Genetic requirements for Staphylococcus aureus nitric oxide resistance and virulence.
Genetic requirements for Staphylococcus aureus nitric oxide resistance and virulence.
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DOI：
10.1371/journal.ppat.1006907
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发表时间：
2018-03
期刊：
影响因子：
6.7
通讯作者：
Richardson AR
中科院分区：
文献类型：
--
作者：
Grosser MR;Paluscio E;Thurlow LR;Dillon MM;Cooper VS;Kawula TH;Richardson AR
金黄色葡萄球菌表现出多种针对宿主先天免疫的防御能力，包括在一氧化氮 (NO·) 存在的情况下复制的能力。金黄色葡萄球菌 NO· 抗性是一个复杂的性状，取决于该病原体代谢适应 NO· 存在的能力。在这里，我们利用 USA300 LAC 生成的文库中的转座子连接深度测序 (Tn-Seq) 来定义金黄色葡萄球菌 NO· 抗性所需的完整基因集。我们将 NO·抗性基因列表与 LAC 在小鼠皮肤感染 (SSTI) 中持续存在所需的基因组进行了比较。总共，我们鉴定了 168 个对于完全 NO· 抗性至关重要的基因，其中 49 个基因也是金黄色葡萄球菌在 SSTI 中持续存在所必需的。许多这些NO·抗性基因先前被证明是在这种免疫自由基存在下生长所必需的。然而，新定义的基因，包括那些编码SodA、MntABC、RpoZ、参与Fe-S簇修复/稳态的蛋白质、UvrABC、硫氧还蛋白样蛋白质和F1F0 ATP酶的基因，之前尚未报道有助于金黄色葡萄球菌NO·抗性。最引人注目的发现是，编码 F1F0 ATP 酶成分的任何基因的丢失导致突变体无法在 NO· 或任何其他抑制细胞呼吸的条件下生长。此外，这些突变体在小鼠 SSTI 中高度减毒。我们发现，在金黄色葡萄球菌中，F1F0 ATP 酶以 ATP 水解模式运行，挤出质子并贡献质子动力。 ΔatpG 突变体中有效质子挤出的丧失导致细胞质酸化。虽然呼吸细胞可以耐受这种酸度，但发酵所需的酶在 pH ≤ 7.0 时无法有效运行，并且 ΔatpG 突变体无法生长。因此，金黄色葡萄球菌NO·抗性需要弱碱性胞质，如果没有活性F1F0 ATP酶复合物就无法实现这一条件。人类病原体金黄色葡萄球菌对宿主免疫反应的许多方面具有显着的抵抗力，包括抗菌自由基一氧化氮 (NO·)。这种耐药性的机制很复杂，包含许多基因产物。在这里，我们采用一种涉及转座子诱变与下一代测序（称为 Tn-Seq）相结合的方法来鉴定金黄色葡萄球菌 NO· 抗性和毒力所需的完整基因集。虽然我们鉴定了许多先前报道的NO·抗性决定因素，但从这种非靶向方法中发现了新的基因产物。具体来说，我们确定F1F0 ATP酶在NO·胁迫和毒力期间是必需的，但在正常培养条件下是可有可无的。这种条件适应性贡献的原因源于以下事实：在发酵条件下，F1F0 ATP酶以ATP水解模式发挥作用，有效地挤出质子并将细胞内pH提高到8.0以上。这恰好是许多发酵酶的最佳 pH 值。如果没有 F1F0 ATP 酶，质子挤出就会受到限制，细胞内 pH 值仍然太低，无法继续有效发酵。因此，在感染期间，当金黄色葡萄球菌由于发炎组织的性质而必须发酵时，F1F0 ATP酶成为一种必需的酶复合物和开发新抗菌剂的有效靶标。
Staphylococcus aureus exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). S. aureus NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for S. aureus NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for S. aureus to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F1F0 ATPase, have not been previously reported to contribute to S. aureus NO· resistance. The most striking finding was that loss of any genes encoding components of the F1F0 ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in S. aureus, the F1F0 ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the ΔatpG mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the ΔatpG mutant cannot thrive. Thus, S. aureus NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F1F0 ATPase enzyme complex. The human pathogen Staphylococcus aureus is remarkably resistant to many facets of the host immune response, including the antibacterial radical nitric oxide (NO·). The mechanism underlying this resistance is complex and comprises many gene products. Here we employ an approach that involves transposon mutagenesis coupled to next-generation sequencing (known as Tn-Seq) to identify the complete set of genes required for S. aureus NO· resistance and virulence. While we identified many previously reported NO·-resistance determinants, new gene products were discovered from this untargeted approach. Specifically, we identified the F1F0 ATPase as being essential during NO· stress and virulence yet dispensable under normal culture conditions. The reason for this conditional fitness contribution stems from the fact that under fermentative conditions, the F1F0 ATPase functions in the ATP hydrolysis mode, effectively extruding protons and raising the intracellular pH above 8.0. This happens to be the optimal pH for many fermentation enzymes. Without the F1F0 ATPase, proton extrusion is limited and the intracellular pH remains too low for efficient fermentation to continue. Thus, during infection when S. aureus must ferment due to the nature of inflamed tissue, the F1F0 ATPase becomes an essential enzyme complex and a valid target for the development of new antimicrobials.
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影响因子：
4.4
作者：
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