TolCV1 inhibition by NPPB renders Vibrio vulnificus 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 Vibrio vulnificus. In this context, we speculated that TolCV1 blockers would have a dual effect of enhancing susceptibility to antibiotics and suppressing virulence of V. vulnificus. Here, we show that the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) increases susceptibility to antibiotics and suppresses cytotoxicity of V. vulnificus through inhibition of TolCV1. NPPB significantly decreased TolCV1 in V. vulnificus 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 V. vulnificus. Moreover, NPPB inhibited the secretion of RtxA1 toxin and protected host cells from V. vulnificus-induced cytotoxicity. In addition, NPPB markedly suppressed V. vulnificus growth in the presence of bile salts and enhanced the therapeutic effect of tetracycline in V. vulnificus-infected mice. The safety and efficacy of NPPB were confirmed at the cellular and animal levels. Collectively, TolCV1 inhibition by NPPB renders V. vulnificus less virulent and more susceptible to antibiotics.