The type VI secretion system protein VgrG of Vibrio splendidus promotes pathogen internalization by interacting with IQGAP1

To establish a successful infection, bacteria have evolved highly efficient strategies that enable them to enter host cells and disrupt host transport pathways, thereby creating a favorable intracellular niche for survival. With the occurrence of the phagocytic process, Vibrio cholerae releases the effector VgrG-1 of its type VI secretion system (T6SS), which disrupts the host actin cytoskeleton. It is precisely because of this destruction that a bystander protection effect for extracellular bacterial communities can be established. However, VgrG proteins exist in multiple isoforms. Those lacking additional effector domains are generally considered to function primarily as structural components for T6SS assembly, and their roles in pathogenesis are poorly defined. In this study, we characterized a gene from Vibrio splendidus with a cDNA sequence of 1983 base pairs, designated as the valine-glycine repeat protein G ( VsVgrG ) without actin-crosslinking domain. The expression analysis revealed that both mRNA and protein levels of VsVgrG were significantly enhanced following stimulation with Apostichopus japonicus coelomic fluid. Flow cytometry and bacterial plate count assays demonstrated that VsVgrG knockout significantly reduced V. splendidus internalization. Furthermore, immunofluorescence analysis revealed that, compared to cells treated with the Δ VsVgrG mutant, the actin cytoskeleton of sea cucumber coelomocytes exhibited significant dissociation following infection by the wild-type strain. Mechanistically, we identified A. japonicus IQ Motif Containing GTPase Activating Protein 1 (AjIQGAP1) as a direct host binding partner of VsVgrG. Combined overexpression and inhibitor treatment, we demonstrated that VsVgrG promotes pathogen internalization by targeting AjIQGAP1 via actin-dependent endocytosis pathway. Our findings reveal a potential mechanism for V. splendidus internalization and advance our understanding of universal biological mechanisms in pathogen-host interactions.