Potential use of Sodium Butyrate (SB) as an anti-virulence agent againstVibrio choleraetargeting ToxT virulence protein

Abstract

ABSTRACTCholera, a diarrhoeal disease caused by gram-negative bacteriumVibrio choleraeremains a global health threat in developing countries owing to its high transmissibility and increase in antibiotic resistance. The current issue is to overcome the problem of resistance by antimicrobial therapy. There is a need for alternative strategies with an emphasis on anti-virulent approaches to alter the outcome of bacterial infections.Vibrio choleraecauses cholera by secreting virulence factors in the intestinal epithelial cells. Virulence factors help in cholera toxin production and colonisation during infection. Here, we show that sodium butyrate (SB), a small molecule, had no effect on bacterial viability but was effective in suppressing the virulence attributes ofV. cholerae. The production of cholera toxin (CT) was downregulated in a standardV. choleraeEl Tor strain and two clinical isolates when grown in presence of sodium butyrate. Analysis of mRNA and protein levels further demonstrated that sodium butyrate reduced the expression of the ToxT-dependent virulence genes liketcpAandctxAB. DNA-protein interaction assays conducted at cellular (ChIP) and inin vitroconditions (EMSA) indicated that sodium butyrate weakens the binding between ToxT and its downstream promoter DNA, likely by blocking DNA binding. Furthermore, the efficacy of sodium butyrate was confirmed by showing its anti-virulence activity and tissue damage recovery in animal models. Collectively, these findings suggest that sodium butyrate (SB) has the potential to be developed as an anti-virulence agent againstV. choleraein place of conventional antibiotics or as an adjunctive therapy to combat cholera.IMPORTANCEThe world has been facing an upsurge in cholera cases since 2021 with a similar trend continuing into 2022 with over 29 countries reporting cholera outbreaks (World Health Organization 16 December 2022 Disease Outbreak News; Cholera – Global situation). Treatment of cholera involves oral rehydration therapy coupled with antibiotics to reduce the duration of the illness. However, over the last few years, there has been indiscriminate use of antibiotics that contributed largely to the reservoir of antibiotic-resistant strains. In this study, we have addressed the problem of antibiotic resistance by targeting virulence factors. The screening of several compounds led to the identification of a small molecule, sodium butyrate that inhibits the virulence cascade inV. cholerae. We demonstrated that (i) sodium butyrate intervened with ToxT protein-DNA binding and subsequently affected the expression of ToxT-regulated virulence genes (ctxABandtcpA) (ii) Sodium Butyrate is a potential therapeutic candidate for development of novel antimicrobial agents.