ArcB initiates quorum sensing to regulate T3SS in Vibrio alginolyticus by recognizing bacterial and host-derived autoinducer-2 as a kinase

BackgroundThe marine pathogen Vibrio alginolyticus employs its type III secretion system (T3SS), a syringe-like secretion apparatus, to kill eukaryotic cells. Although the cascade regulation of the T3SS encoding ExsACDE operon has been described in detail for the V. alginolyticus T3SS, little is known about the signals and signaling pathways that regulate the operon.MethodsTo investigate the regulation of T3SS by V. alginolyticus quorum sensing (QS) components, we measured lactate dehydrogenase (LDH) release following infection of Fathead minnow cells. A bioinformatics approach was employed to identify potential sensor kinases interacting with LuxU-LuxO. Bacterial two-hybrid assays were conducted to further elucidate interactions between these components. Phosphorylation and site-directed mutagenesis analyses were performed to delineate the phosphorelay system. The response of ArcB to autoinducer-2 (AI-2) or an AI-2 mimic was assessed using a Vibrio luminescence assay, and their interactions were quantitatively analyzed via microscale thermophoresis (MST) assays.ResultsWe observed that none of the three previously annotated sensing kinases in the V. alginolyticus QS system could regulate T3SS. Instead, the hybrid sensing kinase ArcB forms a signaling cascade with LuxU and LuxO to modulate T3SS expression. Furthermore, we confirmed that ArcB acts as a kinase in this pathway. Additionally, we found that ArcB can sense the LuxS-dependent autoinducer AI-2. Interestingly, host-derived AI-2 mimics produced during infection were also recognized by ArcB. Both signaling molecules activate the T3SS regulatory pathway.ConclusionsOur findings establish the ArcB-LuxU-LuxO signaling pathway as essential for the regulation of T3SS in V. alginolyticus. We further demonstrated that ArcB initiates this cascade by acting as a sensor for bacterial autoinducer-2, a signaling molecule involved in inter-species communication. Moreover, we show that host cells produce an AI-2 mimic during infection, which is also sensed by ArcB and can activate T3SS gene expression.