Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China; Department of Emergency Medicine, Xinqiao Hospital, Army Medical University (Third Military Medical University), Chongqing 400037, China.
Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China.
J Adv Res. 2022 Sep;40:167-178. doi: 10.1016/j.jare.2021.11.015. Epub 2021 Nov 26.
Vancomycin-intermediate Staphylococcus aureus (VISA) is typically associated with a decline in virulence. We previously reported a WalK(S221P) mutation that plays an important role in mediating vancomycin resistance in VISA XN108. Whether this mutation is implicated in bacterial virulence remains unknown.
This study aimed to investigate the effect of WalK(S221P) mutation on the virulence of VISA and the underlying mechanism of this effect.
The influence of WalK(S221P) mutation on VISA virulence and its underlying mechanism were explored using animal models, RNA-seq analysis, RT-qPCR, hemolytic assay, slide coagulase test, Western blot, β-galactosidase assay, and electrophoresis mobility shift assay (EMSA).
Compared with XN108, WalK(S221P)-reverted strain XN108-R exacerbated cutaneous infections with increased lesion size and extensive inflammatory infiltration in mouse models. The bacterial loads of S. aureus XN108-R in murine kidney increased compared with those of XN108. RNA-seq analysis showed upregulation of a set of virulence genes in XN108-R, which exhibited greater hemolytic and stronger coagulase activities compared with XN108. Introduction of WalK(S221P) to methicillin-resistant S. aureus USA300 and methicillin-susceptible strain Newman increased the vancomycin resistance of the mutants, which exhibited reduced hemolytic activities and decreased expression levels of many virulence factors compared with their progenitors. WalK(S221P) mutation weakened agr promoter-controlled β-galactosidase activity. EMSA results showed that WalK-phosphorylated WalR could directly bind to the agr promoter region, whereas WalK(S221P)-activated WalR reduced binding to the target promoter. Inactivation of agr in S. aureus did not affect their vancomycin susceptibility but mitigated the virulence alterations caused by WalK(S221P) mutation.
The results of our study indicate that WalK(S221P) mutation can enhance vancomycin resistance in S. aureus of diverse genetic backgrounds. WalK(S221P)- bearing S. aureus strains exhibit reduced virulence. WalK(S221P) mutation may directly impair the activation of the agr system by WalR, thereby decreasing the expression of virulence factors in VISA.
耐万古霉素中间金黄色葡萄球菌（VISA）通常与毒力下降有关。我们之前报道过一个 WalK（S221P）突变，该突变在介导 VISA XN108 中的万古霉素耐药性方面发挥着重要作用。然而，这种突变是否与细菌毒力有关尚不清楚。
本研究旨在探讨 WalK（S221P）突变对 VISA 毒力的影响及其作用机制。
通过动物模型、RNA-seq 分析、RT-qPCR、溶血试验、玻片凝固酶试验、Western blot、β-半乳糖苷酶试验和电泳迁移率变动分析（EMSA）等方法，探讨 WalK（S221P）突变对 VISA 毒力的影响及其潜在机制。
与 XN108 相比，WalK（S221P）回复突变株 XN108-R 在小鼠模型中加重了皮肤感染，导致病变面积增大和炎症浸润广泛。与 XN108 相比，金黄色葡萄球菌 XN108-R 在小鼠肾脏中的细菌负荷增加。RNA-seq 分析显示，XN108-R 中一组毒力基因上调，其溶血活性和凝固酶活性均强于 XN108。将 WalK（S221P）引入耐甲氧西林金黄色葡萄球菌 USA300 和耐甲氧西林敏感株 Newman 中，增加了突变体的万古霉素耐药性，与亲本相比，其溶血活性降低，许多毒力因子的表达水平降低。WalK（S221P）突变削弱了 agr 启动子控制的β-半乳糖苷酶活性。EMSA 结果表明，WalK 磷酸化的 WalR 可以直接结合 agr 启动子区域，而 WalK（S221P）激活的 WalR 减少了与靶启动子的结合。金黄色葡萄球菌 agr 的失活不影响其对万古霉素的敏感性，但减轻了 WalK（S221P）突变引起的毒力改变。
本研究结果表明，WalK（S221P）突变可增强不同遗传背景下金黄色葡萄球菌的万古霉素耐药性。携带 WalK（S221P）的金黄色葡萄球菌菌株表现出较低的毒力。WalK（S221P）突变可能直接损害 WalR 对 agr 系统的激活，从而降低 VISA 中毒力因子的表达。
