TolCV1 inhibition by NPPB renders <i>Vibrio vulnificus</i> less virulent and more susceptible to antibiotics

Bacterial efflux pumps play important roles in the antibiotic resistance and excretion of virulence factors. We previously characterized that TolCV1, a component of efflux pumps, plays critical roles in resistance to antibiotics and bile and also RtxA1 toxin secretion of <i>Vibrio vulnificus</i>. In this context, we speculated that TolCV1 blockers would have a dual effect of enhancing susceptibility to antibiotics and suppressing virulence of <i>V. vulnificus</i>. Here, we show that the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) increases susceptibility to antibiotics and suppresses cytotoxicity of <i>V. vulnificus</i> through inhibition of TolCV1. NPPB significantly decreased TolCV1 in <i>V. vulnificus</i> cells by liberating the protein from the cell body. Checkerboard assay showed that NPPB enhanced the antimicrobial activities of antibiotics such as kanamycin, tetracycline, erythromycin, and ampicillin against <i>V. vulnificus</i>. Moreover, NPPB inhibited the secretion of RtxA1 toxin and protected host cells from <i>V. vulnificus</i>-induced cytotoxicity. In addition, NPPB markedly suppressed <i>V. vulnificus</i> growth in the presence of bile salts and enhanced the therapeutic effect of tetracycline in <i>V. vulnificus</i>-infected mice. The safety and efficacy of NPPB were confirmed at the cellular and animal levels. Collectively, TolCV1 inhibition by NPPB renders <i>V. vulnificus</i> less virulent and more susceptible to antibiotics.