半乳糖基转移酶gttA基因缺失对4b型单增李斯特菌致病性的影响

Effect of Galactosyltransferase gttA Gene Deletion on Pathogenicity of Listeria monocytogenes Serotype 4b

【目的】探究半乳糖基转移酶gttA基因缺失对单增李斯特菌表面蛋白锚定及致病性的影响,以期为单增李斯特菌致病机制研究提供参考。【方法】通过同源重组方法构建gttA基因缺失株,分析亲本株ScottA、缺失株ΔgttA、回补株CΔgttA的生长与运动能力;通过Western blotting探究gttA基因缺失对单增李斯特菌2种表面毒力蛋白InlB、ActA锚定的影响。采用结肠腺癌细胞系(Caco-2)进行黏附与侵袭试验及小鼠感染试验评估gttA基因缺失后对单增李斯特菌黏附与侵袭能力和致病性的影响。【结果】单增李斯特菌正常生长与运动能力不受gttA基因缺失的影响。Western blotting结果显示,gttA基因缺失导致毒力蛋白InlB和ActA在细菌细胞壁表面的锚定量极显著降低(P<0.01)。细胞黏附与侵袭试验结果显示,ΔgttA对Caco-2细胞的黏附和侵袭能力极显著低于ScottA和CΔgttA(P<0.01)。小鼠感染试验结果显示,gttA基因缺失导致单增李斯特菌在小鼠肝脏及脾脏中的定植能力极显著或显著减弱(P<0.01;P<0.05)。将gttA基因回补后,细菌对Caco-2细胞的黏附与侵袭水平,以及在肝脏、脾脏中的定植能力均能恢复至亲本株ScottA水平。【结论】gttA基因缺失并不影响单增李斯特菌生长与运动能力,但会影响主要毒力蛋白在单增李斯特菌表面的锚定,减弱该菌对Caco-2细胞系的黏附与侵袭能力及对小鼠的致病性。

【Objective】This study was aimed to investigate the effect of deletion of galactosyltransferase gttA gene on the anchoring of surface proteins and pathogenicity of Listeria monocytogenes,so as to provide a reference to the pathogenic mechanism research of Listeria monocytogenes.【Method】Homologous recombination methods were used to construct the gttA gene deletion strain,the growth and motility of the parental strain ScottA,the deletion strainΔgttA and the complement strain CΔgttA were analyzed.The effects of gttA gene deletion on the anchoring of two surface virulence proteins InlB and ActA in Listeria monocytogenes were investigated by Western blotting.The adhesion and invasion test of colorectal adenocarcinoma cell line(Caco-2)and mouse infection test were used to evaluate the effect of gttA gene deletion on the adhesion and invasion ability and pathogenicity of Listeria monocytogenes.【Result】The growth and motility of Listeria monocytogenes were not affected by the deletion of gttA gene.Western blotting results showed that after the deletion of gttA gene,the anchor quantities of virulence proteins InlB and ActA on the bacterial cell wall surface were extremely significantly decreased(P<0.01).The results of cell adhesion and invasion assay showed thatΔgttA had an extremely significantly lower adhesion and invasion ability to Caco-2 cells than ScottA and CΔgttA(P<0.01).The results of mouse infection test showed that the colonization ability of Listeria monocytogenes in liver and spleen of mice was extremely significantly or significantly weakened due to the deletion of gttA gene(P<0.01 or P<0.05).After the gttA gene was replenished,the level of adhesion and invasion in Caco-2 cells,and the colonization ability of the bacteria in liver and spleen were restored to the level of the parental strain ScottA.【Conclusion】The deletion of gttA gene did not affect the growth and motility of Listeria monocytogenes,but affected the anchoring of the major virulence proteins on the surface of Listeria monocytogenes,which in turn weakened the ability of the bacterium to invade Caco-2 cell line and the pathogenicity of the bacterium in mice.