Discovery of a Small-Molecule Inhibitor Targeting the Biofilm Regulator BrpT in Vibrio vulnificus

Abstract

Vibrio vulnificus, an opportunistic human pathogen, employs biofilm formation as a key survival and virulence mechanism. BrpT, a transcriptional regulator, is essential for V. vulnificus biofilm development by regulating the expression of biofilm-related genes. In this study, we aimed to identify a small molecule inhibitor of BrpT to combat V. vulnificus biofilm formation. High-throughput screening of 7,251 compounds using an Escherichia coli reporter strain carrying the arabinose-inducible brpT gene and a BrpT-activated promoter fused to the luxCDABE operon identified a hit compound, BTI (BrpT Inhibitor). BTI potently inhibited BrpT activity in V. vulnificus (EC₅₀ of 6.48 μM) without affecting bacterial growth or host cell viability. Treatment with BTI significantly reduced the expression of the BrpT regulon and impaired biofilm formation and colony rugosity in V. vulnificus, thus increasing its susceptibility to antibiotics. In vitro biochemical analyses revealed that BTI directly binds to BrpT and inhibits its transcriptional regulatory activity. The identification of BTI as a specific inhibitor of BrpT that effectively diminishes V. vulnificus biofilm formation provides a promising foundation for the development of novel anti-biofilm strategies, with the potential to address the growing challenge of antibiotic resistance and improve the treatment of biofilm-associated infections.

Keywords: BrpT; Vibrio vulnificus; biofilm; high-throughput screening; small-molecule inhibitor; transcription regulation.

Fig. 1. High-throughput screening for BrpT inhibitors.

(A) Schematic representation of BrpT’s role in activating biofilm-related genes in V. vulnificus. (B) E. coli-based screening system. BrpT expression (pJN1602) was induced by 0.0002% L-arabinose. Active BrpT drives lux operon expression (pWW2202), producing luminescence. Inhibitors block this process are screened. (C) BrpT activity in E. coli measured by relative luminescence (RLU). Hit compounds significantly reduced BrpTinduced luminescence are shown. (D) V. vulnificus reporter strain for hit validation. Native BrpT activates P_cabA promoter (pWW2201), leading to lux operon expression. (E) BrpT activity in V. vulnificus. Hit compounds significantly reduced luminescence in a parental wild-type V. vulnificus. JN111, a parental wild-type strain, and ΔbrpT, a brpT deletion mutant, were used. Data represent means ± SEMs from three biological replicates. Statistical significance was determined by Student's t-test (****, p < 0.0001; ***, p < 0.0005). Vv, V. vulnificus.

Fig. 2. BTI inhibits BrpT activity without affecting V. vulnificus growth or cellular toxicity.

(A) Chemical structure of BTI (1-{[2-(5-methylfuran-2-yl)-1,3-thiazol-4-yl]methyl}-1,2-dihydropyridin-2-one). (B and C) Dosedependent inhibition of BrpT activity by BTI in V. vulnificus reporter strains. (B) Schematic of V. vulnificus reporter system in which active BrpT induces luminescence via P_cabA-controlled lux operon. RLU measurements at varying BTI concentrations are shown. (C) Quantification of BrpT activity. RLU of wild-type strain without BTI set as 100% and ΔbrpT as 0%. JN111, a parental wild-type strain, and ΔbrpT, a brpT deletion mutant, were used. (D) Growth of V. vulnificus wild-type strain in LBS in the presence of various BTI concentrations up to 100 μM. (E) Assessment of cytotoxicity of BTI towards human epithelial HeLa cells measured by released LDH activities after 3 h of incubation at 37°C. LDH activity from cells lysed with 3% Triton X-100 set as 100%. Cytotoxicity of the wild-type V. vulnificus at an MOI of 10 was shown for comparison. Data represent means ± SEMs from three independent experiments. Statistical significance was determined by Student’s t-test (ns, not significant). RLU, relative luminescence unit; MOI, multiplicity of infection; Vv, V. vulnificus.

Fig. 3. BTI reduces biofilm-related phenotypes of V. vulnificus.

(A) Relative mRNA expression levels of brpA, brpN, cabA, cabH, and brpT relative to those in the parental wild-type (set as 1). (B) Quantification of biofilm formation in V. vulnificus strains grown with varying BTI concentrations. Biofilms were stained with crystal violet and measured at A₅₇₀ after elution. (C) Representative image of colony rugosity of V. vulnificus strains in the presence or absence of 100 μM BTI after 24 h incubation. Scale bar represents 1 mm. (D) Survival of V. vulnificus against antibiotics. Biofilms were grown with 100 μM BTI (or 1% DMSO for control) for 24 h and then treated with various concentrations of ampicillin for 8 h. Viable cells after ampicillin treatment were counted. JN111, a parental wild-type strain, and ΔbrpT, a brpT deletion mutant, were used. Data represent means ± SEMs from three independent experiments. Statistical significance was determined by Student’s t-test (****, p < 0.0001; ***, p < 0.0005; **, p < 0.005; *, p < 0.05; ns, not significant). wt, wild-type; Vv, V. vulnificus.

Fig. 4. BTI directly binds to BrpT and attenuates transcription in vitro.

(A) Microscale thermophoresis analysis demonstrating the direct interaction between BTI and BrpT. The normalized thermophoresis values of BrpT (F_norm) at varying BTI concentrations are shown. (B) In vitro transcription assay determining the effect of BTI on BrpT-mediated transcriptional activation of the brpN promoter. A template DNA containing P_brpN was transcribed in vitro with 8 μM BrpT in the presence or absence of 1 mM BTI as indicated. Relative levels of the transcripts were determined using the heights of the transcript peaks measured in arbitrary fluorescent units, shown with that of BrpT without BTI set as 1. Error bars represent the SEM from three independent experiments. Statistical significance was determined by Student’s t-test (***, p < 0.0005; *, p < 0.05).