Identification and Validation of an Antivirulence Agent Targeting HlyU-Regulated Virulence in Vibrio vulnificus

Abstract

Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance. Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality. Here, we developed an inhibitor-screening reporter platform to target HlyU, a master transcriptional regulator of virulence factors in V. vulnificus by assessing rtxA1 transcription under its control. The inhibitor-screening platform includes wild type and ΔhlyU mutant strains of V. vulnificus harboring the reporter construct PrtxA1::luxCDABE for desired luminescence signal detection and control background luminescence, respectively. Using the inhibitor-screening platform, we identified a small molecule, fursultiamine hydrochloride (FTH), that inhibits the transcription of the highly invasive repeat-in-toxin (rtxA1) and hemolysin (vvhA) along with other HlyU regulated virulence genes. FTH has no cytotoxic effects on either host cells or pathogen at the tested concentrations. FTH rescues host cells from the necrotic cell-death induced by RtxA1 and decreases the hemolytic activity under in vitro conditions. The most important point is that FTH treatment does not induce the antivirulence resistance. Current study validated the antivirulence strategy targeting the HlyU virulence transcription factor and toxin-network of V. vulnificus and demonstrated that FTH, exhibits a potential to inhibit the pathogenesis of deadly, opportunistic human pathogen, V. vulnificus without inducing AMR.

Identification of 2′,4′-Dihydroxychalcone as an Antivirulence Agent Targeting HlyU, a Master Virulence Regulator in Vibrio vulnificus

“…The EMSA results showed the precise targeting of the known master virulence factor, HlyU ( Figure 4 A–C). We established a G. mellonella (wax-worm) infection model for studying V. vulnificus pathogenesis in an earlier study [ 12 ]. In this model, 2′,4′-DHC protected ~50% wax-worm larvae from V. vulnificus infection ( Figure 4 D,D’).…”

“…All recombinant DNA techniques were performed using various molecular biology kits for plasmid and genomic DNA isolation (Intron Biotech, Seongnam, Korea), and gel purification of DNA (Cosmogenetech LaboPass, Gel extraction kit, Seoul, Korea) as per the manufacturers’ protocols. An expand long PCR amplification kit (Qiagen, Hilden, Germany) was used to amplify the long DNA cassette (P rtxA 1 :: luxCDABE_cm r , 7.997 kb) [ 12 ] harboring the P rtxA1 promoter tagged with luxCDABE genes and chloramphenicol acetyltransferase gene using specific primers ( Table S1 ). The ~8 kb long region was flanked with a λ-red recombinase site for the genomic integration as described earlier [ 28 ].…”

“…The ∆ hlyU V. vulnificus strain was included as the control. RNA was isolated using a QIAGEN RNeasy Mini kit and cDNA was synthesized using the Ecodry Premix (random hexamer) kit (Takara Bio, Mountain View, CA, USA) as described earlier [ 12 ]. The qRT-PCR reactions were performed using Taq Universal SYBR Green supermix (Bio-Rad, Hercules, CA, USA) to determine the relative expression of rtxA1 , vvhA , hlyU , and hns genes wherein the gyrB gene served as an endogenous control.…”

“…The toxicity of lead antivirulence small molecule 2′,4′-DHC was evaluated against HeLa and HEK293 human cell lines using an EZ-Cytox cell viability assay kit (DoGen, Seoul, Korea) as described earlier [ 12 ]. HeLa and HEK293 cells were obtained from the American Type Culture Collection (ATCC) and were cultured in Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum at 37 °C with 5% CO 2 .…”

“…As an alternative approach to bactericidal antibiotics that cause AMR development, the antivirulence strategy is rapidly progressing to treat bacterial infections by disarming their virulence factors and subjecting them to be cleared by the host’s immune system [ 11 , 12 ]. The advantage of this approach is that such a strategy can be used not only for treating the AMR pathogens by targeting new therapeutic targets but also helps in reducing AMR development via antibiotic-mediated clonal selection.…”