[1]饶一凡,毛旭虎,彭华刚,等.WalK(S221P)突变影响万古霉素中等耐药金黄色葡萄球菌毒力的作用[J].第三军医大学学报,2020,42(03):219-228.
 RAO Yifan,MAO Xuhu,PENG Huagang,et al.Role of WalK(S221P) mutation on virulence of vancomycin-intermediate Staphylococcus aureus and underlying mechanism[J].J Third Mil Med Univ,2020,42(03):219-228.
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WalK(S221P)突变影响万古霉素中等耐药金黄色葡萄球菌毒力的作用(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第03期
页码:
219-228
栏目:
基础医学
出版日期:
2020-02-15

文章信息/Info

Title:
Role of WalK(S221P) mutation on virulence of vancomycin-intermediate Staphylococcus aureus and underlying mechanism
作者:
饶一凡毛旭虎彭华刚尚伟龙饶贤才
西南大学生命科学学院现代生物医药研究所,三峡库区生态环境与生物资源省部共建国家重点实验室培育基地;陆军军医大学(第三军医大学):第一附属医院临床微生物学与免疫学教研室,基础医学院微生物学教研室
Author(s):
RAO Yifan MAO Xuhu PENG Huagang SHANG Weilong RAO Xiancai

State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Institute of Modern Biopharmaceuticals, School of Life Sciences, Southwest University, Chongqing, 400715; 2Department of Clinical Microbiology and Immunology, First Affiliated Hospital, 3Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing, 400038, China

关键词:
万古霉素中等耐药金黄色葡萄球菌二元调控系统WalKR WalK(S221P)突变毒力
Keywords:
 
分类号:
R372; R378.11; R394.3
文献标志码:
A
摘要:

目的 探讨WalK(S221P)突变对万古霉素中等耐药金黄色葡萄球菌(vancomycin-intermediate Staphylococcus aureus, VISA)毒力的影响及其机制。方法采用转录组测序比较VISA菌株XN108及其WalK(S221P)回复菌株K65的毒力基因表达差异,利用RT-qPCR检测菌株毒力基因的表达变化,溶血实验比较菌株的溶血能力。采用同源重组技术在金葡菌中引入WalK(S221P)突变,E-test检测菌株的耐药性。凝胶阻滞实验(EMSA)检测WalKR对靶基因调控区的结合能力。结果转录组测序结果显示,K65中多种重要毒力因子基因的表达上调(P<0.01),调控系统Agr的活性增强,溶血能力恢复。将WalK(S221P)突变引入USA300菌株后,其耐药性升高,毒力因子表达水平及溶血活性降低;EMSA证实WalKR对金葡菌Agr有直接调控作用;敲除agrA基因后,引入WalK(S221P)突变仍可使USA300agrA对万古霉素的耐药性升高,但毒力基因的表达水平及溶血活性改变不显著。结论WalK(S221P)突变影响VISA毒力是通过Agr实现的,该突变导致WalKR的活性降低,激活Agr的能力下降,最终影响VISA菌株的毒力。

Abstract:

Objective To explore the role and mechanism of WalK(S221P) mutation affecting the virulence of vancomycin-intermediate Staphylococcus aureus (VISA).  MethodsRNA sequencing (RNA-seq) was used to compare the differentially expressed genes (DEGs) between VISA strain XN108 and its isogenic WalK(S221P) mutation cured strain K65. The expression of selected DEGs in strains of interest were detected by RT-qPCR. Hemolytic experiment was employed to detect the hemolytic activities of S. aureus strains. WalK(S221P) mutation carried strains (K-USA300 and K-USA300ΔagrA) were constructed by homologous recombination technologies. E-test was applied to detect the vancomycin sensibilities of S. aureus strains. The electrophoretic mobility shift assay (EMSA) was performed to detect the binding ability of WalKR on the promoter regions of target gene. ResultsRNA-seq revealed that the expression levels of several important virulence factors were upregulated in WalK(S221P) mutation cured K65 when compared with those in the wild-type XN108 (P<0.01). The activity of Agr and the hemolytic activity in K65 were also enhanced. Introduction of WalK(S221P) mutation into MRSA strain USA300 resulted in the reduced vancomycin susceptibility of K-USA300 than that of the wild-type USA300, however, K-USA300 presented decrease in the expression levels of these important virulence factors, and the hemolytic activity of K-USA300 was also reduced. EMSA showed that WalK-phosphorylated WalR could directly bind to the agr promoter region. Introduction of WalK(S221P) into USA300ΔagrA reduced vancomycin susceptibility of K-USA300ΔagrA, while the expression levels of selected virulence genes and hemolytic activities were comparable between K-USA300ΔagrA and USA300ΔagrA. ConclusionWalK(S221P) mutation affects the virulence of VISA by directly contacting the agr promoter region. WalK(S221P) mutation induces decreased WalKR activity and lowered activation to Agr, and thus affects the virulence of VISA.

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更新日期/Last Update: 2020-02-06