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Colonization of the human nose by Staphylococcus aureus in one-third of the population represents a major risk factor for invasive infections. The basis for adaptation of S. aureus to this specific habitat and reasons for the human predisposition to become colonized have remained largely unknown. Human nasal secretions were analyzed by metabolomics and found to contain potential nutrients in rather low amounts. No significant differences were found between S. aureus carriers and non-carriers, indicating that carriage is not associated with individual differences in nutrient supply. A synthetic nasal medium (SNM3) was composed based on the metabolomics data that permits consistent growth of S. aureus isolates. Key genes were expressed in SNM3 in a similar way as in the human nose, indicating that SNM3 represents a suitable surrogate environment for in vitro simulation studies. While the majority of S. aureus strains grew well in SNM3, most of the tested coagulase-negative staphylococci (CoNS) had major problems to multiply in SNM3 supporting the notion that CoNS are less well adapted to the nose and colonize preferentially the human skin. Global gene expression analysis revealed that, during growth in SNM3, S. aureus depends heavily on de novo synthesis of methionine. Accordingly, the methionine-biosynthesis enzyme cysteine-γ-synthase (MetI) was indispensable for growth in SNM3, and the MetI inhibitor DL-propargylglycine inhibited S. aureus growth in SNM3 but not in the presence of methionine. Of note, metI was strongly up-regulated by S. aureus in human noses, and metI mutants were strongly abrogated in their capacity to colonize the noses of cotton rats. These findings indicate that the methionine biosynthetic pathway may include promising antimicrobial targets that have previously remained unrecognized. Hence, exploring the environmental conditions facultative pathogens are exposed to during colonization can be useful for understanding niche adaptation and identifying targets for new antimicrobial strategies.

Phytochelatins are small cysteine-rich non-ribosomal peptides that chelate soft metal and metalloid ions, such as cadmium and arsenic. They are widely produced by plants and microbes; phytochelatin synthase genes are also present in animal species from several different phyla, but there is still little known about whether these genes are functional in animals, and if so, whether they are metal-responsive. We analysed phytochelatin production by direct chemical analysis in Lumbricus rubellus earthworms exposed to arsenic for a 28 day period, and found that arsenic clearly induced phytochelatin production in a dose-dependent manner. It was necessary to measure the phytochelatin metabolite concentrations directly, as there was no upregulation of phytochelatin synthase gene expression after 28 days: phytochelatin synthesis appears not to be transcriptionally regulated in animals. A further untargetted metabolomic analysis also found changes in metabolites associated with the transsulfuration pathway, which channels sulfur flux from methionine for phytochelatin synthesis. There was no evidence of biological transformation of arsenic (e.g. into methylated species) as a result of laboratory arsenic exposure. Finally, we compared wild populations of earthworms sampled from the field, and found that both arsenic-contaminated and cadmium-contaminated mine site worms had elevated phytochelatin concentrations.

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.