Role of Rab5b in the defense of Cynoglossus semilaevis against Vibrio vulnificus infection.

Cynoglossus semilaevis, an important mariculture species in China, faces significant losses due to Vibrio infections. Current antibiotic treatments are constrained by efficacy and environmental considerations. While Rab5b is known to regulate phagosome maturation in immunity, its direct antimicrobial function and therapeutic potential have not been explored. This study characterized rab5b (651 bp ORF, 216 aa) through bioinformatics, expressed recombinant Rab5b prokaryotically, and evaluated its roles using antimicrobial assays, membrane integrity tests (FITC/PI, SEM, Crystal Violet Staining, TEM), transcriptomics (RNA-seq, WGCNA, STEM), and in vivo challenges. Rab5b exhibited widespread tissue expression, with significantly higher expression in the gut (approximately 8.5-fold) and blood (approximately 6.2-fold) compared to the liver (p < 0.05), and showed significant upregulation following V. vulnificus infection. Recombinant Rab5b demonstrated broad-spectrum activity against 8 pathogenic bacteria, such as Aeromonas spp. and V. vulnificus, inhibiting biofilms and disrupting membrane integrity through binding and permeabilization. Transcriptomic analysis revealed that Rab5b coordinates DNA repair-immune crosstalk by activating ATM/ATR pathways to upregulate FA/BER genes (fen1, pcna, rad51, etc.) and collaborates with AURKA for cell cycle progression. In vivo experiments demonstrated that Rab5b reduced mortality by 46.7% in infected fish and improved bacterial clearance. This study establishes Rab5b's dual role as an antimicrobial peptide and immunomodulator, offering a novel therapeutic approach against biofilm-associated infections and a host-directed strategy to address antibiotic resistance in aquaculture.