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

Vibrio fluvialis is an emerging enteric pathogen of increasing public health threat. Two quorum sensing (QS) systems, VfqI-VfqR and CqsA/LuxS-HapR, and two type VI secretion systems (T6SSs), VflT6SS1 and VflT6SS2, have been identified in V. fluvialis. VflT6SS2 is regulated by environmental signals and intracellular regulators. In Vibrio species, QS systems were frequently reported to regulate various physiological functions and systems. Therefore, we wonder if QS systems can function as functional regulators of VflT6SS2. Here, we investigated the effects of QS circuit on VflT6SS2. Our results showed that the QS response regulator LuxO represses while the major regulator HapR activates VflT6SS2. The effect of LuxO is more pronounced at low cell density and is HapR-dependent. Deletion of hapR abolished Hcp expression and alleviated antibacterial virulence. However, these effects were rescued by introducing HapR-expressing plasmid. Reporter fusion analyses showed that HapR is required for the promoter activities of VflT6SS2. Sequence inspection of the major cluster promoter revealed two potential Motif 1 HapR binding sites, and their direct bindings were confirmed by both electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay. Meanwhile, two single Motif 2 HapR binding sites were identified in each of tssD2_a (hcpA) and tssD2_b (hcpB) promoter regions of the orphan cluster which are less conserved and displayed lower affinities to HapR. Together, our current study demonstrated that VflT6SS2 expression was under the control of QS circuit in V. fluvialis, and this finding will enhance our understanding of possible crosstalk between T6SS and QS in different microbes.