Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species

Increasing antibiotic resistance has led to the development of new strategies to combat bacterial infection. Anti-virulence strategies that impair virulence of bacterial pathogens are one of the novel approaches with less selective pressure for developing resistance than traditional strategies that impede viability. In this study, a small molecule CM14 [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide] that inhibits the activity of HlyU, a transcriptional regulator essential for the virulence of the fulminating human pathogen Vibrio vulnificus, has been identified. Without affecting bacterial growth or triggering the host cell death, CM14 reduces HlyU-dependent expression of virulence genes in V. vulnificus. In addition to the decreased hemolysis of human erythrocytes, CM14 impedes host cell rounding and lysis caused by V. vulnificus. Notably, CM14 significantly enhances survival of mice infected with V. vulnificus by alleviating hepatic and renal dysfunction and systemic inflammation. Biochemical, mass spectrometric, and mutational analyses revealed that CM14 inhibits HlyU from binding to target DNA by covalently modifying Cys30. Remarkably, CM14 decreases the expression of various virulence genes of other Vibrio species and thus attenuates their virulence phenotypes. Together, this molecule could be an anti-virulence agent against HlyU-harboring Vibrio species with a low selective pressure for the emergence of resistance.


Identification of CM14 as an inhibitor of the HlyU activity.
To identify a specific inhibitor of HlyU, we constructed an Escherichia coli reporter strain containing pKK1306 (carrying an arabinose-inducible hlyU of V. vulnificus) and pZW1608 (carrying a promoterless lux operon fused to a promoter P VVMO6_00539 ) 40 . Because the VVMO6_00539 gene is directly repressed by HlyU ( Fig. 1a; Supplementary Fig. S1a,b), the resulting E. coli strain remains non-luminescent in an arabinose-containing media unless a potential hit molecule inhibits either the expression or function of HlyU (Fig. 1a). By using this HlyU-repressed lux reporter system instead of the HlyUactivated system, we could eliminate the false identification of luciferase-inhibiting and/or luminescence-absorbing molecules as hits. Due to the lack of a previously discovered ligand or a putative ligand-binding site in HlyU, a random chemical library containing 8,385 small molecules was screened using the E. coli reporter strain. From the screening, three hit molecules (1025E12, 1030B04, and 1040E12) were identified as putative HlyU inhibitors (Fig. 1b). These hit molecules were reexamined using the V. vulnificus reporter strains containing the same reporter plasmid pZW1608 (Fig. 1c) or pZW1609 (Fig. 1d), respectively. In contrast to pZW1608, pZW1609 carries the promoterless lux operon fused to a promoter of the rtxA gene, P rtxA , which is directly induced by HlyU 26 . With each of the hit molecules, the wild-type V. vulnificus containing pZW1608 was more luminescent than the negative control (dimethyl sulfoxide, DMSO) (Fig. 1c), while V. vulnificus containing pZW1609 was less luminescent than the negative control (Fig. 1d). The use of these two distinct V. vulnificus reporter strains verified that the hit inhibitor molecules function directly on HlyU, not on other components such as a luciferase enzyme.
Among the hit molecules, 1025E12, N-(4-oxo-4H-thieno [3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide (C 20 H 11 NO 3 S, molecular weight of 345.37) was most effective in the HlyU inhibition, and thus selected as a HlyU inhibitor and renamed 'CM14' (Fig. 2a). The structure of CM14 was confirmed by 1 H NMR, 13 C NMR, and mass spectrometric analyses (see Supplementary Information Methods). The HlyU activities were assessed using the wild-type V. vulnificus containing pZW1609 in the presence of various concentrations of CM14, and the half maximal effective concentration (EC 50 ) of the molecule was determined as 30.97 μM (Fig. 2b). It is noteworthy that CM14 in the range of 20 to 200 μM did not alter the HlyU levels in V. vulnificus cells (Fig. 2c), suggesting that CM14 inhibits the activity rather than the cellular levels of HlyU. In addition, CM14 did not affect the growth of V. vulnificus (up to 2 mM) and was not toxic to the human epithelial INT-407 cells (up to 500 μM) (Fig. 2d,e). Therefore, these results suggested that CM14 is a small-molecule inhibitor of HlyU activity having a potential to be developed as an anti-virulence agent against V. vulnificus. Next, we examined if CM14 affects the expression of vvhA, rtxA, and plpA in V. vulnificus. Consistent with the previous result that CM14 inhibits HlyU activity, the transcript levels of vvhA, rtxA, and plpA of the wild-type V. vulnificus strain were significantly reduced in the presence of the molecule at 20 μM ( Fig. 3a; WT + DMSO vs. WT + CM14). The reduced expression levels of the genes were close to those of the hlyU mutant strain ZW141 ( Fig. 3a; WT + CM14 vs. hlyU + DMSO). We further investigated whether the reduced expression of the virulence genes is reflected in the virulence-related phenotypes. It was reported that V. vulnificus VvhA has a hemolytic activity against erythrocytes 31 . Thus, we compared the hemolytic activities in the culture supernatants of the V. vulnificus strains grown in the presence or absence of CM14. When incubated with human erythrocytes, the culture supernatant of the wild-type V. vulnificus grown in the presence of DMSO control showed robust hemolytic activity (Fig. 3b). In contrast, the culture supernatant of the wild-type V. vulnificus grown in the presence of CM14 exhibited significantly reduced (at 20 μM) or nearly no hemolytic activities (at 50 μM) similar to that of the hlyU mutant (Fig. 3b). Collectively, these results indicated that the effect of CM14 on the decreased expression of virulence genes is also represented as a reduced virulence-related phenotype of V. vulnificus in vitro.

CM14 attenuates the virulence of V. vulnificus ex vivo.
The effects of CM14 on the V. vulnificusmediated cytopathic changes of the host cells were assessed ex vivo. Since CM14 significantly decreased the rtxA transcript level in V. vulnificus (Fig. 3a), we first examined whether the molecule prevents the actin cytoskeleton dysregulation primarily caused by the MARTX toxin 35,36 . To this end, we monitored a rapid rounding phenotype of the HeLa cells infected with the V. vulnificus strains in the presence or absence of CM14. HeLa cells became round at 1 h post infection of the wild type 41 (Fig. 3c; WT + DMSO). However, the rounding of HeLa cells was significantly attenuated in the presence of CM14 at 50 μM ( Fig. 3c; WT + CM14), and thus the morphology of the cells was comparable to that of the cells with phosphate buffered saline (PBS, vehicle control) or the hlyU mutant ( Fig. 3c; PBS + DMSO or hlyU + DMSO).
Furthermore, the effects of CM14 on the cytotoxicity of V. vulnificus were evaluated. For this purpose, lactate dehydrogenase (LDH) release from the INT-407 cells infected with the bacteria was determined. As shown in Fig. 3d, CM14 reduced LDH release from the cells infected with the wild-type V. vulnificus in a dose-dependent manner. Notably, 100 μM of CM14 almost abolished the LDH-releasing activity of V. vulnificus (Fig. 3d). Taken together, these results revealed that CM14 successfully attenuates the cytopathicity and cytotoxicity of V. vulnificus ex vivo.
CM14 attenuates the pathogenesis of V. vulnificus in mice. To investigate the in vivo efficacy of CM14, mortality of mice infected with V. vulnificus was evaluated with or without co-administration of the molecule (Fig. 4a). All of the mice infected subcutaneously with the wild type strain were succumbed within 15 h Figure 1. High-throughput screening for HlyU inhibitors. (a) Schematic demonstration of high-throughput screening of small molecules. An E. coli reporter strain contains pKK1306 expressing HlyU under arabinoseinducible promoter P BAD and pZW1608 carrying the luxCDABE genes under HlyU-repressed promoter P VVMO6_00539 . (b-d) Each bar represents RLU of the E. coli reporter strain (b) and V. vulnificus reporter strains containing pZW1608 (c) or pZW1609 (d) in the presence of hit molecules as indicated. Error bars represent the standard deviation (SD) from biological triplicates. Statistical significance was determined by multiple comparisons after one-way analysis of variance (ANOVA) (***p < 0.0005). 1025E12, 1030B04, and 1040E12, hit molecules; Positive, RLUs from E. coli without arabinose (b) or V. vulnificus hlyU mutant (c,d); Negative, RLUs from E. coli with arabinose (b) or V. vulnificus wild type (c,d); RLU, relative luminescence unit.
www.nature.com/scientificreports www.nature.com/scientificreports/ post infection ( Fig. 4a; WT + DMSO). In contrast, 80% of the mice survived until the end of experiment (36 h post infection) when CM14 was co-administered at 1.125 mM concentration (1.4 mg/kg body weight) ( Fig. 4a; WT + CM14). These results revealed that co-administration of CM14 significantly prolonged the survival of mice infected with V. vulnificus (p < 0.0001, log rank test). Markedly, the survival rate of the mice infected with the wild type in the presence of CM14 was not statistically different from that of mice infected with the hlyU mutant ( Fig. 4a; hlyU + DMSO). The combined results indicated that CM14 effectively inhibits the pathogenesis of V. vulnificus during murine infection.
To examine not only survival but also pathophysiological changes, especially in the degrees of hepatic and renal dysfunction, we analyzed the biochemical parameters in the blood of the mice infected with V. vulnificus in the presence or absence of CM14. When mice were infected with the wild type (WT + DMSO), the blood plasma levels of total protein (TP) and albumin (ALB) were decreased, while the levels of aspartate aminotransferase (AST) and blood urea nitrogen (BUN) were increased, compared to the uninfected control mice injected with the vehicle (Fig. 4b; PBS + DMSO). However, the levels of biochemical parameters in mice infected with wild type in the presence of CM14 (WT + CM14) were comparable to those in the control groups such as mice injected with the vehicle or hlyU mutant ( Fig. 4b; PBS + DMSO or hlyU + DMSO). The levels of alanine aminotransferase (ALT) and creatine (CREA) did not show any significant differences among the groups in the conditions tested (Fig. 4b).
Since severe inflammation is accompanied with V. vulnificus infection 38,42 , we next assessed immune responses in the V. vulnificus-infected mice either co-administered with or without CM14. The pro-inflammatory cytokines interleukin (IL)-1β and IL-6 levels in mouse blood plasma were significantly elevated upon infection of the wild type (Fig. 4c,d; PBS + DMSO vs. WT + DMSO). However, co-administration of CM14 alleviated the secretion of these pro-inflammatory cytokines (Fig. 4c,d; WT + CM14). Consistent with this, the recruitment of F4/80 + macrophages to the infection site was also reduced by the administration of CM14 (Fig. 4e). Remarkably, the percentage of F4/80 + cells over 4' ,6-diamidino-2-phenylindole (DAPI) + cells at the site infected with the wild www.nature.com/scientificreports www.nature.com/scientificreports/ type in the presence of CM14 was not significantly different from that with the hlyU mutant (Fig. 4e). Meanwhile, CM14 did not appear to be toxic to mice, as the levels of blood parameters and macrophage infiltration of the mice injected with CM14 were comparable to those of the mice injected with the vehicle (Fig. 4b to e; CM14 vs. PBS + DMSO). Furthermore, none of the mice injected with CM14 died (Fig. 4a). Taken together, these results indicated that CM14 attenuates the virulence of V. vulnificus in vivo and is not toxic toward mice.
CM14 inhibits the binding of HlyU to its target promoter DNA. As a transcriptional regulator, HlyU functions by binding directly to its target DNA 14,26,30,43 . Thus, we examined whether CM14 inhibits the activity of HlyU by altering the DNA binding of HlyU. Electrophoretic mobility shift assays (EMSAs) revealed that HlyU bound to the target P rtxA DNA and resulted in a retarded band of the DNA-HlyU complex in a HlyU concentration-dependent manner (Fig. 5a, DMSO). When 20 μM of CM14 was added, however, the HlyU binding to the DNA decreased, as less amount of retarded bands were detected compared to the DMSO-added control ( Fig. 5a; CM14). In contrast, a random molecule that showed no HlyU-inhibiting activity in the screening did not affect HlyU binding to the DNA ( Fig. 5a; Control). To determine the effect of CM14 on the dissociation constant (K d ) for HlyU, additional EMSA experiments were performed (Fig. 5b,c). Based on the concentration of HlyU required to bind 50% of the DNA probe, the K d for HlyU without CM14 was estimated as 25.16 nM, while that with 2.5 µM of CM14 was estimated as 54.83 nM (Fig. 5d), indicating that the molecule significantly affects the equilibrium between free and DNA-bound HlyU proteins in the binding reaction. Indeed, the addition of increasing amounts of CM14 resulted in a concentration-dependent inhibition of HlyU binding to the DNA, and 50 μM of CM14 completely abolished the formation of the DNA-HlyU complex (Fig. 5e). Together, the results suggested that inhibition of HlyU binding to its target DNA is a possible mechanism of CM14. vulnificus strains along with CM14 as indicated and expressed using the LDH activity from the cells completely lysed by 5% Triton X-100 as 100%. Error bars represent the SD from three independent experiments (a,b) and from the representative of three independent experiments (d). Statistical significance was determined by the Student's t-test (a) and by one-way ANOVA (b,d) (***p < 0.0005; **p < 0.005; *p < 0.05; ns, not significant). WT, wild type; hlyU, hlyU mutant.
www.nature.com/scientificreports www.nature.com/scientificreports/ Chemical modification of HlyU by CM14. The possible mechanism of CM14 to inhibit the DNA-binding activity of HlyU was further investigated at a molecular level. To this end, tandem mass spectrometric analysis was performed for the CM14-treated HlyU sample. Figure 6a clearly showed that the Cys30 residue (C#) in the HlyU peptide, RLQILC#MLHNQELSVGELCAK, was covalently modified by the moiety with molecular mass of 130.042 Da, indicating that a certain part of CM14, probably consisting of C 9 H 7 O, is attached to the Cys30 of HlyU. Importantly, this modification seems to occur in vivo as well, because the freshly purified HlyU protein from the CM14 (50 μM)-treated E. coli cells also revealed the same result ( Supplementary Fig. S2a). To verify this modification on the Cys30, a mutant HlyU protein with Cys to Ser substitution at Cys30 (HlyU C30S ) was prepared and reacted with CM14. When the resulting mixture was analyzed by tandem mass spectrometry, a spectrum corresponding to the HlyU peptide containing a substituted serine, but not containing the covalently modified moiety, was detected ( Supplementary Fig. S2b), indicating that the thiol group of Cys30 is important for the covalent modification. Consistent with this, the mutant HlyU C30S became resistant to CM14, as supported by the observations that the DNA-binding activity of HlyU C30S was less affected by the molecule in vitro (Fig. 6b) and that the expression of rtxA was not attenuated by the molecule in vivo (Fig. 6c).
According to the previously determined crystal structure of HlyU, there is another Cys residue, Cys96, near the Cys30 (Supplementary Fig. S2c, PDB code: 3JTH). To examine the role of Cys96 on the CM14-mediated modification of Cys30, this residue was also substituted with Ser. The resulting HlyU C96S was also resistant to CM14 in vitro and in vivo, as was HlyU C30S (Fig. 6b,c; HlyU C96S ). Notably, however, Cys96 residue was detected unmodified in the above tandem mass spectrometric analysis of CM14-treated HlyU sample. Taken together, the , and CM14 (n = 6, control)] were determined by blood biochemical analysis. The data represent the mean ± SD. Statistical significance was determined by multiple comparisons after one-way ANOVA (*p < 0.05 relative to PBS + DMSO; # p < 0.05 relative to WT + DMSO). (c,d) The cytokine levels of IL-1β (c) and IL-6 (d) in the blood plasma of each group (n = 7) were quantified by enzyme-linked immunosorbent assay (ELISA). (e) Infiltration of macrophages at the injection sites was determined using skin tissue samples that were immune-stained with F4/80 antibody (for macrophages, red) and DAPI (for nucleus, blue) for counter staining. The percentage of F4/80 + cells in DAPI + cells was analyzed by using MetaMorph software. Scale bars, 10 μm (n = 4). Error bars represent the SD. Statistical significance was determined by log rank test (a) and by multiple comparisons after one-way ANOVA (c-e) (****p < 0.0001; ***p < 0.0005; **p < 0.005; *p < 0.05; ns, not significant; ND, not detected). WT, wild type; hlyU, hlyU mutant.
www.nature.com/scientificreports www.nature.com/scientificreports/ results indicated that CM14 reacts with the thiol group of Cys30 of HlyU via a putative chemical reaction involving Cys96, and consequently inhibits the DNA-binding activity of HlyU.
To gain insights into the structural influence of CM14 on HlyU, we determined the crystal structure of CM14-treated HlyU protein at 2.1 Å resolution and compared it with the previously determined apo-HlyU structure 44 (PDB code: 3JTH) (Fig. 6d,e). The overall structure of the CM14-treated HlyU is similar to that of apo-HlyU (Fig. 6d). However, there is an extra electron density map around Cys30 of the CM14-treated HlyU suggesting a potential chemical modification of Cys30 (Fig. 6e). Although the moiety attached to Cys30 is partially visible presumably due to the high flexibility, this observation is consistent with the above result that CM14 modifies the Cys30 of HlyU (Fig. 6a). Notably, further comparison revealed that CM14 induces a conformational change of HlyU, thereby substantially decreasing the distance between Cys30 and Cys96 from 8.4 Å to 4.1 Å (Fig. 6f,g). In addition, we found that the distance between two DNA-binding -helices ( 4) in HlyU dimer by 2.9 Å (Fig. 6d), which may account for the impaired DNA-binding activity of HlyU (Fig. 5).
CM14 exhibits anti-virulence effects against other Vibrio species. HlyU proteins are highly conserved in Vibrio species and show high degree of sequence similarity. Especially, the residues Cys30 and Cys96 are well conserved in HlyU homologues of common pathogenic Vibrios, including V. parahaemolyticus, V. alginolyticus, and V. cholerae 43 (Supplementary Fig. S3). Thus, we hypothesized that CM14 would be effective against other Vibrio species harboring HlyU homologue. Unfortunately, the homologues of rtxA and vvhA are absent in V. parahaemolyticus and V. alginolyticus, while the plpA homologue is present. However, the plpA homologues have not been reported to be regulated by HlyU. Accordingly, we examined the expression of exsA in V. parahaemolyticus which is directly induced by HlyU 14 . As expected, CM14 significantly reduced the exsA expression in V. parahaemolyticus (Fig. 7a). Since ExsA positively regulates multiple T3SS1-associated genes 14 , we further examined the expression of T3SS1 genes 37,45 in the presence or absence of CM14. Again, the expression of tested T3SS1 genes, such as vp1668, vopQ, vopS, and vopR was significantly attenuated by CM14 treatment (Fig. 7a). Moreover, this molecule reduced the cytotoxicity of V. parahaemolyticus against the INT-407 cells in a dose-dependent manner (Fig. 7b).
Next, the effects of CM14 on V. alginolyticus and V. cholerae were examined. Since V. alginolyticus possesses T3SS which is particularly similar to that of V. parahaemolyticus 16 , we assumed that HlyU may also regulate T3SS in V. alginolyticus. In V. cholerae, HlyU activates the expression of hlyA by directly binding to the promoter region 43 . As shown in Fig. 7c to f, CM14 markedly inhibited the expression of exsA and T3SS genes (val1668, vopQ, vopS, and vopR) in V. alginolyticus and two divergently transcribed hemolysin genes (hlyA and tlh) in V. www.nature.com/scientificreports www.nature.com/scientificreports/ cholerae, thereby attenuating cytotoxicity or hemolytic activity of the Vibrios. Notably, CM14 did not hamper the growth of V. parahaemolyticus, V. alginolyticus, and V. cholerae ( Supplementary Fig. S4), as in the case of V. vulnificus (Fig. 2d).

Discussion
Numerous bacterial genes encoding virulence factors required for overall success in the pathogenesis have been identified 46,47 . Many of these genes are coordinately regulated by a common global regulatory protein(s) to obtain their effective cooperation during infection 48,49 . Therefore, inhibiting the activity of global regulatory proteins is a promising strategy that can prevent the production of virulence factors simultaneously and thereby impede bacterial pathogenesis efficiently 1,2,6 . HlyU homologue in Vibrio species is a key regulatory protein that induces the expression of various virulence genes, suggesting that it could be an attractive target to develop the anti-virulence strategies against the pathogenic Vibrios. In the present study, we have identified and characterized a small molecule, CM14, that specifically inhibits HlyU activity, thus attenuating the pathogenesis of V. vulnificus without suppressing its growth. As expected, it also attenuated virulence phenotypes of other pathogenic Vibrios.
Among the genes regulated by HlyU in V. vulnificus, the expressions of VVMO6_00539 and VVMO6_03281 which are directly repressed by the protein (Supplementary Fig. S1a to c) were significantly induced in the www.nature.com/scientificreports www.nature.com/scientificreports/ presence of CM14 ( Supplementary Fig. S1d,e). These results indicated that CM14 inhibits HlyU activity regardless of its regulatory mode, and also suggested that the molecule functions at a stage of HlyU binding to the target promoter DNA rather than other stages such as interaction of the protein with RNA polymerase. Indeed, the EMSA results revealed that CM14 directly inhibits DNA-HlyU interaction (Fig. 5). This inhibitory mode of action is advantageous in controlling pathogenesis of the bacteria because it blocks the production of virulence factors at the earliest step 2,5 .
To the best of our knowledge, CM14 is the first compound that covalently modifies HlyU and inhibits the virulence of V. vulnificus in a mammalian infection model. Although two compounds, fursultiamine hydrochloride and 2′,4′-dihydroxychalcone, have been identified as HlyU inhibitors, their mode of action was barely demonstrated 24,25 . Moreover, both of them failed to show in vivo efficacy in an animal model, and the latter even impeded bacterial growth at the low concentration of 15 μM. From the structural point of view, compared to the Ten microliters of the concentrated supernatants were spotted onto 7% horse blood agar plate. Three different culture supernatants were spotted and monitored after incubation at 37 °C for 24 h. Error bars represent the SD from more than three biological replicates (a,c,e) and from the representative of three independent experiments (b,d). Statistical significance was determined by the Student's t-test (a,c,e) and by one-way ANOVA (b,d) (***p < 0.0005; **p < 0.005; *p < 0.05; ns, not significant). (2019) 9:4346 | https://doi.org/10.1038/s41598-019-39554-y www.nature.com/scientificreports www.nature.com/scientificreports/ two compounds, CM14 is endowed with a novel keto-alkyne moiety that is required for the covalent modification of Cys30 in HlyU (see below).
Acute failures of liver and kidney in V. vulnificus-infected patients are the key pathophysiological features associated with fatal death 50,51 . Our study revealed that the inhibition of HlyU activity by CM14 suppressed the hepatic and renal dysfunction (Fig. 4b) and subsequently increased the survival rate of mice infected with V. vulnificus (Fig. 4a). In addition, our data showed that CM14 reduces both the production of pro-inflammatory cytokines in the blood plasma (Fig. 4c,d) and the massive recruitment of macrophages to the infection site (Fig. 4e). Because the MARTX toxin and VvhA induce pro-inflammatory cytokine production in mice 38 and these cytokines trigger the recruitment of immune cells such as macrophages 52,53 , the in vivo results indicate that CM14 alleviates the clinical manifestations related to the V. vulnificus-induced septicemia by down-regulating the virulence factors. Since these virulence factors are also crucial for the invading pathogen to combat against residing immune cells and thus to proliferate/disseminate in the host 30,33,38 , V. vulnificus cells attenuated by the molecule might be readily cleared out of the mice.
Given the clear mass spectrometric evidence and biochemical data (Fig. 6a to c), we concluded that the Cys30 residue of HlyU was covalently modified with a certain part of CM14 consisting of C 9 H 7 O, and the Cys96 residue participated in this modification reaction. Based on these observations, we propose a possible chemical reaction mechanism for the covalent modification of HlyU by CM14 ( Fig. 8; see the blue dashed box on the right). In the proposed reaction, the sulfur atom of Cys96 may first attack a carbon atom of the carbon-carbon triple bond of CM14. The second attack by the sulfur atom of Cys30 would release the amine group with the bulky rings, remaining a part with the phenyl group of CM14. Subsequently, a nucleophile (e.g. His92; Supplementary  Fig. S2c) around the reaction site would cleave the sulfur-carbon bond between Cys96 and the remaining part of CM14, and the carbon is protonated, leaving the C 9 H 7 O moiety on Cys30.
Notably, CM14 seems specific for HlyU among various thiol-dependent transcriptional regulators, because only the HlyU regulon was differentially regulated by CM14 in the whole transcriptome sequencing analysis ( Supplementary Fig. S5a). Indeed, samples of WT + CM14, hlyU + DMSO, hlyU + CM14 were clustered into a certain group that is distinct from the WT + DMSO samples in a principal component analysis ( Supplementary  Fig. S5b). We thus hypothesize that the bulky rings of CM14 may be involved in the specific interaction with HlyU at the early steps of binding, but the details of interactions including binding constant remain to be studied in the future. The effects of CM14 on thiol groups of other proteins such as those in the host should also be clarified by future studies.
Nonetheless, how does this modification affect the DNA-binding activity of HlyU protein? Intriguingly, a previous simulation study on the V. cholerae HlyU protein revealed that a distance between Cys38 and Cys104, which correspond to the Cys30 and Cys96 of V. vulnificus HlyU, respectively, has a correlation with the target DNA binding. Specifically, the distance between Cys38 and Cys104 is 8.67 Å when the protein is expected to bind to a target DNA 43 . From the comparison of the crystal structure of CM14-treated HlyU with that of apo-HlyU (Fig. 6d), we found that the distance between Cys30 and Cys96 residues was significantly shortened from 8.4 Å to 4.1 Å upon CM14 treatment (Fig. 6f,g). Furthermore, the distance between two DNA-binding -helices ( 4) in HlyU dimer was also decreased by 2.9 Å (Fig. 6d). Altogether, the results indicate that CM14-mediated Cys30 modification substantially changes the HlyU conformation, and thus inhibits HlyU binding to target DNA (Figs 5  and 8).
Increasing number of studies have reported small molecules that can inhibit the activity or expression of virulence factors without affecting bacterial growth. For instance, Virstatin precludes dimerization of V. cholerae ToxT and prevents the expression of cholera toxin and toxin coregulated pilus 54,55 . Similarly, LED209 inhibits QseC activity, reducing the QseC-dependent virulence gene expression and virulence of multiple Gram-negative pathogens 56,57 . ITC-12 covalently modifies a cysteine residue of LasR, inhibits quorum sensing-mediated gene Figure 8. Proposed molecular mechanism underlying the CM14-mediated inhibition of HlyU binding to target DNA. A possible reaction mechanism for the Cys30 modification of HlyU by CM14 is shown in a blue dashed box on the right. First, the sulfur atom of Cys96 of HlyU reacts with a carbon atom (asterisk) of CM14, a Michael reaction acceptor site. Then, a sulfur atom of Cys30 of HlyU attacks a carbonyl carbon of CM14 and releases an amine group with bulky rings. Subsequently, a nucleophile (Nu, e.g. His92) around the reaction site cleaves the sulfur-carbon bond between Cys96 and the remaining part of CM14, protonating the carbon to create the carbon-carbon double bond. The remaining part of CM14 (represented by a green rounded box) is covalently linked to the sulfur atom of Cys30. Active HlyU can bind to target promoter DNA, leading to the production of virulence factors and making V. vulnificus fully virulent. In contrast, inactivation of HlyU by CM14 inhibits the DNA binding of HlyU, resulting in reduced expression of HlyU-regulated virulence genes. These events eventually attenuate the virulence of V. vulnificus.
www.nature.com/scientificreports www.nature.com/scientificreports/ expression, and attenuates virulence of Pseudomonas aeruginosa 58 . Ebselen binds to an active cysteine residue in the cysteine protease domain and thereby inhibits the autoproteolytic cleavage of TcdA and TcdB, the Clostridium difficile major toxins 59 . Interestingly, CM14, in addition to ITC-12 and Ebselen, also covalently modifies Cys30 of V. vulnificus HlyU (Fig. 6a), supporting the present idea that cysteine residues, along with their scarcity and enhanced reactivity, can be good targets for the development of selective inhibitors of proteins 60 .
CM14 successfully inhibited the expression of various virulence genes in Vibrio species, including vvhA, rtxA, and plpA of V. vulnificus (Fig. 3a), T3SS1 genes of V. parahaemolyticus (Fig. 7a), T3SS genes of V. alginolyticus (Fig. 7c), and hlyA and tlh of V. cholerae (Fig. 7e). Consistent with the previous report that the promoter region of rtxA in V. cholerae is not directly bound by the HlyU protein 61 , the expression of rtxA in V. cholerae was not affected by CM14 (Fig. 7e). This is noteworthy because it further supports that CM14 specifically affects the HlyU protein. Nevertheless, these results suggest that CM14 has a broad-spectrum anti-virulence effect against pathogenic Vibrio species harboring HlyU homologue to regulate the expression of diverse virulence genes.
In conclusion, we identified a small molecule CM14 which inhibits HlyU activity by covalently modifying Cys30 and thus attenuates the virulence of Vibrio species. CM14 exhibited its anti-virulence effect even at the post-infection treatment, although it was ex vivo case ( Supplementary Fig. S6). Further studies are needed to explore the potential of CM14 as a therapeutic agent against V. vulnificus infection, including the evaluation of CM14 analogues with improved bioavailability. Since CM14 does not hamper the bacterial growth, it would present no or low selective pressure for the development of resistance.  Table S1), a HlyU-activated reporter plasmid, was exposed to various concentrations (10 −10 to 10 −3 M) of CM14. Luminescence and growth (A 600 ) of the reporter strain were measured after 1.5 h incubation using a microplate reader (Infinite ™ M200 microplate reader, Tecan, Männedorf, Switzerland), and RLUs were calculated by dividing luminescence with A 600 62 . The HlyU activities were expressed using the RLU observed in the absence of CM14 (in the presence of 2% DMSO) as 100%. The EC 50 was calculated by plotting the relative HlyU activities versus the CM14 concentrations using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA).
Western blot and transcript analyses. The V. vulnificus strains along with CM14 or 2% DMSO were grown to A 600 of 0.5 and used to analyze either the HlyU protein or the vvhA, rtxA, and plpA transcript levels. HlyU and DnaK in the cell lysates were detected using rabbit anti-V. vulnificus HlyU antibody and mouse anti-E. coli DnaK antibody (Enzo lifescience, Farmingdale, NY) by Western blot analysis. Expression of specific genes was determined by qRT-PCR with a pair of specific primers (Supplementary Table S3). Relative expression levels of each gene were calculated by using the 16S rRNA expression level as the internal reference for normalization.

Virulence assays.
To determine hemolytic activity in vitro, the V. vulnificus strains grown to A 600 of 1.0 along with CM14 or 2% DMSO (control) were harvested and fractionated into cells and supernatants by centrifugation. The culture supernatants were purified through Puradisc TM 25 mm syringe filter (pore size 0.2 μm; GE healthcare, Menlo Park, CA) and concentrated using Amicon Ultra-15 (cut-off 10 kDa; Millipore, Temecula, CA). An aliquot of the supernatants was mixed with an equal volume of human erythrocytes (10% in PBS; Innovative Research, Novi, MI) and incubated at 37 °C for 3 h. The hemolytic activity was measured by spectrophotometry as described previously 63 .
Two different assays were performed to determine cytopathicity and cytotoxicity of the V. vulnificus strains ex vivo. To examine the cytopathic changes, HeLa cells grown in a µ-slide 4-well plates (Ibidi, Germany) were infected with the V. vulnificus strains at an MOI of 2 along with 50 μM of CM14 or 1% DMSO (control). After 1 h incubation at 37 °C, the cells were washed and fixed, and nuclei and actin of the cells were stained with Hoechst ® 33342 (final 5 μg/ml; Thermo Fisher Scientific, Waltham, MA) and with rhodamine-phalloidin (one unit per microscope slide; Thermo Fisher Scientific), respectively. Cell morphological changes were photographed using a laser scanning confocal microscope (C2plus, Nikon, Japan) and analyzed using NIS-Elements software (Nikon). To examine cytotoxicity, the monolayers of INT-407 cells (HeLa cell-derived epithelial cells) grown in a 96-well tissue culture plate (Nunc, Roskilde, Denmark) were infected with V. vulnificus strains at an MOI of 10 along with various concentrations of CM14 or 1% DMSO (control). After 2.5 h incubation at 37 °C, the LDH activities in the supernatant were measured as described previously 30 . (2019) 9:4346 | https://doi.org/10.1038/s41598-019-39554-y www.nature.com/scientificreports www.nature.com/scientificreports/ Mouse infection assays. All manipulations for mouse infection assay were performed following the National Institutes of Health Guidelines for Humane Treatment and approved by the Animal Care and Use Committee of Seoul National University (SNU-170417-26-2). Mouse mortality, blood biochemical parameters, pro-inflammatory cytokine production, and macrophage infiltration were evaluated to determine the virulence of V. vulnificus in vivo. For the mouse mortality test, the V. vulnificus strains grown to A 600 of 0.5 were harvested and suspended in PBS to 7.5 × 10 6 CFU/ml. Groups of Institute of Cancer Research (ICR) female mice (7-week-old, specific-pathogen-free; Orient Bio, Seongnam, Republic of Korea) were injected with 100 μl of the bacterial suspension along with CM14 (to achieve 1.4 mg/kg body weight) or 10% DMSO subcutaneously under the dorsal skin. Survival of the mice was monitored for 36 h as described previously 30 .
To examine the levels of blood biochemical parameters, pro-inflammatory cytokine production, and macrophage infiltration to the injection sites, the mice injected as described above were sacrificed at 7 h post infection to obtain blood and skin tissue samples, respectively. For blood biochemical analysis, the blood samples were collected using cardiac puncture in heparin-coated tube (IDEXX Laboratories, Westbrook, ME) and analyzed as described previously 30 . Breifly, the levels of TP, ALB, AST, ALT, BUN, and CREA in the blood plasma were measured by using a biochemistry autoanalyzer (Hitachi 7180 autoanalyzer, High-Technologies Corp., Tokyo, Japan). The remaining blood samples were fractionated by centrifugation for 10 min at 1,000 × g to obtain the blood plasma. Cytokine levels of IL-1β and IL-6 in the blood plasma were determined by ELISA using commercially available ELISA kits for IL-1β (R&D systems, Minneapolis, MN) and IL-6 (AbFrontier, Seoul, Republic of Korea). For immunohistochemical analysis, the mouse skin tissue samples around injection sites were embedded in optimum cutting temperature (O.C.T.) compound (Sakura Finetek, Torrance, CA) and stored at −80 °C. Frozen tissue samples were cryo-sectioned to a 20-μm thickness and then mounted on silane-coated slides (Muto Pure Chemicals, Tokyo, Japan). Tissue samples on slides were fixed with 80% acetone for 10 min, washed twice with PBS, and blocked in 5% normal goat serum (Sigma-Aldrich, St. Louis, MO) for 20 min. Slides were incubated with F4/80 antibody (1:100 dilution; Santa Cruz, Paso Robles, CA) for 2 h at room temperature. After washing three times with PBS, the slides were incubated with Alexa Fluor 488 ® -conjugated goat anti-rabbit secondary antibody (1:200 dilution; Thermo Fisher Scientific) for 1 h. Subsequently, all slides were incubated with DAPI solution (5 μg/ml; Thermo Fisher Scientific) in PBS for 5 min at room temperature. All immunofluorescence images were obtained by Eclipse Ts2 ® fluorescence microscopy (Nikon, Tokyo, Japan), and colocalization of F4/80 with DAPI was analyzed by MetaMorph software (Universal Imaging, West Chester, PA).
Protein purification, site-directed mutagenesis, and EMSA. The purification of recombinant HlyU was performed by affinity chromatography followed by size exclusion chromatography. Site-directed mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit as described previously 64 . For EMSA, the 264-bp [γ-32 P]ATP-labeled DNA fragment of P rtxA was amplified and incubated with the purified HlyU. Electrophoretic analysis of the DNA-protein complexes was performed as described previously 30 . When necessary, various concentrations of CM14 or DMSO were added to reaction mixture before incubation. As a control, a chemical randomly chosen from libraries that had no HlyU-inhibiting activity was added to the reaction mixture instead of CM14.
Mass spectrometric analysis of the HlyU modification. The gel slices corresponding to HlyU protein treated with CM14 were destained and followed by in-gel reduction and alkylation of cysteine residues. The resulting samples were washed, digested by sequencing-grade trypsin, subjected to C18-SPE clean up, and reconstituted for LC-MS/MS analysis. The acquired datasets from LC-MS/MS experiment were initially searched to find the unknown cysteine modification and subjected to MS-GF + analysis 65 to confirm the cysteine modification.
Crystallization, structure determination, and refinement. HlyU protein was incubated with CM14 for 0.5 h at 4 °C and crystallized in a precipitation solution containing 0.1 M HEPES (pH 8.0), 20% (w/v) polyethylene glycol (PEG) 4 K and 10% (v/v) 2-propanol by hanging-drop vapor diffusion method at 14 °C. The HlyU-CM14 crystals were flash-frozen using 20% (w/v) sorbitol as a cryoprotectant in a nitrogen stream at −173 °C. An X-ray diffraction dataset was collected at Pohang Accelerator Laboratory beamline 5 C. The structure was determined and refined at a 2.1 Å resolution with an R factor of 23.8% and an R free of 26.8%. Further details on the structure determination and refinement are given in Supplementary Table S4. statistical analysis. Statistical analyses were performed as indicated in figure legends using GraphPad Prism 7.0 (GraphPad Software). For mouse lethality, mouse infection experiments were repeated twice to ensure reproducibility.