SigB(Q225P)突变促进金黄色葡萄球菌生物被膜形成

SigB(Q225P) mutation promotes formation of Staphylococcus aureus biofilm

目的探讨金黄色葡萄球菌Sig B(Q225P)突变对其生物被膜形成能力的影响。方法比较临床分离金葡菌(XQ株)与实验室传代获得菌落颜色变白之XQW株的生物学特性,通过结晶紫染色法以及激光共聚焦检测菌株的生物被膜形成。进行XQ及XQW全基因组测序,比较相关基因,寻找突变位点。分别扩增sigB(Q225P)基因及其上游片段,经酶切后连接穿梭载体p BT2,构建同源重组质粒p BT2-sigB(Q225P),电转化至金葡菌RN4220,再将经RN4220修饰后的质粒电转入Newman。利用p BT2质粒对温度敏感的特点筛选Newman-sigB(Q225P)。利用同样的方法构建sigB敲除菌株Newman-△sigB,并构建回补菌株Newman-△sigB/sigB以及Newman-△sigB/sigB(Q225P)。比较这些菌株的生物被膜形成差异。结果 XQW株在液体培养基中呈絮状生长,结晶紫法以及激光共聚焦检测结果显示其生物被膜形成能力显著强于野生株,全基因组测序和比较发现XQW的sigB基因发生(Q225P)突变。构建的同源重组质粒p BT2-sigB(Q225P)及p BT2-△sigB转化金葡菌Newman后,经筛选,获得Newman-sigB(Q225P)及Newman-△sigB菌株;回补质粒p LI50-sigB及p LI50-sigB(Q225P)转化Newman-△sigB,筛选获得回补菌株Newman-△sigB/sigB及Newman-△sigB/sigB(Q225P)。经过结晶紫染色法及激光共聚焦测定生物被膜,发现Newman-sigB(Q225P)较野生株的生物被膜形成能力显著升高,与XQW的生物被膜形成能力强于XQ野生型的表型相一致;Newman-△sigB/sigB(Q225P)的生物被膜形成能力明显高于Newman-△sigB/sigB。结论 Sig B(Q225P)具有促进金葡菌生物被膜形成的能力。

Objective To investigate the role of SigB （Q225P） in regulating biofilm formation of Staphylococcus aureus （S. aureus）. Methods The biological characteristics of a clinically isolated S. aureus strain （XQ） and its mutant strain XQW obtained by passage were compared. The biofilm formation was assessed by crystal violet staining and laser confocal microscopy. Genome sequencing of the 2 strains was performed to compare the relevant genes and identify the mutation sites. Homologous recombination method was used to construct different mutant S. aureus strains, namely NewmansigB （Q225P） with sigB （Q225P） gene replacement, Newman-△sigB with sigB deletion, and the complementary strains Newman-△sigB/sigB and Newman-△sigB/sigB （Q225P）, and biofilm formations of these strains were compared. ResultsXQW strain grew to form flocculent structures in liquid culture medium and showed a significantly greater ability of biofilm formation than its wild-type strain XQ as determined by crystal violet staining and laser confocal microscopy. Genome comparison suggested that a SigB （Q225P） mutation occurred in XQW strain. The constructed Newman-sigB （Q225P） strain showed a much stronger ability of biofilm formation than wildtype Newman strain, a result consistent with the finding in XQW strain. The biofilm formation ability of Newman-△sigB/sigB （Q225P） strain was also stronger than that of Newman-△sigB/sigB strain. Conclusion SigB （Q225P） mutation promotes biofilm formation in S. aureus.