FliC蛋白R91S突变对肠炎沙门菌鞭毛形态和小鼠体内定植的影响

The Effect of R91S Mutation in FliC on the Flagellar Shape and Salmonella Enteritidis Colonization in BALB/c Mice

沙门菌血清D群3个血清型FliC蛋白氨基酸序列比对分析表明肠炎沙门菌与鸡伤寒沙门菌完全相同,二者与鸡白痢沙门菌存在第91位氨基酸位点差异。本研究旨在探究肠炎沙门菌FliC蛋白第91位精氨酸突变对鞭毛形态、细菌运动性和小鼠体内定植能力的影响。运用λ-Red同源重组技术删除肠炎沙门菌CICC10467 fliC基因,构建系列反式回补突变株,通过体外生长特性试验和Western blot试验分析各菌株生长和FliC蛋白表达情况,运动性试验分析各菌株在半固体琼脂中的泳动能力,电子显微镜观察各菌株鞭毛形态,细胞感染试验分析各菌株的细胞黏附和入侵能力,动物感染试验分析各菌株的组织侵染能力。结果表明,fliC基因缺失株及点突变回补株与野生株的体外生长能力无显著差异(P≥0.05)。fliC基因缺失后肠炎沙门菌不表达鞭毛蛋白,各点突变回补株与野生株鞭毛蛋白表达量无明显差异。FliC蛋白R91S突变导致肠炎沙门菌鞭毛形态由超螺旋形态转变为钝直、柔韧度减弱,运动性显著降低(P<0.0001),对RAW264.7和HCT116细胞的黏附入侵能力显著下降(P<0.001),对BALB/c小鼠的器官侵染能力显著减弱(P<0.001)。综上表明,FliC蛋白第91位精氨酸对维持细菌运动性至关重要,第91位精氨酸突变能够显著改变肠炎沙门菌鞭毛形态,减弱肠炎沙门菌在小鼠体内定植能力。

FliC amino acid sequences alignment of three serovars in Salmonella enterica serogroup D revealed that S.Enteritidis sharing the same pattern with S.Gallinarum displayed different amino acid residue at the 91th position.The objective of this work is to investigate the effect of arginine mutation at the 91th position of FliC protein on flagellar shape and S.Enteritidis colonization in liver and spleen in BALB/c mice.fliC gene was deleted from the genome of CICC10467 usingλ-Red homologous recombination system and a series of trans-complemented strains were constructed.The growth characteristics and FliC expression were evaluated through growth curve analysis and Western blot assay.Flagellar motility and shape were analyzed with soft agar plates and transmission electron microscope,respectively.The ability of cellular adhesion and invasion of wild type and its derivates in vitro and in vivo were tested.The result suggested that there was no difference on bacterial growth and FliC expression between wild type and complements besides loss of expression in fliC mutant.R91S substitution changed the flagellar shape of S.Enteritidis into blunt and straight and weaken flexibility and nearly abrogated bacterial motility(P<0.0001),the ability of adhesion and invasion to HCT116 and RAW264.7 and the load of mutant in liver and spleen of BALB/c mice was decreased comparison with wild type(P<0.001).Taken together,arginine at the 91th position is essential for bacterial motility,R91S substitution remarkably changes the flagellar shape of S.Enteritidis and impairs bacterial colonization in BALB/c mice.