VfqI-VfqR quorum sensing circuit modulates type VI secretion system VflT6SS2 in Vibrio fluvialis

V. fluvialis is an emerging foodborne pathogen and could cause cholera-like gastroenteritis syndrome and poses a potential threat to public health. VflT6SS2 is a functionally active type VI secretion system (T6SS) in V. fluvialis which confers bactericidal activity. VflT6SS2 is composed of one major cluster and three hcp-vgrG orphan clusters. Previously, we identified two quorum sensing (QS) systems CqsA/LuxS-HapR and VfqI-VfqR in V. fluvialis and demonstrated that the former regulates VflT6SS2. However, whether VfqI-VfqR QS regulates VflT6SS2 is unknown. In this study, we showed that the mRNA abundances of VflT6SS2 tssD2 (hcp), tssI2 (vgrG) and tssB2 (vipA) were all significantly decreased in VfqI or/and VfqR deletion mutant(s). Consistently, Hcp expression/secretion was reduced too in these mutants. Complementation assay with VfqR mutant further confirmed that the reduced Hcp expression/secretion and impaired antibacterial virulence are restored by introducing VfqR-expressing plasmid. Reporter fusion analyses revealed that VfqR modulates the promoter activities of VflT6SS2. Bioinformatical prediction and further reporter fusion assay in E. coli supported that VfqR acts as a transcriptional factor to bind and regulate the gene expression of the VflT6SS2 major cluster. However, VfqR seems to promote transcription of hcp (tssD2) in the orphan clusters through elevating the expression of vasH which is encoded by the VflT6SS2 major cluster. Additionally, we found that the regulation intensity of VfqR on VflT6SS2 is weaker than that of HapR. In conclusion, our current study disclosed that in V. fluvialis, VfqI-VfqR circuit upregulates the expression and function of VflT6SS2 by directly or indirectly activating its transcription. These findings will enhance our understanding of the complicated regulatory network between QS and T6SS in V. fluvialis.