Guanylate-binding proteins protect the sea cucumbers Apostichopus japonicus against infection of Vibrio splendidus

Guanylate-binding proteins (GBPs) are interferon-induced innate immune effector molecules belonging to the GTPase superfamily and are crucial components of cell-autonomous immunity. To date, most research on GBPs has focused on mammals, with limited studies in marine invertebrates. In this study, we identified three GBP genes from the sea cucumber Apostichopus japonicus, designated as AjGBP1, AjGBP2, and AjGBP3. Phylogenetic analysis revealed that AjGBPs cluster with GBPs from other invertebrates. Multiple sequence alignment and protein structural analysis indicated that the AjGBPs possess a conserved GTPase-binding domain at the N-terminus and an effector domain composed of alpha-helices at the C-terminus. These AjGBPs are ubiquitously expressed in the body wall, muscle, intestine, respiratory tree, and coelomocytes of A. japonicus, although their expression levels vary among different tissues. Under Vibrio splendidus challenge, the expression of all three AjGBPs was significantly upregulated in coelomocytes. Recombinant AjGBP proteins with GTPase activity were produced through prokaryotic expression, and bacterial binding assays demonstrated that they could bind V. splendidus in vitro in a GTP hydrolysis-dependent manner. Interference with AjGBPs expression suppressed the antibacterial capacity of coelomocytes. These findings suggest that AjGBPs target V. splendidus and play an important role in the antibacterial immune response of A. japonicus.