Integration Host Factor Modulates the Expression and Function of T6SS2 in Vibrio fluvialis

Abstract

Vibrio fluvialis, an emerging foodborne pathogen of increasing public health concern, contains two distinct gene clusters encoding type VI secretion system (T6SS), the most newly discovered secretion pathway in Gram-negative bacteria. Previously we have shown that one of the two T6SS clusters, namely VflT6SS2, is active and associates with anti-bacterial activity. However, how its activity is regulated is not completely understood. Here, we report that the global regulator integration host factor (IHF) positively modulates the expression and thus the function of VflT6SS2 through co-regulating its major cluster and tssD2-tssI2 (also known as hcp-vgrG) orphan clusters. Specifically, reporter gene activity assay showed that IHF transactivates the major and orphan clusters of VflT6SS2, while deletion of either ihfA or ihfB, the genes encoding the IHF subunits, decreased their promoter activities and mRNA levels of tssB2, vasH, and tssM2 for the selected major cluster genes and tssD2 and tssI2 for the selected orphan cluster genes. Subsequently, the direct bindings of IHF to the promoter regions of the major and orphan clusters were confirmed by electrophoretic mobility shift assay (EMSA). Site-directed mutagenesis combined with reporter gene activity assay or EMSA pinpointed the exact binding sites of IHF in the major and orphan cluster promoters, with two sites in the major cluster promoter, consisting with its two observed shifted bands in EMSA. Functional studies showed that the expression and secretion of hemolysin-coregulated protein (Hcp) and the VflT6SS2-mediated antibacterial virulence were severely abrogated in the deletion mutants of ΔihfA and ΔihfB, but restored when their trans-complemented plasmids were introduced, suggesting that IHF mostly contributes to environmental survival of V. fluvialis by directly binding and modulating the transactivity and function of VflT6SS2.

CqsA/LuxS-HapR Quorum sensing circuit modulates type VI secretion system VflT6SS2 in Vibrio fluvialis

“…Overnight cultures of V. fluvialis 85003 WT and its derivative mutants were inoculated at 1:100 into 20 mL LB and incubated to respective OD 600 of 0.2, 0.5, 1.0 and 1.5 with shaking at 30°C. Protein samples from cell pellets and cell-free supernatants were prepared as previously described [ 23 , 24 ]. The protein concentration was determined using the BCA TM protein assay kit (Thermo Fisher Scientific, United States).…”

“…Integration host factor (IHF) and σ 54 -dependent VasH encoded in VflT6SS2 major cluster transcriptionally activate the expression of VflT6SS2. IHF targets both the major and orphan clusters whereas VasH acts on the orphan cluster [ 23 , 24 ]. In addition to IHF and VasH, many other global transcriptional regulators such as sigma factor 54 (σ 54 , RpoN), cAMP receptor protein (CRP), and TsrA have also been reported to manipulate T6SS expression and function [ 25–27 ].…”

The nucleoid-associated protein IHF acts as a ‘transcriptional domainin’ protein coordinating the bacterial virulence traits with global transcription

“…In various Salmonella species IHF is shown to profoundly influence the expression of virulence genes, facilitate invasion associated with transition of bacterial cells to stationary phase ( 29 ) and promote biofilm formation ( 30 ). IHF activates the expression of virulence traits in human pathogens Vibrio cholerae ( 31 ) and Vibrio fluvialis ( 32 ) as well as in plant pathogens Erwinia amylovora ( 33 ) and Dickeya zeae ( 34 ). In the soil bacterium Pseudomonas putida , IHF activates the genes involved in adaptation to the post-exponential phase with limited effect on genes involved in central metabolism ( 35 ).…”

Confirmed and Potential Roles of Bacterial T6SSs in the Intestinal Ecosystem

“…In Citrobacter freundii , T6SS plays a wide-ranging role in the regulation of the flagellar system and in motility; it is also involved in the adherence and cytotoxicity to host cells (Liu et al, 2015). T6SS2 in Vibrio fluvialis is associated with anti-bacterial activity and contributes to bacteria survival in highly competitive environments (Pan et al, 2018). The T6SS effector VgrG1 from A. hydrophila induces host cell toxicity by ADP ribosylation of actin (Suarez et al, 2010).…”