Vpa1463, a DUF1566-domain protein, is indispensable for bile resistance and biofilm formation in Vibrio parahaemolyticus and is required for its adaptation to food-related environments.

Vibrio parahaemolyticus is a notorious seafood-borne pathogen associated acute gastroenteritis, posing increasing challenge to global food security and public health. Bile represents one of the most important factors that affect its intestinal colonization. And bacteria usually employ shared strategies to resist bile salts alongside other environmental stresses. To identify the essential genes for stresses adaptation in V. parahaemolyticus, we screened a genome-wide transposon-insertion library for bile hypersensitive mutants and identified vpa1463, encoding a conserved but uncharacterized DUF1566-domain protein, as essential for bile tolerance. The deletion of vpa1463 drastically reduced the bile salts minimum inhibitory concentration MIC from 2.5% (wild-type) to 0.3% (Δvpa1463), and inhibited swarming and swimming motilities. Mechanistic analyses revealed that the deletion of vpa1463 increases outer-membrane (OM) permeability, compromises its integrity, and decreases efflux activity. Data-independent acquisition (DIA)-based proteomics identified 1156 differentially expressed proteins in the vpa1463 mutant compared to the wild type, including key proteins associated with efflux pumps and porins, as well as those involved in flagellar biosynthesis. Moreover, the mutation of vpa1463 led to attenuated biofilm formation and decrease its colonization on food processing contact materials or food surfaces by approximately one order of magnitude. These membrane defects led to a cascade of phenotypic failures in Δvpa1463, impaired motility, heightened susceptibility to antibiotics, food-grade disinfectants and organic acids. Collectively, Our data demonstrate that Vpa1463 is a vital contributor to membrane homeostasis and environmental fitness, making it a promising target for strategies to mitigate V. parahaemolyticus contamination in the food industry and prevent human infection.