WalK(S221P), a naturally occurring mutation, confers vancomycin resistance in VISA strain XN108
WalK(S221P), a naturally occurring mutation, confers vancomycin resistance in VISA strain XN108
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WalK(S221P) 是一种自然发生的突变，使 VISA 菌株 XN108 具有万古霉素抗性。
DOI：
10.1093/jac/dkw518
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发表时间：
2017-04-01
影响因子：
5.2
通讯作者：
Rao, Xiancai
中科院分区：
文献类型：
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作者：
Peng, Huagang;Hu, Qiwen;Rao, Xiancai
目的：万古霉素中间金黄色葡萄球菌(VISA)菌株已在全球范围内传播。我们先前分离的ST239 VISA(XN108)对万古霉素的最低抑菌浓度为12 mg/L，本研究探讨了XN108对万古霉素耐药的机制。利用等位基因置换实验对新的突变步态(S221P)进行了鉴定和研究。测定了菌株对万古霉素的敏感性、自溶活性和菌体形态。[k-32P]ATP法检测Walk及其突变体S221P的自磷酸化活性，凝胶迁移率分析法检测Walk(S221P)激活的Walk R与其靶基因lytM启动子区域的结合活性。结果：基因组比对发现3个突变：GRAS(T136I)、RpoB(H481N)和Walk(S221P)，它们可能与XN108对万古霉素耐药有关。将WAK(S221P)引入万古霉素敏感菌株N315，使其万古霉素MIC从1.5 mg/L增加到8 mg/MIC，而将WAK(S221P)等位基因替换为XN108中天然的N315 WAK等位基因，使其万古霉素MIC从12 mg/L降低到4 mg/L。VISA菌株的细胞壁增厚，自溶能力降低，这与细胞壁代谢和毒力调节相关基因的表达变化一致。Walk(S221P)的自身磷酸化水平降低，这可能导致WalR的磷酸化水平降低。Walk(S221P)-磷酸化的WalR与lytM启动子的结合能力也降低。结论：自然发生的Walk(S221P)突变在XN108对万古霉素耐药中起关键作用。
Objectives: Vancomycin-intermediate Staphylococcus aureus (VISA) strains have spread globally. We previously isolated an ST239 VISA (XN108) with a vancomycin MIC of 12 mg/L. The mechanism for XN108 resistance to vancomycin was investigated in this study.Methods: Genome comparison was performed to characterize mutations that might contribute to the XN108 resistance phenotype. The novel mutation WalK(S221P) was identified and investigated using allelic replacement experiments. Vancomycin susceptibilities, autolytic activities and morphologies of the strains were examined. Autophosphorylation activities of WalK and the WalK(S221P) mutant were determined in vitro with [k-32P] ATP, and binding activity of WalK(S221P)-activated WalR to the promoter region of its target gene lytM was determined by electrophoretic mobility shift assay.Results: Genome comparison revealed three mutations, GraS(T136I), RpoB(H481N) and WalK(S221P), which might be responsible for vancomycin resistance in XN108. The introduction of WalK(S221P) to the vancomycin-susceptible strain N315 increased its vancomycin MIC from 1.5 to 8 mg/L, whereas the allelic replacement of WalK(S221P) with the native N315 WalK allele in XN108 decreased its vancomycin MIC from 12 to 4 mg/L. The VISA strains have thickened cell walls and decreased autolysis, consistent with observed changes in the expression of genes involved in cell wall metabolism and virulence regulation. WalK(S221P) exhibited reduced autophosphorylation, which may lead to reduced phosphorylation of WalR. WalK(S221P)-phosphorylated WalR also exhibited a reduced capacity to bind to the lytM promoter.Conclusions: The naturally occurring WalK(S221P) mutation plays a key role in vancomycin resistance in XN108.