Regulation mechanism of the quorum sensing regulator AphA on the type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis
10.3760/cma.j.cn112338-20231215-00354
- VernacularTitle:群体感应调控子AphA对河弧菌Ⅵ型分泌系统VflT6SS2的调控研究
- Author:
Qian CHENG
1
;
Yu HAN
;
Yuanming HUANG
;
Saisen JI
;
Jie LI
;
Baowei DIAO
;
Weili LIANG
Author Information
1. 传染病溯源预警与智能决策全国重点实验室，中国疾病预防控制中心传染病预防控制所，北京 102206
- Keywords:
Vibrio fluvialis;
Quorum sensing regulator;
Type Ⅵ secretion system;
Transcriptional regulation
- From:
Chinese Journal of Epidemiology
2024;45(4):566-573
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis. Methods:Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), Δ aphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter- lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2, hcp ( tssD2) and vgrG ( tssI2), and the quorum sensing regulator HapR in WT and Δ aphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results:The mRNA expression levels of tssB2, hcp( tssD2), vgrG ( tssI2), and hapR as well as the protein expression and secretion levels of Hcp in Δ aphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD2a, tssI2a, and hapR were higher in Δ aphA strain than in the WT strain, while the promoter activity of tssD2b showed the opposite trend. The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD2b. The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR. Conclusions:AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR. AphA showed opposite regulation patterns for tssD2a and tssD2b, and AphA could positively regulate the expression of tssD2b by directly binding to the tssD2b promoter region (-335 bp to -229 bp).
