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, rshSyn, codY and rshSyn, 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, rshSyn and rshSyn, 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 rshSyn mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis.
Highlights
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
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)
We aimed to delineate the impact of (p)ppGpp synthesis induced by amino acid starvation on gene expression in S. aureus under defined nutrient limitation rather than after the addition of inhibitors such as mupirocin
Summary
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. 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 reprogramming of the bacterial cellular machinery under stringent conditions were mostly studied in Escherichia coli. In E. coli, (p)ppGpp binds, with the help of the DksA protein, directly to the RNA polymerase (RNAP). 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]
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