分子伴侣prsA2基因缺失对单增李斯特菌致病性的影响

Effect of Chaperone prsA 2 Gene Deletion on the Pathogenicity of Listeria monocytogenes

【目的】构建单增李斯特菌分子伴侣prsA2基因缺失株与回补株,探究其对单增李斯特菌内化素B(InlB)锚定能力以及致病性的影响。【方法】利用穿梭质粒pKSV7和回补质粒pIMK2分别构建prsA2基因缺失株与回补株;通过生长曲线分析亲本株10403S、缺失株ΔprsA2和回补株CΔprsA2生长能力;利用Western blotting检测prsA2基因缺失对单增李斯特菌InlB锚定的影响;通过肠上皮细胞(Caco-2)黏附侵袭试验与免疫荧光试验、小鼠毒力试验分析prsA2基因缺失对单增李斯特菌黏附侵袭能力、在宿主细胞间迁移能力及小鼠致病性的影响。【结果】PCR扩增结果显示,试验成功构建了缺失株ΔprsA2和回补株CΔprsA2。生长曲线结果显示,prsA2基因缺失不影响单增李斯特菌在BHI培养基中的生长能力(P>0.05)。Western blotting结果显示,缺失株ΔprsA2细菌表面的InlB锚定量极显著低于10403S和CΔprsA2(P<0.01),细菌培养上清中InlB锚定量极显著高于10403S和CΔprsA2(P<0.01)。细胞试验结果显示,缺失株ΔprsA2对Caco-2细胞的黏附率、侵袭率均极显著低于10403S和CΔprsA2(P<0.01);prsA2基因缺失减弱了单增李斯特菌募集肌动蛋白形成肌动蛋白尾巴在细胞间迁移的能力。小鼠毒力试验结果显示,缺失株ΔprsA2在小鼠肝脏与脾脏中的细菌载量均显著低于10403S和CΔprsA2(P<0.05)。【结论】分子伴侣prsA2基因缺失不影响单增李斯特菌在BHI培养基中的生长能力,但缺失株ΔprsA2表面的InlB锚定量显著下降,缺失prsA2基因减弱单增李斯特菌对Caco-2细胞的黏附侵袭能力、在细胞间迁移能力以及对小鼠的致病性。

【Objective】This study was aimed to construct the chaperone prsA 2 gene deletion strain and its complemental strain,and explore the roles of prsA 2 gene in the anchoring of virulent factor internalin B(InlB)and pathogenicity of Listeria monocytogenes.【Method】The deletion strainΔprsA 2 and complemental strain CΔprsA 2 were constructed with shuttle vector pKSV7 and pIMK2,respectively.The growth ability of wild type 10403S,ΔprsA 2 and CΔprsA 2 was analyzed by growth curve.The effect of prsA 2 gene deletion on the anchoring of InlB was evaluated by Western blotting.The effect of prsA 2 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 strainΔprsA 2 and its complemental strain CΔprsA 2 were constructed successfully.Growth curve results showed that the growth ability of wild type 10403S,ΔprsA 2 and CΔprsA 2 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 bacteriaΔprsA 2 was extremely significantly lower than 10403S and CΔprsA 2(P<0.01),and the InlB anchor content in the bacterial culture supernig was extremely significantly higher than 10403S and CΔprsA 2(P<0.01).The cell test results showed that the adhesion rate and invasion rate of deletion strainΔprsA 2 on Caco-2 cells were extremely significantly lower than 10403S and CΔprsA 2(P<0.01).Deletion of prsA 2 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 strainΔprsA 2 in liver and spleen of mice was significantly lower than that of 10403S and CΔprsA 2(P<0.05).【Conclusion】The deletion of chaperone prsA 2 gene didn’t affect the growth ability of Listeria monocytogenes in BHI broth,but the InlB anchor quantity on the surface ofΔprsA 2 strain was significantly decreased,and deletion of prsA 2 gene weakened the adhesion and invasion ability on Caco-2 cells,the ability of intercellular migration and the pathogenicity in mice of Listeria monocytogenes.