Effects of single and double deletion of Vibrio anguillarum histone-like protein HU subunits on biofilm formation and pathogenicity in flounder (Paralichthys olivaceus).

Abstract

Vibrio anguillarum is a major bacterial pathogen in marine aquaculture, which causes significant economic losses. The histone-like protein HU, a conserved nucleoid-associated protein, plays crucial roles in chromosome organization, global gene regulation, and stress adaptation. In many bacteria, HU exists as both a homodimer and a heterodimer composed of two subunits, HUα and HUβ, which are encoded by hupA and hupB, respectively. However, the functional roles and divergence of HU subunits in V. anguillarum remain poorly understood. In this study, we successfully constructed the single-gene deletion mutants (ΔhupA and ΔhupB) and the hupA/hupB double-gene deletion mutant (ΔHU). Compared with wild-type (WT) strain, ΔhupA and ΔHU exhibited impaired growth, whereas ΔhupB showed enhanced growth. Deletion of hupB completely abolished biofilm formation and autoaggregation, whereas the ΔHU mutant partially restored biofilm formation. Interestingly, ΔHU and ΔhupA displayed enhanced autoaggregation. All mutants displayed significantly reduced motility. Notably, extracellular DNase production was markedly decreased in ΔHU. In addition, ΔHU showed the highest susceptibility to DNA-mediated killing, while ΔhupB displayed enhanced resistance to this stress. Moreover, the virulence of ΔhupA and ΔHU was significantly attenuated, with a 10.00-fold and a 12.60-fold increase in LD50, respectively, compared to the WT strain, while ΔhupB showed no significant change in virulence. Collectively, our findings reveal the functional divergence between HUα and HUβ in regulating growth, motility, biofilm formation, quorum-sensing, DNase production, and DNA resistance in V. anguillarum, and provide a theoretical basis and novel insights for the development of live attenuated vaccines against vibriosis in aquaculture.