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

Abstract

The emergence of antimicrobial resistance and rapid acclimation allows Vibrio vulnificus to rapidly propagate in the host. This problematic pathological scenario can be circumvented by employing an antivirulence strategy, treating Vibrio infections without hindering the bacterial growth. We developed a genome-integrated orthogonal inhibitor screening platform in E. coli to identify antivirulence agents targeting a master virulence regulator of V. vulnificus. We identified 2′,4′-dihydroxychalcone (DHC) from the natural compound library and verified that it decreases the expression of the major toxin network which is equivalent to the ∆hlyU deletion mutant. 2′,4′-DHC also reduced the hemolytic activity of V. vulnificus which was tested as an example of virulence phenotype. The electrophoretic mobility shift assay confirmed that 2′,4′-DHC specifically targeted HlyU and inhibited its binding to PrtxA1 promoter. Under in vivo conditions, a single dose of 2′,4′-DHC protected ~50% wax-worm larvae from V. vulnificus infection at a non-toxic concentration to both V. vulnificus and wax-worm larvae. In the current study, we demonstrated that an orthogonal reporter system is suitable for the identification of antivirulence compounds with accuracy, and identified 2′,4′-DHC as a potent antivirulence agent that specifically targets the HlyU virulence transcriptional regulator and significantly reduces the virulence and infection potential of V. vulnificus.

Spatiotemporal Regulation of Vibrio Exotoxins by HlyU and Other Transcriptional Regulators

“…No such DNA binding defect has been shown in the fursultiamine hydrochloride-treated HlyU, but the expression of the rtxA gene was also significantly affected in that case [147]. Consistent with HlyU's global regulatory function in Vibrio species, these HlyU inhibitors, especially CM14, exhibited strong anti-virulence effects against various pathogenic Vibrio species by inhibiting expression of multiple exotoxin genes [148,149].…”

“…Recently, three small molecules have been identified as HlyU inhibitors [147][148][149]. Although they have no structural similarity, both N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3phenylprop-2-ynamide (CM14) and 2 ,4 -dihydroxychalcone interfere in the binding between HlyU and the V. vulnificus rtxA gene promoter thus decrease MARTX toxin expression [148,149]. No such DNA binding defect has been shown in the fursultiamine hydrochloride-treated HlyU, but the expression of the rtxA gene was also significantly affected in that case [147].…”

Active Antioxidant Phenolics from Brazilian Red Propolis: An Optimization Study for Their Recovery and Identification by LC-ESI-QTOF-MS/MS

“…Isoliquiritigenin (compound 12 with [M ₋ H]¯ at m / z 255.0676) was previously described in the literature and tentatively identified herein based on product ions at m / z 119.0496 and 135.0082 [ 41 ]. The compound 2′,4′-Dihydroxychalcone (compound 13 ), detected with [M–H] − at m / z 239.0723, was identified based on fragment ions at m / z 239.0709, 197.0609 and 135.0085 [ 42 ] This compound was previously reported as an efficient antiviral targeting HlyU in Vibrio vulnificus [ 43 ]. Compound 14 was assigned as 7-hydroxyflavanone ( m / z 239.0719), yielding the m / z 197.0610 fragment [ 37 ].…”

Complex regulatory networks of virulence factors in Vibrio vulnificus

“…Moreover, the selective attenuation of the certain pathogen would prevent unintended side effects of antibiotics such as the elimination of commensal bacteria. Recently, substantial efforts have been conducted to identify small molecules that attenuate the virulence of V. vulnificus by inhibiting the activity of its transcription factors [86][87][88][89].…”

“…Since HlyU is widely conserved in other pathogenic Vibrio species, HlyU is another attractive target to inhibit the virulence of Vibrio species. Three HlyU inhibitors are currently reported: CM14 [N-(4-oxo-4H-thieno [3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide], DHC (2′,4′-dihydroxychalcone), and FTH (fursultiamine hydrochloride) (Figure 3B) [86,87,89].…”