christiane Goerke 1 , Bernhard Krismer 1 , Jacques Schrenzel 2 , Michael Lalk 3 , Christiane Wolz 1 * 1 Interfaculty Institute of Microbiology and Infection Medicine, University of Tu¨ bingen, Tu¨ bingen, Germany, 2 Genomic Research Laboratory, Infectious Diseases Service, Geneva University Hospitals and the University of Geneva, Geneva, Switzerland, 3 Institute of Pharmaceutical Biology, Ernst-Moritz-Arndt University of Greifswald, Greifswald, Germany, 4 Department of Microbiology, Biocenter, University of Wu¨ rzburg, Wu¨ rzburg, Germany 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 psma1-4 and/or psmb1,2 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. Citation: Geiger T, Francois P, Liebeke M, Fraunholz M, Goerke C, et al. (2012) The Stringent Response of Staphylococcus aureus and Its Impact on Survival after Phagocytosis through the Induction of Intracellular PSMs Expression. PLoS Pathog 8(11): e1003016. doi:10.1371/journal.ppat.1003016 Editor: Frank R. DeLeo, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States of America Received May 22, 2012; Accepted September 22, 2012; Published November 29, 2012 Copyright: ß 2012 Geiger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by grants to C. Wolz and M. Lalk from the Deutsche Forschungsgemeinschaft TR34. T. Geiger received a grant from the fortune program (2068-0-0) of the medical faculty of Tu¨ bingen. M. Liebeke was a recipient of a fellowship from the Alfred Krupp von Bohlen and Halbach-Stiftung: ‘‘A functional Genomics Approach in Infection Biology’’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: christiane.wolz@med.uni-tuebingen.de ¤ Current address: Department of Surgery and Cancer, Biomolecular Medicine, Faculty of Medicine, Imperial College London, London, United Kingdom Introduction In most bacteria, nutrient limitations provoke the so-called stringent response, which is initiated by the rapid synthesis of the alarmones pppGpp and/or ppGpp, here referred to as (p)ppGpp. Under stringent conditions, (p)ppGpp results in the shut-down of proliferation-related activities, including the transcriptional re- pression of genes coding for major components of the protein synthesis apparatus (rRNA, ribosomal proteins and translation factors) as well as the inhibition of replication [1,2,3]. Typically genes that are presumed to be important for maintenance and stress-defence are activated under stringent conditions. However, the stringent phenotype resulting from (p)ppGpp synthesis seems to be bacteria species specific and may be mediated by fundamentally different molecular mechanisms [3]. The molecular mechanisms leading to the profound reprogram- ming of the bacterial cellular machinery under stringent conditions were mostly studied in Escherichia coli. Here, (p)ppGpp can be synthesized by either one of the two homologous enzymes: RelA and SpoT. The RelA-synthase is activated by sensing uncharged tRNAs that are bound to the ribosome. SpoT is a bifunctional enzyme that not only produces (p)ppGpp in response to diverse signals but also contains a (p)ppGpp hydrolase domain important for (p)ppGpp turnover. In E. coli, (p)ppGpp binds, with the help of the DksA protein, directly to the RNA polymerase (RNAP). However, even in this model organism, there is still much debate concerning how (p)ppGpp eventually leads to different promoter activities, how (p)ppGpp influences the stability of open complex formation at the initial phase of transcription and which of the promoters are indirectly regulated via secondary regulatory circuits such as alternative sigma factors or other transcription factors [1]. In other organisms, such as the gram-positive Bacillus subtilis, (p)ppGpp probably does not interact with the RNAP, and no PLOS Pathogens | www.plospathogens.org 1 November 2012 | Volume 8 | Issue 11 | e1003016