分子伴侣prsA2基因缺失对单增李斯特菌致病性的影响OACSTPCD

[Objective]This study was aimed to construct the chaperone prsA2 gene deletion strain and its complemental strain,and explore the roles of prsA2 gene in the anchoring of virulent factor internalin B(InlB)and pathogenicity of Listeria monocytogenes.[Method]The deletion strain ΔprsA2 and complemental strain CΔprsA2 were constructed with shuttle vector pKSV7 and pIMK2,respectively.The growth ability of wild type 10403S,ΔprsA2 and CΔprsA2 was analyzed by growth curve.The effect of prsA2 gene deletion on the anchoring of InlB was evaluated by Western blotting.The effect of prsA2 gene deletion on the adhesion and invasion ability,migration ability between host cells and pathogenicity of Listeria monocytogenes were analyzed by intestinal epithelial cell(Caco-2)adhesion and invasion test,immunofluorescence test and mouse virulence test.[Result]PCR amplification results showed deletion strain ΔprsA2 and its complemental strain CΔprsA2 were constructed successfully.Growth curve results showed that the growth ability of wild type 10403S,ΔprsA2 and CΔprsA2 in BHI broth exhibit no significant difference(P＞0.05).The results of Western blotting showed that the InlB anchor content on the surface of the bacteria ΔprsA2 was extremely significantly lower than 10403S and CΔprsA2(P＜0.01),and the InlB anchor content in the bacterial culture supernig was extremely significantly higher than 10403S and CΔprsA2(P＜0.01).The cell test results showed that the adhesion rate and invasion rate of deletion strain ΔprsA2 on Caco-2 cells were extremely significantly lower than 10403S and CΔprsA2(P＜0.01).Deletion of prsA2 gene reduced the ability to recruit actin to form an actin tail for intercellular migration of Listeria monocytogenes.The mouse virulence test showed that the bacterial load of deletion strain ΔprsA2 in liver and spleen of mice was significantly lower than that of 10403S and CΔprsA2(P＜0.05).[Conclusion]The deletion of chaperone prsA2 gene didn't affect the growth ability of Listeria monocytogenes in BHI broth,but the InlB anchor quantity on the surface of ΔprsA2 strain was significantly decreased,and deletion of prsA2 gene weakened the adhesion and invasion ability on Caco-2 cells,the ability of intercellular migration and the pathogenicity in mice of Listeria monocytogenes.