Bacterial persister cells are phenotypic variants that exhibit a transient non-growing state and antibiotic tolerance. Here, we provide in vitro evidence ofStaphylococcus aureuspersisters within infected host cells. We show that the bacteria surviving antibiotic treatment within host cells are persisters, displaying biphasic killing and reaching a uniformly non-responsive, non-dividing state when followed at the single-cell level. This phenotype is stable but reversible upon antibiotic removal. IntracellularS. aureuspersisters remain metabolically active but display an altered transcriptomic profile consistent with activation of stress responses, including the stringent response as well as cell wall stress, SOS and heat shock responses. These changes are associated with multidrug tolerance after exposure to a single antibiotic. We hypothesize that intracellularS. aureuspersisters may constitute a reservoir for relapsing infection and could contribute to therapeutic failures.

Temperate bacteriophages play an important role in the pathogenicity of Staphylococcus aureus , for instance, by mediating the horizontal gene transfer of virulence factors. Here we established a classification scheme for staphylococcal prophages of the major Siphoviridae family based on integrase gene polymorphism. Seventy-one published genome sequences of staphylococcal phages were clustered into distinct integrase groups which were related to the chromosomal integration site and to the encoded virulence gene content. Analysis of three marker modules (lysogeny, tail, and lysis) for phage functional units revealed that these phages exhibit different degrees of genome mosaicism. The prevalence of prophages in a representative S. aureus strain collection consisting of 386 isolates of diverse origin was determined. By linking the phage content to dominant S. aureus clonal complexes we could show that the distribution of bacteriophages varied remarkably between lineages, indicating restriction-based barriers. A comparison of colonizing and invasive S. aureus strain populations revealed that hlb -converting phages were significantly more frequent in colonizing strains.

Most of the dissimilarity between Staphylococcus aureus strains is due to the presence of mobile genetic elements such as bacteriophages or pathogenicity islands. These elements provide the bacteria with additional genes that enable them to establish a new lifestyle that is often accompanied by a shift to increased pathogenicity or a jump to a new host. S. aureus phages may carry genes coding for diverse virulence factors such as Panton-Valentine leukocidin, staphylokinase, enterotoxins, chemotaxis-inhibitory proteins, or exfoliative toxins. Phages also mediate the transfer of pathogenicity islands in a highly coordinated manner and are the primary vehicle for the horizontal transfer of chromosomal and extra-chromosomal genes. Here, we summarise recent advances regarding phage classification, genome organisation and function of S. aureus phages with a particular emphasis on their role in the evolution of the bacterial host.

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.

The repressor CodY is reported to inhibit metabolic genes mainly involved in nitrogen metabolism. We analyzed codY mutants from three unrelated Staphylococcus aureus strains (Newman, UAMS-1, and RN1HG). The mutants grew more slowly than their parent strains in a chemically defined medium. However, only codY mutants were able to grow in medium lacking threonine. An excess of isoleucine resulted in growth inhibition in the wild type but not in the codY mutants, indicating that isoleucine plays a role in CodY-dependent repression. Prototypic CodY-repressed genes including the virulence regulator agr are repressed after up-shift with isoleucine. The CodY-dependent repression of agr is consistent with the concomitant influence of CodY on typical agr -regulated genes such as cap , spa , fnbA , and coa . However, some of these virulence genes (e.g., cap , fnbA , and spa ) were also regulated by CodY in an agr- negative background. Microarray analysis revealed that the large majority of CodY-repressed genes were involved in amino acid metabolism; CodY-activated genes were mainly involved in nucleotide metabolism or virulence. In summary, CodY in S. aureus not only acts as a repressor for genes involved in nitrogen metabolism but also contributes to virulence gene regulation by supporting as well as substituting for agr function.