副溶血弧菌VPA1045和VPA1049调节Hcp2蛋白转位

VPA1045 and VPA1049 of Vibrio parahaemolyticus regulate translocation of Hcp2

摘要:

摘要:【目的】副溶血弧菌通过分泌多种毒力因子引起急性胃肠炎，其中VI型分泌系统(T6SS1和T6SS2)是近年来新发现的一种毒力因子分泌系统，但该系统如何受到调控的、如何影响细胞粘附等问题在副溶血弧菌中尚不清楚。本研究的目的是为了阐明副溶血弧菌调控因子VPA1045和VPA1049对T6SS2分泌系统在转录、翻译和翻译后水平的调控作用。【方法】利用同源重组方法构建Vpa1045和Vpa1049缺失株，荧光定量方法检测T6SS2转位因子hcp2的转录水平差异，免疫印迹分析细菌菌体中Hcp2的表达与上清中Hcp2的跨膜转位差异。【结果】副溶血弧菌VPA1045和VPA1049的基因结构中均含有Che-Y 结构域，属于二元调控因子的反应调节因子。缺失Vpa1045和Vpa1049后，菌体中的Hcp2表达量和基因组中hcp2的转录水平差异不显著，细菌上清中的Hcp2转位量和对HeLa细胞的细胞粘附率与亲本株HZ相比显著下降。【结论】二元调控因子VPA1045和VPA1049通过上调Hcp2的转位量从而翻译后上调副溶血弧菌T6SS2。

Abstract:

Abstract:［Objective］ Vibrio parahaemolyticus causes acute gastroenteritis by secreting a number of virulence factors including two sets of type VI secretion systems (T6SS1 and T6SS2)．However，it remains unknown how T6SS is regulated in the bacterium and what kinds of effects the systems have on adhesion onto and invasion into the host cells． This study was attempted to examine if VPA1045 and VPA1049 regulate T6SS2．［Methods］Vpa1045 and Vpa1049 deletion mutants were constructed by homologous recombination． Real-time PCR was performed to examine the transcription level of the translocon protein gene hcp2 in V．parahaemolyticus clinical strain HZ and its mutants． Western blot was used to analyze the levels of expression and translocation of Hcp2．［Results］Both VPA1045 and VPA1049 contain the Che-Y domain，and belong to the two-component regulators． Deletion of either Vpa1045 or Vpa1049 did not affect expression and transcription of Hcp2，but decreased translocation of Hcp2 in the supernatant of V． parahaemolycus． Bacterial adherence to the HeLa monolayers was also significantly reduced as compared with their parent strains．［Conclusion］ The twocomponent regulators VPA1045 and VPA1049 regulate T6SS2 of V． parahaemolyticus post-translationally by up-regulating Hcp2 translocation．