The stringent response of Staphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression.
The stringent response of Staphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression.
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DOI：
10.1371/journal.ppat.1003016
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Wolz C
中科院分区：
文献类型：
--
作者：
Geiger T;Francois P;Liebeke M;Fraunholz M;Goerke C;Krismer B;Schrenzel J;Lalk M;Wolz C
严格响应由快速（p）ppGpp合成启动，这导致大多数细菌中基因表达的深刻重编程。严格的表型似乎是物种特异性的，可能是由根本不同的分子机制介导的。在金黄色葡萄球菌中，氨基酸剥夺后的（p）ppGpp合成通过双功能酶RSH（RelA/SpoT同系物）的合酶结构域实现。在一些厚壁菌中，严格的反应和CodY调节子之间的直接联系被提出。通过转录组分析来分析野生型菌株HG 001、rshSyn、codY和rshSyn、codY双突变体，以描绘通过氨基酸剥夺诱导严格响应后的RH依赖性（p）ppGpp合成的不同结果。在这些条件下，编码蛋白质合成机制和核苷酸代谢的主要成分的基因仅在rsh阳性菌株中下调。在（p）ppGpp诱导后被激活的基因大多通过GTP应答性阻遏物CodY的去阻遏而间接调节。只有七个基因，包括那些编码细胞毒性酚可溶性调节蛋白（PSM），被发现通过独立于CodY的RSH上调。qtRT-PCR分析表明，在S.金黄色葡萄球菌在人类多形核中性粒细胞（PMN）。RSH活性反过来对psms的细胞内表达至关重要。因此，rshSyn和rshSyn，codY突变体在类似于psm突变体的吞噬作用后存活的能力较低。吞噬体内诱导psmα1-4和/或psmβ 1，2可以补充rshSyn突变体的存活。因此，细胞内psm表达需要活性RSH合酶，其有助于吞噬后的存活。严格应答是细菌对多种不同环境应激条件的应答，其特征在于信使分子（p）ppGpp的合成。现在有越来越多的证据表明，这些分子在病原体在宿主内的特定表型状态之间转换中也起着关键作用。这对于适应感染期间遇到的不同微环境似乎至关重要，例如在被吞噬细胞摄取后。吞噬细胞的杀伤以及在这些细胞内的生存被认为是人类病原体金黄色葡萄球菌在体内成功传播的主要机制。在目前的研究中，我们证明了严格的反应对整体基因表达的影响，在S。金黄色葡萄球菌及其对人中性粒细胞胞内存活的影响。我们揭示了一个严格的反应后，吸收的S。金黄色葡萄球菌在中性粒细胞中的表达和RSH活性对于细胞内诱导psm表达至关重要，编码细胞毒性酚可溶性调节蛋白（PSM）。最后，我们表明，这反过来介导细菌的生存和逃避吞噬作用后。这些发现有助于了解PSM如何以及在何处可以作为有效的细胞溶解分子，并强调（p）ppGpp作为细胞内信号分子的重要性。
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. 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.
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