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 P _rtxA1::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.

Keywords: Vibrio vulnificus; drug identification and repositioning; fursultiamine hydrochloride; hemolysin; hlyU; rtxA1.

Figure 1

Overview of the study. The upstream and downstream regulatory network of HlyU (Panel I), the design of the reporter platform (Panel II), the workflow for the identification, validation, and assessment of antivirulence characteristics of small molecule inhibitor, FTH targeting HlyU in V. vulnificus (Panels III–V), and adaptive evolution demonstrating the avoidance of antimicrobial and antivirulence resistance development during antivirulence approach (Panel VI).

Figure 2

Steps to identify the antivirulence agent fursultiamine hydrochloride (FTH). (A) Primary screening of small molecule libraries (1,840 chemicals at 20 μM) using V. vulnificus reporter. A total of 150 chemicals were shown as representative for inhibition of relative light units (RLU) per unit optical density (OD₆₀₀) after 6 h of incubation. The RLU/OD₆₀₀ ratio and the percent inhibition of normalized luminescence signal allowed subdivision of chemicals into four groups. Complete (100%) RLU inhibition was observed with chemicals that allowed no or minimal growth of bacteria (“bacterial growth cessation”), and these were excluded from further analysis. The group showing more than 30% RLU inhibition is labeled “potential antivirulence hits.” (B) Dose-dependent response of selected chemicals from the group labeled “potential antivirulence hits” using V. vulnificus reporter. ZH, zuclopenthixol hydrochloride; BH, benzamil hydrochloride; OME, omeprazole; FTH, fursultiamine hydrochloride; VAT, vatalanib; GBR, GBR 12909 dihydrochloride; and MND, mebhydroline 1,5-naphtalenedisulfonate. (C) Quantitative RT-PCR of rtxA1 gene of WT V. vulnificus following the treatment with ZH, BH, OME, and FTH (20 μM), which were selected because these chemicals did not inhibit V. vulnificus growth. (D) HeLa cell proliferation as a measure of the in vitro cytotoxicity of FTH using EZ-Cytox cell viability kit. A₄₅₀ values were measured after 48 h, and IC₅₀ values were calculated with GraphPad prism 6.01. (E) Structure of fursultiamine hydrochloride.

Figure 3

Concentration-dependent expression of genes in the HlyU regulatory network in response to FTH treatment of wild type V. vulnificus. The relative expression of genes in response to varying concentrations of FTH is depicted as follows: (A) rtxA1, encoding the RtxA1 toxin; (B) vvhA, encoding hemolysin; (C) hlyU, encoding HlyU transcriptional regulator, and (D) hns, encoding a global repressor. Respective DMSO controls showed no difference in gene expression of WT sample. (E) A transcriptional fusion of P_hlyU-hlyU::luxCDABE gene showing the level of luminescence in wild type and ΔhlyU V. vulnificus; (F) Revalidation of hlyU transcription with varying concentration of FTH depicts the unchanged level of hlyU transcript in wild type V. vulnificus harboring the transcriptional fusion P_hlyU-hlyU::luxCDABE. The percent RLU per unit OD₆₀₀ was calculated based on the RLU values shown in (E).

Figure 4

The effect of FTH on the hemolytic activity of V. vulnificus. Untreated and FTH-treated (10, 20, 40, and 60 μM) 3 h culture supernatants of WT V. vulnificus were used for hemolysis visualization and hemolytic unit calculation (see method for details). (A) Visualization of hemolysis by WT V. vulnificus in the absence and presence of FTH, and (B) the absorption spectra showing the characteristic hemolysis peaks in various treatments. (C) Quantitative estimation of hemolysis inhibition activity of FTH expressed as hemolytic unit.

Figure 5

Rescue of cytoskeletal destabilization by FTH treatment using a human cell line. (A) HeLa cells were treated with WT V. vulnificus with and without FTH (100 and 200 μM), along with ΔhlyU, ΔrtxA1 and PBS controls. V. vulnificus control strains (ΔhlyU and ΔrtxA1) lacking toxins show a discrete cytoskeleton network (red) spread throughout the cytoplasm, while the WT V. vulnificus possessing the RtxA1 toxin totally destabilized the cytoskeletal network and cellular morphology. The presence of FTH (200 μM) with WT V. vulnificus rescued the cytoskeletal destabilization and protected cells from rounding in a concentration-dependent manner. Scale bar is equal to 20 micron. (B) Frequency of cytoskeletal destabilization and consequent rounding of HeLa cells as a measure of RtxA1 activity. A total of 1,000 cells were counted in random microscopic fields for each sample, and percentages were calculated based on round vs. intact shaped cells. FTH treatment inhibited RtxA1 at the transcriptional level. Thus, the cytoskeleton was found to be intact in the FTH-treated wild type sample (normal cell shape at 200 μM) but not in the untreated WT sample (rounded cells).

Figure 6

Laboratory-induced adaptive evolution of antimicrobial vs. antivirulence resistance in V. vulnificus. (A) Schematic diagram showing laboratory-induced adaptive evolution by continuous transferring of V. vulnificus exposed to norfloxacin (0.155 μg/ml) or FTH (40 and 60 μM). The numbers below the tubes represent the number of transfers. Yellow color in tubes denotes the growth. (B) Evaluation of norfloxacin MIC against non-transferred V. vulnificus; untreated V. vulnificus transferred 12 times (V. vulnificus × 12 transfers) and adaptively evolved, norfloxacin-resistant V. vulnificus (Nor^R V. vulnificus). For MIC calculations, an initial bacterial inoculum of 1 × 10⁵ CFU/ml was used for Nor^R V. vulnificus, V. vulnificus, and V. vulnificus × 12 transfers. Nor^R V. vulnificus adapted to grow at 0.077 μg/ml, in contrast to the WT V. vulnificus control strain without any treatment. This data showed that V. vulnificus can evolve and acquire resistance in order to survive sublethal antibacterial concentrations in a very short amount of time. (C) Verification of identity of the adaptively evolved, putative Nor^R V. vulnificus strain. (I) Various numbers (1-6) on TCBS agar plate represent the original stock wild type V. vulnificus (1), the untreated V. vulnificus × 12 transfers (2), FTH-treated V. vulnificus (3), the adaptively evolved Nor^R V. vulnificus (4), a sugar-fermenting V. alginolyticus strain acts as a positive control of histochemical plate for sugar fermenting Vibrio (5), and E. coli DH5α as negative control (6). All the Vibrio strains (but not E. coli DH5α) grew on the TCBS agar plates. (II) Reconfirmation of norfloxacin resistance of adaptively evolved Nor^R strain on TCBS agar plate supplemented with 0.155 μg/ml norfloxacin. Growth of the adaptively evolved Nor^R strain (4), on TCBS-norfloxacin plate while the controls (1–3) did not show any sign of growth confirms its identity and its evolved norfloxacin resistance in Vibrio species. (D) The antivirulence resistance of FTH-exposed strains were analyzed by gene expression of rtxA1 and vvhA to verify resistance against FTH. The qRT-PCR results showed the same level of rtxA1 and vvhA transcriptional inhibition in the FTH-exposed strain and in the original stock culture of V. vulnificus.