生物被膜形成关键基因csgD对鼠伤寒沙门菌黏附侵袭上皮细胞能力的影响

Effects of the key gene csgD related biofilm formation on the abilities of Salmonella Typhimurium of adhesion to and invasion into epithelial cells

为了探究生物被膜形成关键基因csgD与鼠伤寒沙门菌黏附侵袭上皮细胞能力的关系,本研究利用Red同源重组系统构建鼠伤寒沙门菌S025株csgD基因缺失株(S025ΔcsgD),利用原核表达载体构建回复株(S025ΔcsgDR);运用结晶紫染色定量、共聚焦显微镜和扫描电镜观察、平板表型鉴定等方法检测鼠伤寒沙门菌生物被膜形成能力,并比较野生株、缺失株和回复株黏附侵袭Caco-2细胞和HeLa细胞的差异。结晶紫染色定量、共聚焦显微镜和扫描电镜观察结果显示,与野生株S025和回复株S025ΔcsgDR相比,S025ΔcsgD生物被膜形成能力显著降低;生物被膜成分分析发现,野生株呈红色干燥粗糙型菌落,而S025ΔcsgD呈白色光滑型菌落,表明其卷曲菌毛和纤维素成分减少,回复株的菌落与野生株相似。黏附与侵袭试验结果显示,与野生株与回复株相比,S025ΔcsgD的黏附率和侵袭率显著降低。结果表明,生物被膜形成关键基因csgD在鼠伤寒沙门菌黏附和侵袭宿主上皮细胞方面发挥关键作用,这为进一步阐明生物被膜状态下的鼠伤寒沙门菌的致病机理奠定了理论基础。

In this study,the relationship between the key gene csgD related to biofilm formation and the abilities of Salmonella Typhimurium of adhesion to and invasion into epithelial cells was investigated.The Red recombination system was used to construct a mutant(S025ΔcsgD)with deletion of csgD gene.Meanwhile,the prokaryotic expression vector was used to construct a csgD-complemented strain(S025ΔcsgDR).The biofilm formation of S.Typhimurium was detected using the crystal violet staining method,confocal laser scanning microscope,scanning electron microscope,and congo red and calcofluor plates.In addition,the abilities of adhesion and invasion of these three strains,including the wild-type S025,csgD mutant,and csgD-complemented strain,were compared in Caco-2 and HeLa cells.The results of crystal violet staining,confocal laser scanning microscope,and scanning electron microscope showed that the biofilm formation of the csgD mutant was significantly decreased,compared with that of the wild strain and the revertant.The biofilm composition analysis showed that the colonies of the wild strain were dry,rough and red,while the colonies of the csgD mutant were smooth and white,indicating that the components of curli fimbriae and cellulose were much reduced.The colonies of the csg D-complemented strains were similar to that of the wild strain.In addition,the adhesion and invasion rates of the csg D mutant were significantly lower than those of the wild strain and the revertant.These results indicated that the csg D gene,a key biofilm formation gene,played a vital role in the adhesion and invasion of S.Typhimurium into the host epithelial cells,which laid a theoretical foundation for further elucidating the pathogenic mechanism of S.Typhimurium with biofilm formation.