组氨酸激酶EnvZ调节副溶血弧菌群集性爬动和生物被膜形成的作用机制

The histidine kinase EnvZ regulates the swarming motility and biofilm formation of Vibrio parahaemolyticus

摘要:

目的 探究组氨酸激酶EnvZ调控副溶血弧菌群集性爬动和生物被膜形成的作用机制。方法 利用含有诱导型启动子的pBAD24与pMal质粒，构建相应基因的过表达质粒，并将其导入野生株和envZ基因缺失株中，通过运动性培养基比较各菌株的群集性爬动能力，采用结晶紫染色法检测各菌株的生物被膜形成能力，并结合RT-qPCR与荧光报告系统检测下游基因的表达水平，以探究EnvZ调节副溶血弧菌群集性爬动和生物被膜形成的分子机制。结果 ΔenvZ菌株的群集性爬动能力显著低于野生株，而导入pBAD24-envZ回补质粒的ΔenvZ菌株能够恢复其群集性爬动能力。这些菌株的群集性爬动能力与侧生鞭毛相关基因的表达水平呈正相关。在envZ基因缺失的菌株中，Scr系统启动子的转录活性显著低于野生株和ΔompR菌株。在envZ基因缺失的菌株中过表达Scr系统，可以显著恢复其群集性爬动；然而，在scrABC基因缺失的菌株中过表达EnvZ，并不能改变其群集性爬动能力。ΔenvZ菌株的生物被膜形成能力显著低于野生株，而pMal-envZ质粒能够使ΔenvZ菌株的生物被膜厚度恢复至野生株水平，但pMal-scrABC重组质粒则无此功能。在ΔenvZ菌株中，胞外多糖操纵子(epsA-J)的启动子活性与关键基因的转录水平均显著低于野生株。结论 组氨酸激酶EnvZ可通过调控Scr系统的表达来增强副溶血弧菌的群集性爬动能力，同时也可通过调控胞外多糖的表达来促进该菌的生物被膜形成。

Abstract:

Objective Vibrio parahaemolyticus is a major foodborne pathogen that causes acute gastroenteritis in humans. Here, we aim to decipher the mechanisms by which the histidine kinase EnvZ regulates the swarming motility and biofilm formation of V. parahaemolyticus.Methods The plasmids pBAD24 and pMal carrying inducible promoters were used to construct the plasmids carrying target genes for overexpression. The recombinant plasmids were introduced into the wild-type strain (WT) and envZ-deleted strain (ΔenvZ) of V. parahaemolyticus. Swarming plates were prepared to measure swarming motility, while biofilm formation was detected by crystal violet staining. The RT-qPCR and bioluminescence reporter assays were employed to explore the mechanisms by which EnvZ regulated the expression of downstream genes.Results The swarming motility of ΔenvZ was significantly lower than that of WT, while the introduction of the pBAD24-envZ plasmid into ΔenvZ restored its swarming ability. The transcription levels of the lateral flagellar genes were positively correlated with the swarming motility. The promoter activities of P_scrABC-lux in ΔenvZ and ΔenvZΔompR were lower than those in WT and ΔompR. The swarming ability of ΔenvZ was significantly increased when the Scr system was overexpressed via the pMal-scrABC plasmid, while the overexpression of EnvZ in the strain without scrABC did not change the swarming motility. In addition, ΔenvZ exhibited a significant decrease in biofilm formation compared with WT. The pMal-envZ plasmid restored the biofilm formation of ΔenvZ to the level of WT, whereas the pMal-scrABC plasmid did not have this effect. The promoter activity of the extracellular polysaccharide operon (epsA-J) and the transcription levels of the extracellular polysaccharide genes in ΔenvZ were both significantly lower than those in WT.Conclusion The histidine kinase EnvZ enhances the swarming motility of V. parahaemolyticus by regulating the expression of the Scr system and promotes the biofilm formation by regulating the expression of extracellular polysaccharides.