The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of staphylococcus aureus

The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of <i>Staphylococcus aureus</i>, causing a wide range of infectious diseases. Here, we characterized <i>cwrA</i> as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed <i>cwrA</i> deletion mutants in the genetic background of methicillin-resistant <i>S. aureus</i> (MRSA) and methicillin-sensitive <i>S. aureus</i> (MSSA) strains. Phenotypic analyses indicated that deletion of <i>cwrA</i> led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the <i>ica</i> operon activity in <i>S. aureus</i>. Furthermore, <i>cwrA</i> deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that <i>cwrA</i> deletion mutant more virulence than the parental strain because of its enhanced haemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and haemolytic activity in <i>S. aureus</i>.

形成致密生物膜并分泌多样化毒力因子的能力，是金黄色葡萄球菌（*Staphylococcus aureus*）的关键致病决定因素，该菌可引发多种感染性疾病。本研究通过电泳迁移率变动分析（electrophoretic mobility shift assays），将*cwrA*鉴定为受VraR调控的基因，其编码一种细胞壁抑制应答蛋白（CwrA）。我们在耐甲氧西林金黄色葡萄球菌（methicillin-resistant *S. aureus*, MRSA）与甲氧西林敏感金黄色葡萄球菌（methicillin-sensitive *S. aureus*, MSSA）的遗传背景中构建了*cwrA*基因缺失突变株。表型分析结果显示，*cwrA*基因缺失会导致生物膜形成受损，该现象与细胞间多糖粘附素（polysaccharide intercellular adhesin, PIA）密切相关。此外，实时定量聚合酶链式反应（real-time quantitative PCR, RT-qPCR）与β-半乳糖苷酶活性测定结果表明，在金黄色葡萄球菌中，CwrA可通过调控*ica*操纵子的活性促进生物膜形成。进一步研究发现，与野生型（wild-type, WT）菌株相比，*cwrA*缺失突变株的自溶活性降低，因此其生物膜内的胞外DNA（extracellular DNA, eDNA）释放量更少。我们还观察到，*cwrA*缺失突变株的溶血活性增强，其毒力强于亲本菌株。从机制上来说，该表型改变与SaeRS双组分系统的激活相关，该系统可正向调控编码膜损伤毒素的基因的转录水平。综上，本研究结果表明，CwrA在金黄色葡萄球菌的生物膜形成及溶血活性调控中发挥重要作用。