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Resveratrol inhibits the virulence of Vibrio harveyi by reducing the activity of Vibrio harveyi hemolysin
Aquaculture 522 (2020) 735086
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Resveratrol inhibits the virulence of Vibrio harveyi by reducing the activity of Vibrio harveyi hemolysin
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Xiaoran Zhaoa,b,1, Yi Guoa,b,1, Ping Nia,b, Jiannan Liua,b, Feng Wanga, Zhenyu Xinga, ⁎ Shigen Yea,b, a b
Dalian Key Laboratory of Marine Animal Disease Prevention and Control, Dalian Ocean University, Dalian, China Key Laboratory of Environment Controlled Aquaculture (KLECA), Ministry of Education, Dalian, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Vibrio harveyi VHH Anti-virulence Resveratrol
Vibrio harveyi is a kind of pathogenic bacteria that occurs widely in the ocean and exhibits multiple drug resistance. V. harveyi hemolysin (VHH) is the essential virulence factor of V. harveyi, and its main function is to promote the entry and spread of V. harveyi by lysing the cell membrane, which plays a critical role in the pathogen infection of the host. In this study, we used VHH as the target protein of novel virulence factor inhibitors and found that resveratrol (RSV) significantly inhibited its hemolytic activity. Furthermore, we investigated the mechanism of action of RSV and evaluated its therapeutic effect in V. harveyi-infected Takifugu rubripes. Remarkably, RSV downregulated the transcription of the vhh gene within a concentration range that did not inhibit bacterial growth. In addition, RSV antagonized its hemolytic activity by directly binding to the active center of VHH. RSV could effectively inhibit the damage caused by VHH and V. harveyi both in vitro and in vivo. These results suggest that RSV is a promising agent for the treatment of V. harveyi infection.
1. Introduction Takifugu rubripes is an economically important cultured species in Asia, especially in China and Japan. Due to its delicious taste and high nutritional value, the annual production of this cultured fish has increased in recent years (Koizumi and Hiratsuka, 2009). However, with the rapid development of the T. rubripes aquaculture industry, the problem posed by diseases such as V. harveyi infection has become increasingly serious (Kyoungmi et al., 2010; Mohi et al., 2010). V. harveyi is a Gram-negative bacterium that is ubiquitous in marine environments. It is a causative agent of vibriosis that is capable of infecting a wide range of aquatic animals (Ransangan et al., 2012). The main clinical symptoms of infected T. rubripes are deep skin ulcers, visceral hyperemia and enteritis (Kyoungmi et al., 2010; Peng et al., 2019). The lesions inside organ tissues include multiple nodules on the surface of the branchiostegite and granuloma in the viscera (Mohi et al., 2010). The disease breaks out quickly and is difficult to control, causing huge economic losses to the T. rubripes industry. To complete its intracellular life cycle, V. harveyi uses a number of surface proteins and virulence factors to subvert host cell functions and
gain access to the cytosol, including casease, gelatinase, phospholipase, V. harveyi hemolysin (VHH), metalloproteinase and chitinase (Ruwandeepika et al., 2010a), which function in different phases of the infection to facilitate bacterial entry, reproduction and cell-cell spreading. Among these activities, the dissolution of erythrocyte membranes and the release of hemoglobin are mediated by VHH, a member of the thermolabile hemolysin (TLH) family (Arachchige et al., 2012). This toxin, encoded by the vhh gene, is an essential virulence factor of V. harveyi. In a case of “bright-red” syndrome in Pacific white shrimp caused by V. harveyi infection, researchers first noted erythema on the ventral cuticle of the infected shrimp. Histological examination showed that the bacteria infecting the host diffused from the blood cavity to other tissues, leading to systemic vibriosis, and one of the responsible factors was VHH (Soto-Rodriguez et al., 2010). Zhang and Austin (Zhang and Austin, 2010; Zhang et al., 2001) found that the most pathogenic V. harveyi VIB645 strain harbored two hemolysin genes (i.e., vhhA and vhhB), while other strains exhibited only one hemolysin gene or no such genes. Numerous studies have established that VHH is critical in the process of V. harveyi infection. While the use of antibiotics has effectively controlled many serious
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Corresponding author at: Dalian Key Laboratory of Marine Animal Disease Prevention and Control, Dalian Ocean University, 52 Heishijiao Street, Dalian 116023, Liaoning Province, China. E-mail address: [email protected] (S. Ye). 1 These authors contributed equally. https://doi.org/10.1016/j.aquaculture.2020.735086 Received 3 November 2019; Received in revised form 28 December 2019; Accepted 5 February 2020 Available online 07 February 2020 0044-8486/ © 2020 Elsevier B.V. All rights reserved.
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Table 1 Primers used in this experiment. Purpose
Primers
Sequence
Gene amplification
vhh-F vhh-R vhh-RT-F vhh-RT-R luxS-RT-F luxS-RT-R toxR-RT-F toxR-RT-R L247A-F L247A-R Y368A-F Y368A-R
5′-GCGGATCCATGAATAAAACTATTACGTTACT-3′ 5′-GGCTCGAGGAAAGGATGGTTTGACAATTGA-3′ 5′-CAGCCAGACCAAATCAACAAG-3′ 5′-TGAGAAGTGTCCCAAGAACCA-3′ 5′-CTGCTCCAAACAAAGACATCCT-3′ 5′-CTCATGTAGAAACCAGTACGG-3′ 5′-GAAGCAGCACTCACCGAT-3′ 5′-GGTGAAGACTCATCAGCA-3′ 5′-CTTGAGTTTGGTGCGAATGACTTCATG-3′ 5′-CATGAAGTCATTCGCACCAAACTCAAG-3′ 5′-CTTCATCGGTGGATGCGCTATACCATCATG-3′ 5′-CATGATGGTATAGCGCATCCACCGATGAAG-3′
qRT-PCR
Site-directed mutagenesis
Note: The restriction enzyme cutting sites are indicated by single underline; the mutation sites are indicated by double underline.
Hope Biotech) with 15‰ salinity at 28 °C. The striped-fin loach epithelial cell line, provided by Liaoning Provincial Key Laboratory of Marine Biological Resource Restoration and Habitat Restoration, was grown in DMEM/F-12 (HyClone) with 10% heat-inactivated fetal bovine serum (FBS, AllBio), 100 μg/mL penicillin and 100 μg/mL streptomycin at 25 °C in a humidified chamber with 5% CO2 and was passaged every four days. Normal T. rubripes (about 4.5 ± 1 g in weight and 8.0 ± 0.4 cm in length) were purchased from the Dalian Fugu Group CO. LTD. The fish had no history of untoward mortalities or abnormalities and were acclimatized at 19 °C for two weeks prior to experiments. RSV (purity 99.80%) was purchased from Chengdu Herbpurify CO. LTD, dissolved in dimethyl sulfoxide (DMSO) and used to produce a 20 mg/mL stock solution, which was then diluted according to the experimental requirements.
infections in the past several decades, the traditional approach has so far shown limited success in preventing or treating bacterial diseases in marine animals and has even promoted the development of bacterial resistance; V. harveyi has not been spared in this scenario (Zhu et al., 2006). According to a survey, V. harveyi in mariculture waters shows severe multidrug resistance, and the drug resistance spectrum is diverse (Stalin and Srinivasan, 2016). In addition, Roque et al. (Roque et al., 2001) found that the minimum inhibitory concentration of V. harveyi isolated from diseased shrimp in the same geographic area had increased at least three-fold over 3–5 years. V. harveyi is now resistant to common antibiotics such as kanamycin, streptomycin, oxytetracycline, ofloxacin, and gentamicin (Tendencia and de la Peña, 2001), so the resistance of V. harveyi has become the most severe obstacle in the treatment of V. harveyi infection, and there in an urgent need to develop new alternative strategies to treat the disease caused by V. harveyi. Resveratrol is a naturally occurring phytoalexin produced by some spermatophytes (such as grapes and the traditional Chinese medicine Polygonum cuspidatum) that is readily available and inexpensive and presents a variety of health care functions (Frémont, 2000). In terms of its anti-tumor properties, RSV exerts anti-mutagenic and anti-inflammatory effects, can induce phase II drug-metabolizing enzymes and inhibit cyclooxygenase and catalase functions and shows good anticancer activity in the three stages of the initiation, promotion and expansion of tumorigenesis (Jang et al., 1997). Regarding the reduction of cardiovascular events, RSV acts as an active protective factor to inhibit the formation of atherosclerosis and thrombosis. RSV at a physiological concentration of 0.1 μM can relax vasodilatation, thereby reducing the incidence of cardiovascular disease (Granzotto and Zatta, 2014). RSV is a natural antioxidant that regulates the activity of antioxidant enzymes by inhibiting the production of free radicals and can significantly inhibit the autooxidation hemolysis of rat red blood cells and the oxidation hemolysis caused by hydrogen peroxide (Mendes et al., 2012). We screened targeted compounds that can inhibit VHH-mediated hemolysis and found that the polyphenolic compound RSV significantly inhibited the hemolytic activity of VHH. In this research, V. harveyi and RSV were used as experimental materials to reveal the mechanism by which RSV inhibits the virulence of V. harveyi and to evaluate the therapeutic effect.
2.2. Preparation of the VHH protein The vhh gene fragment was obtained by PCR (primer sequences are shown in Table 1) and inserted in the pET28a(+) vector, which was then transformed into BL21(DE3) cells, and the vhh-expressing strain pET28a(+)-vhh-BL21(DE3) was obtained. The strain was induced with IPTG and lysed by sonication to obtain inclusion bodies. After a series of purification, renaturation and concentration steps, the recombinant VHH protein was finally obtained. 2.3. Homology modeling and docking In this study, the three-dimensional structure of VHH (GenBank: AAG25957.1) was modeled using the homology model of Modeller 9.18 (Webb and Sali, 2014), and the molecular model of RSV (CAS: 501–360) was constructed using the Gaussian 03 program. The interaction between RSV and VHH receptors was analyzed by molecular docking analysis. Briefly, Modeller 9.18 was first used to search for proteins that are homologous to VHH in the Protein Data Bank (PDB) by superimposing the structure of homologous proteins to determine their structurally conserved regions (SCRs) and LOOP. Second, the model protein was compared with the homologous protein to acquire the SCR and LOOP of the model protein, and the structure of other parts of the model protein was predicted simultaneously. Finally, the flexible parts of VHH and RSV were determined using AutoDock Tools software, and the docking of the protein receptors and ligands was performed. Binding energy was calculated, and the binding sites were classified, with locking into several of the most likely binding sites eventually being observed.
2. Materials and methods 2.1. Materials The V. harveyi strain (NO. 2HWH020) used in the experiment was isolated from cultured fugu in the early stage of the disease at Dalian Tianzheng Industrial Co., Ltd. and showed strong virulence. Stock cultures were maintained at −80 °C in a suspension of nutrient broth (NB, Qingdao Hope Biotech) with 15‰ salinity and 20% glycerol. When required, the isolate was grown on nutrient agar (NA, Qingdao
2.4. Site-directed mutagenesis The L247A and Y368A mutations were introduced into pET28a 2
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corresponding well and cocultured with the cells. After 12 h of incubation, the supernatants from each well were collected for cell viability analysis using the Cell Counting Kit (CCK, TransGen Biotech) according to the manufacturer's directions. To determine the effects of RSV on VHH-triggered cell damage, the cells were cultured in the same manner described above. 2 μL of purified protein (10 μg/mL) was mixed with RSV at different concentrations in advance, and the mixture was placed at 28 °C for 30 min, after which it was added to the correct wells. At the indicated time points, cell viability was detected via the CCK assay at OD450nm.
(+)-vhh by PCR using the Fast Mutagenesis System (TransGen Biotech). The primers used to induce the point mutations are listed in Table 1. The DMT enzyme was added to the PCR product, followed by incubation at 37 °C for 1 h to digest the methylated plasmid template. A 2 μL aliquot of the digestion products was transformed into 50 μL of DMT competent cells, and all mutations were verified by DNA sequencing. The preparation and purification of the mutant proteins were identical to the procedure described in sections “2. vhh gene cloning and prokaryotic expression” and “3. Protein affinity”. 2.5. Hemolysis assay
2.9. Tissue distribution of RSV in T. rubripes The effect of RSV on hemolysis induced by VHH or its mutants was evaluated as described previously with slight modifications (Guo et al., 2020). Briefly, 2 μL of purified proteins (40 ng/mL) was mixed with different doses of RSV and 0.01 M PBS (pH = 7.4) in a total volume of 485 μL. The mixture was incubated at 28 °C for 30 min before the addition of defibrinated erythrocytes at a total volume of 3%. After 4 h, the reaction mixture was centrifuged (1290 g, 1 min) to remove the erythrocytes. Hemolytic activity was determined by measuring the OD543nm of the cell-free supernatant. Samples treated with 1% Triton X100 (Solarbio) were set as the 100% lysis controls. Hemolysis was expressed as the ratio of the OD543nm of each sample to the complete lysis controls.
To explore the distribution of RSV in the fugu liver and muscle, we first drew the standard curve of RSV. The RSV stock solution was diluted with PBS to produce a series of standard solutions (100, 150, and 200 μg/mL). Each RSV standard solution and acetonitrile (Solarbio) were mixed evenly at a ratio of 1:2, followed by centrifugation at 20700 g for 10 min. The supernatant was filtered through a 0.22 μm filtration membrane and injected into a high-performance liquid chromatography (HPLC) system equipped with a C18 column (4.6 mm × 250 mm, 5 μm; Thermo Scientific) and a UV detector set at 303 nm. The mobile phase of 30% acetonitrile and 70% acidified water (0.2%(v/v) phosphate) was run at a flow rate of 1 mL/min with an operating temperature of 25 °C. The area under the time concentration curve (AUC) of each sample was recorded, and the standard curve of the AUC-concentration was drawn. Next, the T. rubripes were administered intramuscularly with 50 μL of RSV at a dose of 100 mg/kg. The T. rubripes were sacrificed at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 8 and 12 h after drug administration. Their livers and muscles were promptly removed, and physiological saline was added to prepare a homogenate. HPLC was carried out as described above. The AUC was taken into the standard line, and the corresponding drug concentration was calculated.
2.6. Minimum inhibitory concentration assay The minimum inhibitory concentration (MIC) of RSV against V. harveyi was determined by the broth dilution method recommended by NCCLS (Bruck, 1980). A single colony of pure V. harveyi was selected and inoculated in fresh NB with 15‰ salinity, then cultured at 28 °C until reaching an OD600nm = 0.1. RSV stock solution was added to the culture medium to a final concentration of 2048 μg/mL, which was then continuously diluted to 1 μg/mL via a two-fold dilution method. Then, the culture was continued for 12 h. Thereafter, the effect of RSV on the growth of V. harveyi was evaluated by measuring the OD600nm value. For the positive control, no RSV was added, and for the negative control, no bacteria were included.
2.10. Animal experiments To assess the toxicity and treatment effects of RSV in T. rubripes fry, we conducted animal infection experiments. Briefly, 120 fry were evenly distributed in 12 tanks sterilized with potassium permanganate and divided into 4 groups: V. harveyi (infection group), RSV + V. harveyi (drug treatment group), RSV alone (drug toxicity group) and no treatment (control group). Infection was carried out by immersion. T. rubripes were immersed for 8 h in filtered sea water inoculated with a fresh bacterial suspension at a final concentration of 5 × 107 CFU/mL, and the challenged fish were transferred to a new site. The fry were administered RSV intramuscularly at 100 mg/kg postinfection (50 μL per fish), and the same doses were administered at 12-h intervals. The mortality of the fish was monitored for 7 d consecutively. The gills and kidneys were prepared for histopathological analysis. Briefly, the samples were fixed in 10% formalin for 24 h, then washed overnight with running water, dehydrated in graded ethanol solutions and fixed in paraffin wax. The tissues were cut to a thickness of 5 μm and stained with hematoxylin and eosin. The histologic sections were observed under a light microscope, and digital images of the histological features were obtained using a Leica microscope imaging system (Leica DM4000, Germany).
2.7. qRT-PCR V. harveyi was cultured in NB (15‰ salinity) with or without various doses of RSV (final concentrations of 16, 8, 4, 2, and 1 μg/mL) to the exponential growth phase (OD600nm = 1.0). The bacterial cells were then collected (4 °C, 1290 g, 5 min), and total RNA was extracted using the RNA Fast 200 kit (Fastagen Biotech). The concentration of the purified RNA was determined using a NanoDrop 2000c UV/Vis spectrophotometer (Thermo Scientific). The quality of the purified RNA was determined by electrophoresis on a 1% agarose gel. The primer pairs used in qRT-PCR are listed in Table 1. RNA was reverse transcribed into cDNA using the Prime Script RT reagent kit (Takara). PCR was performed using a StepOnePlus Real-Time PCR instrument (Applied Biosystems) in a 20 μL volume containing Mon Amp ChemoHS qPCR Mix (Monad) as recommended by the manufacturer. All samples were analyzed in triplicate, and the housekeeping gene toxR (Pang et al., 2006; Torky et al., 2016) was used as an endogenous control. In this study, the expression of the target gene relative to toxR was used to determine the differences in transcription levels between samples.
2.11. Statistical analysis
2.8. Cytotoxicity test and cell damage inhibition test
Statistical analysis and p value calculation were conducted using SPSS 19.0 statistical software. All experimental group data were compared with the corresponding control group data by t-tests. A p value of less than 0.05 was considered to indicate a significant difference, and the difference was considered extremely significant when the p value was less than 0.01. Three parallel plots were generated for all samples
For the detection of RSV cytotoxicity, after digestion, striped-fin loach epithelial cells that had grown to a suitable density were inoculated into 96-well plates at 2 × 104 cells per well in FBS-free medium overnight. RSV with gradient concentrations was added to the 3
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we successfully mutated these residues into Ala residues and performed a hemolysis assay to verify their function and investigated the effect of RSV on the hemolytic activity of wild-type VHH and its mutants. When there was no RSV in the mixed system, the hemolytic activities of WTVHH, L247A and Y368A were almost the same; with an increase in the RSV concentration, the hemolytic activities of all three showed decreases to different degrees, among which the inhibitory effect on VHH was the most obvious. When the concentration was 0.2 μg/mL, the inhibitory effect of RSV on VHH hemolytic activity was extremely significant (p < .01) and continued to increase. At a concentration of 8 μg/mL, the inhibitory effect reached a maximum. Thereafter, the hemoglobin release rate rebounded slightly. Compared with VHH, RSV showed weak inhibition of L247A and Y368A, as the inclusion of 8 μg/ mL of this compound in the reactions resulted in a hemolysis inhibition rate of less than 50% (Fig. 2). These results suggested that Leu247 and Tyr368 were indeed the key sites of RSV binding to VHH.
and were plotted using the mean and standard deviation. 3. Results 3.1. Cloning of the vhh gene and preparation of the protein The vhh gene (GenBank No. MN296414) was amplified by using V. harveyi DNA as a template and vhh-F/R as a primer. The fragment size was 1257 bp, and the sequence was 99% consistent with that of the vhhA gene (GenBank No. AF293430) according to BLAST alignment. pET28a(+)-vhh-BL21 (DE3) was induced by IPTG, sonicated and purified to obtain a VHH recombinant protein with a molecular weight of 47.38 kDa, and the protein existed in the form of inclusion bodies. After renaturation and concentration, the protein could be used in the following experiments. 3.2. Leu247 and Tyr368 are the key sites for the binding of RSV to VHH
3.4. RSV has no effect on the growth of V. harveyi and inhibits vhh transcription by promoting luxS transcription
Modeller 9.18 was used to search the full homologous sequence template of the VHH protein, and active site analysis of the modeled structure was performed. We found that PDB: 6JL2 (Wan et al., 2019) showed the highest degree of matching with the target protein, and the similarity between the amino acid sequences of the catalytic center and VHH was greater than 80%. When this structure was used as a template, the catalytic triad model of Asp390-His393-Ser153 could be obtained with an appropriate location. Therefore, it was used as the template to model the 3D structure of the target protein, and the RSV ligand was molecularly docked with the 3D structure of VHH. The docking energy of RSV and the VHH protein was −6.0 Kcal/ mol. RSV was integrated into the cavity composed of the triplet catalytic center and the oxygen anion hole. In this structure, the metaphenolic hydroxyl group formed a hydrogen bond with Tyr368, and the p-hydroxybenzene ring formed a π-alkyl interaction with Leu247 (Fig. 1). We speculate that RSV inhibited the hemolytic activity of VHH, possibly by acting directly on VHH and inactivating its biological activity.
As shown in Fig. 3A, when the concentration was 32 μg/mL, RSV showed an inhibitory effect on the growth of V. harveyi, and the antibacterial effect was enhanced with an increasing drug concentration. The MIC of RSV was 64 μg/mL, and a concentration of 128 μg/mL was completely bacteriostatic, indicating that RSV has an antibacterial effect at higher concentrations in vitro. To eliminate the interference caused by RSV inhibiting the growth of V. harveyi, we selected the concentration range of 0–16 μg/mL for qRT-PCR experiments to explore the influence of RSV on the transcription level of vhh. The mRNA level of vhh was investigated by qRT-PCR. Previous studies have shown that one of the signal molecules of the V. harveyi density sensing system, AI-2, whose precursor is synthesized by LuxS, plays a negative role in the transcription of vhh (Bai and Zhang, 2010). Therefore, we also detected the transcription of luxS after the RSV treatment of V. harveyi. As the dose of RSV was increased, the transcription level of luxS was increased, and vhh transcription was downregulated in a dose-dependent manner. When V. harveyi was cocultured with RSV at 1, 2, 4, 8 or 16 μg/mL, the transcription level of vhh was reduced 1.15, 1.42, 1.75, 2.29 or 2.67 times, respectively, compared with that in V. harveyi without RSV treatment (Fig. 3B).
3.3. Effect of RSV on the hemolytic activity of the WT-VHH protein and mutant proteins The results of molecular docking showed that Leu247 and Tyr368 played an important role in the binding of VHH to RSV. Subsequently,
Fig. 1. 3D and 2D interaction diagrams of RSV and VHH. The light green stick is RSV, the yellow stick is the amino acid residue of the catalytic center of triplet and oxygen anion hole, and the dark green stick is the amino acid residue involved in the binding. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 4
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Fig. 2. Effect of RSV on hemolytic activity of WT-VHH protein and mutant proteins. The inhibition of RSV on VHH hemolytic activity increased with the increase of RSV concentration, but the effect on the two mutated proteins was not so obvious. N, negative control; P, positive control.
in the liver. In addition, the half-lives of the elimination (t1/2) of RSV in the muscle and liver were 1.25 h and 1.5 h, respectively, and RSV was completely metabolized 4 h after injection. The concentration of RSV in tissues exceeded the minimum effective concentration for the inhibition of hemolysis and cell damage induced by VHH in vitro, so it was feasible to conduct therapeutic experiments at this dose. No deaths occurred during the entire experiment. The infected T. rubripes were accompanied by clinical symptoms such as swimming slowly and loss of appetite. After dissection, it could be observed that the gills, liver and kidneys presented different degrees of bleeding and swelling, and showed severe enteritis. All of the above symptoms were alleviated after treatment with RSV. Since the gill is the first site where the bacteria invade the host and the kidney is an important immune organ of fish, we obtained paraffin sections of these two tissues to further study the pathological changes before and after treatment. In the infected group, edema occurred in the gill filaments, and the gill lamella was expanded, congested and deformed. The renal interstitium became loose, and the original normal structure was destroyed. The glomeruli were atrophied, and the basilar membrane of the renal tubules exhibited edema. In the RSV-treated group, this pathology was discernibly alleviated, with only mild edema and renal interstitial hemorrhage being observed. In the drug toxicity group, no obvious lesions were observed in the gill, and the epithelial cells of the renal tubule showed mild granular degeneration (Fig. 4C). Overall, RSV can alleviate the histopathological damage caused by V. harveyi and has the potential to be used as a new anti-infection drug.
3.5. RSV has a protective effect against VHH-mediated cell damage Tetrazolium salt in CCK solution can be reduced to orange formazan by dehydrogenase, and the color depth shows a good linear relationship with the number of living cells. Therefore, we evaluated the drug toxicity of RSV and its inhibitory effect on VHH-mediated cell damage by measuring the light absorption value at 450 nm. As shown in Fig. 3C, RSV at 32 μg/mL exerted a certain degree of cytotoxicity (p < .05), resulting in 47% cell death. Prior to the application of this concentration, there was some cell death, but the level was not significant, so RSV could be considered safe. When 0.5 μg/mL RSV was added to the VHHcell coculture system, a significant protective effect was observed (p < .05), and the cell survival rate reached a maximum when the concentration was 2 μg/mL, which was 87.34% (Fig. 3D). Although the survival rate decreased as the concentration of RSV increased thereafter, it was still higher than the overall level in the RSV-free group, indicating that RSV could effectively alleviate the cell damage caused by VHH.
3.6. RSV protects T. rubripes from lesions caused by V. harveyi infection The standard curve of the concentration-AUC of RSV was drawn (Fig. 4A) and indicated that the concentration of RSV showed a good linear relationship with the AUC within the range of 0–200 μg/mL. After injection into the T. rubripes, RSV was rapidly distributed in the muscles and liver. From the drug concentration-time in the tissue curve (Fig. 4B), when the drug was administered at a concentration of 100 mg/kg, RSV reached a peak concentration (Cmax) of 64 mg/kg in the muscle at 1 h after injection. However, the peak time (tmax) in the liver occurred 0.25 h earlier, and the Cmax was only 42 mg/kg, indicating that the bioavailability of RSV in muscle was higher than that
4. Discussion As one of the core species of Vibrio, V. harveyi is a conditionally pathogenic bacteria (Austin and Zhang, 2010). When the host organism 5
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Fig. 3. RSV inhibits vhh transcription and VHH-mediated cell damage. A, RSV did not inhibit the extracellular growth of V. harveyi. At low concentrations, RSV had no bacteriostatic effect. However, with the increase in the concentration, the bacteriostatic effect was enhanced, and it was completely bacteriostatic at 128 μg/mL. The OD600nm value was used to determine the concentration of V. harveyi suspension after cultured for 12 h. B, The effects of different concentrations of RSV on gene expression in V. harveyi. With the increase in RSV concentration, the transcription levels of luxS was up-regulated and that was down-regulated of vhh. C, Cytotoxicity of RSV. D, RSV inhibited VHH cell damage. The inhibitory effect was greatest at RSV concentration of 2 μg/mL, although there was a decrease later, the overall survival rate was higher than that of the control group.
et al., 2007). Although these toxins are generally referred to as hemolysins because of their capacity to lyse erythrocytes, they exhibit two different modes of action, involving either a pore-forming protein or a phospholipase enzyme, and VHH belongs to the second group (Arachchige et al., 2012; Rowe and Welch, 1994). Studies by Bai et al. (Fangfang et al., 2010) have shown that VHH has an amphiphilic structure and can induce tubular protrusion formation in the erythrocyte membrane in a short time. After coculture with erythrocytes, VHH was localized by the immunogold staining technique, and it was found that the gold particles were mainly deposited on the membrane, indicating that VHH relies on its phospholipase activity to destroy the cell membrane. However, a small amount of gold particles was deposited inside the cells, suggesting that the cell membrane was not the only target and that VHH may present enzyme activity other than phospholipase activity (Fangfang et al., 2010). VHH belongs to the GDSL hydrolytic enzyme family and is characterized by flexible active sites that bind to multiple substrates by altering conformation (Akoh et al., 2004). Therefore, VHH may also exhibit a flexible exchange site that can be combined with a variety of targets. In addition to dissolving cell membranes, VHH activates caspase-3 and induces apoptosis (Fangfang et al., 2010). In the past, the widespread and frequent use of antibiotics in aquaculture has increased the selective pressure exerted on microorganisms, encouraged the natural emergence of bacterial resistance, and enabled horizontal transfer of resistant genes across organisms through mobile genetic factors such as plasmids, transposons, phages, and integrons (You et al., 2016; Zhu et al., 2006). Reports of multidrug
is immune suppressed or otherwise physiologically stressed, large-scale outbreaks will occur, with the frequency of infection often attributable to intensive culture and adverse environmental conditions (Defoirdt et al., 2007). With the contributions of phospholipase C, hemolysin, lipopolysaccharide, adhesion factor and other virulence factors (Zhang et al., 2007), V. harveyi can cause serious infections in important economic species such as shellfish (Wang et al., 2017), shrimp (Ting et al., 2015), T. rubripes and orange-spotted grouper (Hai et al., 2017). Comparative genomic analysis has revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by deletion and gene acquisitions through duplication or horizontal transfer (Ruwandeepika et al., 2010b). To cope with its complex environment, V. harveyi is very likely to obtain virulence genes from other Vibrio species in the aquatic environment through horizontal gene transfer, thereby increasing the virulence of bacteria to specific hosts and effectively improving their ability to infect aquatic organisms (Ruwandeepika et al., 2010b). In this context, some V. harveyi strains have been transformed from secondary pathogens to a primary pathogens through increases in their virulence (Zhou et al., 2012). In addition, through long-term evolution, V. harveyi has attained strong resistance to the killing effect of host serum by interfering with the P38 MAPK pathway, thereby blocking the phagocytosis of blood cells (Li et al., 2011). V. harveyi is becoming more virulent and harmful, but researchers have unfortunately not yet sufficiently elucidated its pathogenesis. Hemolysin is arguably the most widely distributed exotoxin among pathogenic vibrios and is involved in disease pathogenesis (Boguang 6
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Fig. 4. RSV protects T. rubripes from lesions caused by V. harveyi infection. A, The standard curve of RSV. B, Tissue distribution of RSV in T. rubripes. After inoculation at the concentration of 100 mg/kg, RSV spread rapidly in T. rubripes, reached Cmax in muscle and liver about 1 h, and was completely metabolized at 4 h. C, RSVtreated T. rubripes exhibited less pathological abnormalities in gills and kidneys. After treatment of RSV, pathology was discernibly alleviated, with only mild edema and renal interstitial hemorrhage.
role of the two residues in the binding process. Furthermore, RSV downregulated the transcription of vhh and inhibited the toxicity of VHH to fin epithelial cells in a concentration range that did not affect the growth of V. harveyi (i.e., drug resistance was not easy to achieve). Finally, RSV showed a satisfactory therapeutic effect on tissue lesions caused by V. harveyi infection. RSV was inoculated into the body and spread rapidly, reaching the Cmax in the muscles and liver within approximately 1 h. After RSV treatment, the gills and kidneys of the infected fish showed significant improvement. From the pathological sections, we could see that the organ tissues of the drug toxicity group showed mild kidney injury, similar to the situation reported previously (Cottart et al., 2010; Crowell et al., 2004), which may be due to the toxicity caused by drug accumulation. In the process of the experiment, RSV accumulated to a high dose, resulting in toxic side effects; in practical applications, the dose could be reduced so that the toxicity is weakened or may not even manifest. In future studies, researchers could improve the functional groups of RSV by chemical modification and reduce its toxicity. In summary, our results have demonstrated the effectiveness of this natural compound in inhibiting the cytotoxicity of VHH, which should pave the way for future structural modification of RSV or the generation of RSV derivatives to obtain molecules showing higher potency against VHH and a protective effect, leading to the development of effective and clinically useful VHH-specific therapeutics.
resistance in V. harveyi are common and can be traced back to the last century. In the case described by Karunsagar et al., mass mortality in black tiger shrimp larvae was caused by V. harveyi with multiple resistance to chloramphenicol, erythromycin, cotrimoxazole and streptomycin (Karunasagar et al., 1994). In response to this situation, a variety of new treatments have emerged. Anti-virulence factor therapy is considered one of the promising effective strategies for meeting the challenge posed by widespread drug resistance among bacterial pathogens and could reduce the virulence of pathogens by interfering with the regulation of virulence factor expression or specifically inhibiting a virulence factor (Tom et al., 2011). Compared with vaccines, phage therapy, probiotics and other antibiotic replacement therapy, virulence factor inhibitors are generally small molecular compounds that exist naturally, which are widely available and inexpensive. In the process of use, it can be fed through mixed feeding. In addition, V. harveyi can also infect some invertebrates, for which virulence factor inhibitors are more feasible than others. Here, we provide evidence that RSV is one such agent with the potential to treat infections caused by V. harveyi by specifically antagonizing the activity of VHH. RSV effectively inhibited hemolysis induced by VHH. Molecular docking is increasingly considered as a tool for lead discovery, as the structures of an increasing number of proteins and nucleic acids become available (Shoichet et al., 2002). We obtained the binding sites of RSV and VHH through this technique and selected Leu247 and Tyr368 as the targets for site-directed mutagenesis. The results of the hemolysis assay showed that after the mutation of Leu247 and Tyr368 into Ala, the inhibitory effect of RSV on the hemolytic activity of the mutant protein decreased significantly, revealing the key
Ethical statement In this research, all experimental animals were used according to 7
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the ethical requirements, and experimental procedures were following the guidelines for the Care and Use of Laboratory Animals in China.
963–967. Li, M.F., Wang, C.L., Sun, L., 2011. A pathogenic Vibrio harveyi lineage causes recurrent disease outbreaks in cultured Japanese flounder (Paralichthys olivaceus) and induces apoptosis in host cells. Aquaculture 319, 30–36. Mendes, J.B.E., Riekes, M.K., de Oliveira, V.M., Michel, M.D., Stulzer, H.K., Khalil, N.M., Zawadzki, S.F., Mainardes, R.M., Farago, P.V., 2012. PHBV/PCL microparticles for controlled release of resveratrol: physicochemical characterization, antioxidant potential, and effect on hemolysis of human erythrocytes. Sci. World J. 2012, 1–13. Mohi, M.M., Kuratani, M., Miyazaki, T., Yoshida, T., 2010. Histopathological studies on Vibrio harveyi- infected tiger puffer, Takifugu rubripes (Temminck et Schlegel), cultured in Japan. J. Fish Dis. 33, 833–840. Pang, L., Zhang, X.H., Zhong, Y., Chen, J., Li, Y., Austin, B., 2006. Identification of Vibrio harveyi using PCR amplification of the toxR gene. Lett. Appl. Microbiol. 43, 249–255. Peng, H., Yang, B., Li, B., Cai, Z., Cui, Q., Chen, M., Liu, X., Yang, X., Jiang, C., 2019. Comparative transcriptomic analysis reveals the gene expression profiles in the liver and spleen of Japanese pufferfish (Takifugu rubripes) in response to Vibrio harveyi infection. Fish Shellfish Immunol. 90, 308–316. Ransangan, J., Lal, T.M., Al-Harbi, A.H., 2012. Characterization and experimental infection of Vibrio harveyi isolated from diseased Asian seabass (Lates calcarifer). Malays. J. Microbiol. 8, 104–115. Roque, A., Molina-Aja, A., Bolán-Mejıa, C., Gomez-Gil, B., 2001. In vitro susceptibility to 15 antibiotics of vibrios isolated from penaeid shrimps in northwestern Mexico. Int. J. Antimicrob. Agents 17, 383–387. Rowe, G.E., Welch, R.A., 1994. Assays of hemolytic toxins. Methods Enzymol. 235, 657. Ruwandeepika, H., Defoirdt, T., Bhowmick, P., Shekar, M., Bossier, P., Karunasagar, I., 2010a. Presence of typical and atypical virulence genes in vibrio isolates belonging to the Harveyi clade. J. Appl. Microbiol. 109, 888–899. Ruwandeepika, H.A.D., Defoirdt, T., Bhowmick, P.P., Shekar, M., Bossier, P., Karunasagar, I., 2010b. Presence of typical and atypical virulence genes in vibrio isolates belonging to the Harveyi clade. J. Appl. Microbiol. 109, 888–899. Shoichet, B.K., McGovern, S.L., Wei, B., Irwin, J.J., 2002. Lead discovery using molecular docking. Curr. Opin. Chem. Biol. 6, 439–446. Soto-Rodriguez, S.A., Gomez-Gil, B., Lozano, R., 2010. ‘Bright-red’ syndrome in Pacific white shrimp Litopenaeus vannamei is caused by Vibrio harveyi. Dis. Aquat. Org. 92, 11–19. Stalin, N., Srinivasan, P., 2016. Molecular characterization of antibiotic resistant Vibrio harveyi isolated from shrimp aquaculture environment in the south east coast of India. Microb. Pathog. 97, 110–118. Tendencia, E.A., de la Peña, L.D., 2001. Antibiotic resistance of bacteria from shrimp ponds. Aquaculture 195, 193–204. Ting, C., Nai-Kei, W., Xiao, J., Xing, L., Lvping, Z., Dan, Y., Chunhua, R., Chaoqun, H., 2015. Nitric oxide as an antimicrobial molecule against Vibrio harveyi infection in the hepatopancreas of Pacific white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 42, 114–120. Tom, D., Patrick, S., Peter, B., 2011. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr. Opin. Microbiol. 14, 251–258. Torky, H.A., Abdellrazeq, G.S., Hussein, M.M., Ghanem, N.H., 2016. Molecular characterization of Vibrio Harveyi in diseased shrimp. Alex. J. Vet. Sci. 51. Wan, Y., Liu, C., Ma, Q., 2019. Structural analysis of a vibrio phospholipase reveals an unusual Ser–His–chloride catalytic triad. J. Biol. Chem. 294 (30), 11391–11401 jbc. RA119. 008280. Wang, Y., Barton, M., Elliott, L., Li, X., Abraham, S., O'Dea, M., Munro, J., 2017. Bacteriophage therapy for the control of Vibrio harveyi in greenlip abalone (Haliotis laevigata). Aquaculture 473, 251–258. Webb, B., Sali, A., 2014. Comparative protein structure modeling using MODELLER. Current protocols in. Bioinformatics 47 5.6. 1–5.6. 32. You, K.G., Bong, C.W., Lee, C.W., 2016. Antibiotic resistance and plasmid profiling of Vibrio spp. in tropical waters of peninsular Malaysia. Environ. Monit. Assess. 188, 1–15. Zhang, X.H., Austin, B., 2010. Pathogenicity of Vibrio harveyi to salmonids. J. Fish Dis. 23, 93–102. Zhang, X.H., Meaden, P.G., Austin, B., 2001. Duplication of hemolysin genes in a virulent isolate of Vibrio harveyi. Appl. Environ. Microbiol. 67, 3161. Zhang, X.H., Zhong, Y.B., Chen, J.X., 2007. The biological characteristics, epidemiology and detection techniques of Vibrio Harveyi. Period. Ocean Univ. China 37, 740–748. Zhou, J., Fang, W., Yang, X., Zhou, S., Hu, L., Li, X., Qi, X., Su, H., Xie, L., 2012. A nonluminescent and highly virulent Vibrio harveyi strain is associated with “bacterial white tail disease” of Litopenaeus vannamei shrimp. 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Data availability statement The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Funding information National Key R&D Program of China (2017YFD0701701). Natural Science Foundation of Liaoning Province of China (2019MS-031). Declaration of Competing Interest No conflict of interest was existed. References Akoh, C.C., Lee, G.C., Liaw, Y.C., Huang, T.H., Shaw, J.F., 2004. GDSL family of serine esterases/lipases. Prog. Lipid Res. 43, 534–552. Arachchige, H., Ruwandeepika, D., Sanjeewa, T., Jayaweera, P., Bhowmick, P.P., Karunasagar, I., Bossier, P., Defoirdt, T., 2012. Pathogenesis, virulence factors and virulence regulation of vibrios belonging to the Harveyi clade. Rev. Aquac. 4, 59–74. Austin, B., Zhang, X., 2010. Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Lett. Appl. Microbiol. 43, 119–124. Bai, F.-F., Zhang, X.-H., 2010. Studies on the effect of quorum sensing systems of Vibrio harveyi on the activities of extracellular enzymes. Period. Ocean Univ. China 40, 91–95. Boguang, S., Xiao-Hua, Z., Xuexi, T., Shushan, W., Yingbin, Z., Jixiang, C., Brian, A., 2007. A single residue change in Vibrio harveyi hemolysin results in the loss of phospholipase and hemolytic activities and pathogenicity for turbot (Scophthalmus maximus). J. Bacteriol. 189, 2575. Bruck, E., 1980. National Committee for clinical laboratory standards. Pediatrics 65, 187. Cottart, C.H., Nivet-Antoine, V., Laguillier-Morizot, C., Beaudeux, J.L., 2010. Resveratrol bioavailability and toxicity in humans. Mol. Nutr. Food Res. 54, 7–16. Crowell, J.A., Korytko, P.J., Morrissey, R.L., Booth, T.D., Levine, B.S., 2004. Resveratrolassociated renal toxicity. Toxicol. Sci. 82, 614. Defoirdt, T., Boon, N., Sorgeloos, P., Verstraete, W., Bossier, P., 2007. Alternatives to antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as an example. Trends Biotechnol. 25, 472–479. Fangfang, B., Boguang, S., Woo, N.Y.S., Xiao-Hua, Z., 2010. Vibrio harveyi hemolysin induces ultrastructural changes and apoptosis in flounder (Paralichthys olivaceus) cells. Biochem. Biophys. Res. Commun. 395, 70–75. Frémont, L., 2000. Biological effects of resveratrol. Life Sci. 66, 663–673. Granzotto, A., Zatta, P., 2014. Resveratrol and Alzheimer’s disease: message in a bottle on red wine and cognition. Front. Aging Neurosci. 6, 95. Guo, Y., Xing, Z., He, Z., Zhao, X., Ye, S., 2020. Honokiol inhibits Vibrio harveyi hemolysin virulence by reducing its haemolytic activity. Aquac. Res. 51 (1), 206–214. Hai, T.N., Nguyen, T.T.T., Tsai, M.A., Ya-Zhen, E., Wang, P.C., Chen, S.C., 2017. A formalin-inactivated vaccine provides good protection against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides). Fish Shellfish Immunol. 65, 118–126. Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas, C.F., Beecher, C.W., Fong, H.H., Farnsworth, N.R., Kinghorn, A.D., Mehta, R.G., 1997. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275, 218–220. Karunasagar, I., Pai, R., Malathi, G.R., Karunasagar, I., 1994. Mass mortality of Penaeus monodon larvae due to antibiotic-resistant Vibrio harveyi infection. Aquaculture 128, 203–209. Koizumi, K., Hiratsuka, S., 2009. Fatty acid compositions in muscles of wild and cultured ocellate puffer Takifugu rubripes. Fish. Sci. 75, 1323. Kyoungmi, W., Kihong, K., Sooil, P., 2010. Vibrio harveyi associated with deep skin ulcer in river puffer, Takifugu obscures, exhibited in a public aquarium. Aquac. Res. 40,
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Resveratrol inhibits the virulence of Vibrio harveyi by reducing the activity of Vibrio harveyi hemolysin
T
Xiaoran Zhaoa,b,1, Yi Guoa,b,1, Ping Nia,b, Jiannan Liua,b, Feng Wanga, Zhenyu Xinga, ⁎ Shigen Yea,b, a b
Dalian Key Laboratory of Marine Animal Disease Prevention and Control, Dalian Ocean University, Dalian, China Key Laboratory of Environment Controlled Aquaculture (KLECA), Ministry of Education, Dalian, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Vibrio harveyi VHH Anti-virulence Resveratrol
Vibrio harveyi is a kind of pathogenic bacteria that occurs widely in the ocean and exhibits multiple drug resistance. V. harveyi hemolysin (VHH) is the essential virulence factor of V. harveyi, and its main function is to promote the entry and spread of V. harveyi by lysing the cell membrane, which plays a critical role in the pathogen infection of the host. In this study, we used VHH as the target protein of novel virulence factor inhibitors and found that resveratrol (RSV) significantly inhibited its hemolytic activity. Furthermore, we investigated the mechanism of action of RSV and evaluated its therapeutic effect in V. harveyi-infected Takifugu rubripes. Remarkably, RSV downregulated the transcription of the vhh gene within a concentration range that did not inhibit bacterial growth. In addition, RSV antagonized its hemolytic activity by directly binding to the active center of VHH. RSV could effectively inhibit the damage caused by VHH and V. harveyi both in vitro and in vivo. These results suggest that RSV is a promising agent for the treatment of V. harveyi infection.
1. Introduction Takifugu rubripes is an economically important cultured species in Asia, especially in China and Japan. Due to its delicious taste and high nutritional value, the annual production of this cultured fish has increased in recent years (Koizumi and Hiratsuka, 2009). However, with the rapid development of the T. rubripes aquaculture industry, the problem posed by diseases such as V. harveyi infection has become increasingly serious (Kyoungmi et al., 2010; Mohi et al., 2010). V. harveyi is a Gram-negative bacterium that is ubiquitous in marine environments. It is a causative agent of vibriosis that is capable of infecting a wide range of aquatic animals (Ransangan et al., 2012). The main clinical symptoms of infected T. rubripes are deep skin ulcers, visceral hyperemia and enteritis (Kyoungmi et al., 2010; Peng et al., 2019). The lesions inside organ tissues include multiple nodules on the surface of the branchiostegite and granuloma in the viscera (Mohi et al., 2010). The disease breaks out quickly and is difficult to control, causing huge economic losses to the T. rubripes industry. To complete its intracellular life cycle, V. harveyi uses a number of surface proteins and virulence factors to subvert host cell functions and
gain access to the cytosol, including casease, gelatinase, phospholipase, V. harveyi hemolysin (VHH), metalloproteinase and chitinase (Ruwandeepika et al., 2010a), which function in different phases of the infection to facilitate bacterial entry, reproduction and cell-cell spreading. Among these activities, the dissolution of erythrocyte membranes and the release of hemoglobin are mediated by VHH, a member of the thermolabile hemolysin (TLH) family (Arachchige et al., 2012). This toxin, encoded by the vhh gene, is an essential virulence factor of V. harveyi. In a case of “bright-red” syndrome in Pacific white shrimp caused by V. harveyi infection, researchers first noted erythema on the ventral cuticle of the infected shrimp. Histological examination showed that the bacteria infecting the host diffused from the blood cavity to other tissues, leading to systemic vibriosis, and one of the responsible factors was VHH (Soto-Rodriguez et al., 2010). Zhang and Austin (Zhang and Austin, 2010; Zhang et al., 2001) found that the most pathogenic V. harveyi VIB645 strain harbored two hemolysin genes (i.e., vhhA and vhhB), while other strains exhibited only one hemolysin gene or no such genes. Numerous studies have established that VHH is critical in the process of V. harveyi infection. While the use of antibiotics has effectively controlled many serious
⁎
Corresponding author at: Dalian Key Laboratory of Marine Animal Disease Prevention and Control, Dalian Ocean University, 52 Heishijiao Street, Dalian 116023, Liaoning Province, China. E-mail address: [email protected] (S. Ye). 1 These authors contributed equally. https://doi.org/10.1016/j.aquaculture.2020.735086 Received 3 November 2019; Received in revised form 28 December 2019; Accepted 5 February 2020 Available online 07 February 2020 0044-8486/ © 2020 Elsevier B.V. All rights reserved.
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Table 1 Primers used in this experiment. Purpose
Primers
Sequence
Gene amplification
vhh-F vhh-R vhh-RT-F vhh-RT-R luxS-RT-F luxS-RT-R toxR-RT-F toxR-RT-R L247A-F L247A-R Y368A-F Y368A-R
5′-GCGGATCCATGAATAAAACTATTACGTTACT-3′ 5′-GGCTCGAGGAAAGGATGGTTTGACAATTGA-3′ 5′-CAGCCAGACCAAATCAACAAG-3′ 5′-TGAGAAGTGTCCCAAGAACCA-3′ 5′-CTGCTCCAAACAAAGACATCCT-3′ 5′-CTCATGTAGAAACCAGTACGG-3′ 5′-GAAGCAGCACTCACCGAT-3′ 5′-GGTGAAGACTCATCAGCA-3′ 5′-CTTGAGTTTGGTGCGAATGACTTCATG-3′ 5′-CATGAAGTCATTCGCACCAAACTCAAG-3′ 5′-CTTCATCGGTGGATGCGCTATACCATCATG-3′ 5′-CATGATGGTATAGCGCATCCACCGATGAAG-3′
qRT-PCR
Site-directed mutagenesis
Note: The restriction enzyme cutting sites are indicated by single underline; the mutation sites are indicated by double underline.
Hope Biotech) with 15‰ salinity at 28 °C. The striped-fin loach epithelial cell line, provided by Liaoning Provincial Key Laboratory of Marine Biological Resource Restoration and Habitat Restoration, was grown in DMEM/F-12 (HyClone) with 10% heat-inactivated fetal bovine serum (FBS, AllBio), 100 μg/mL penicillin and 100 μg/mL streptomycin at 25 °C in a humidified chamber with 5% CO2 and was passaged every four days. Normal T. rubripes (about 4.5 ± 1 g in weight and 8.0 ± 0.4 cm in length) were purchased from the Dalian Fugu Group CO. LTD. The fish had no history of untoward mortalities or abnormalities and were acclimatized at 19 °C for two weeks prior to experiments. RSV (purity 99.80%) was purchased from Chengdu Herbpurify CO. LTD, dissolved in dimethyl sulfoxide (DMSO) and used to produce a 20 mg/mL stock solution, which was then diluted according to the experimental requirements.
infections in the past several decades, the traditional approach has so far shown limited success in preventing or treating bacterial diseases in marine animals and has even promoted the development of bacterial resistance; V. harveyi has not been spared in this scenario (Zhu et al., 2006). According to a survey, V. harveyi in mariculture waters shows severe multidrug resistance, and the drug resistance spectrum is diverse (Stalin and Srinivasan, 2016). In addition, Roque et al. (Roque et al., 2001) found that the minimum inhibitory concentration of V. harveyi isolated from diseased shrimp in the same geographic area had increased at least three-fold over 3–5 years. V. harveyi is now resistant to common antibiotics such as kanamycin, streptomycin, oxytetracycline, ofloxacin, and gentamicin (Tendencia and de la Peña, 2001), so the resistance of V. harveyi has become the most severe obstacle in the treatment of V. harveyi infection, and there in an urgent need to develop new alternative strategies to treat the disease caused by V. harveyi. Resveratrol is a naturally occurring phytoalexin produced by some spermatophytes (such as grapes and the traditional Chinese medicine Polygonum cuspidatum) that is readily available and inexpensive and presents a variety of health care functions (Frémont, 2000). In terms of its anti-tumor properties, RSV exerts anti-mutagenic and anti-inflammatory effects, can induce phase II drug-metabolizing enzymes and inhibit cyclooxygenase and catalase functions and shows good anticancer activity in the three stages of the initiation, promotion and expansion of tumorigenesis (Jang et al., 1997). Regarding the reduction of cardiovascular events, RSV acts as an active protective factor to inhibit the formation of atherosclerosis and thrombosis. RSV at a physiological concentration of 0.1 μM can relax vasodilatation, thereby reducing the incidence of cardiovascular disease (Granzotto and Zatta, 2014). RSV is a natural antioxidant that regulates the activity of antioxidant enzymes by inhibiting the production of free radicals and can significantly inhibit the autooxidation hemolysis of rat red blood cells and the oxidation hemolysis caused by hydrogen peroxide (Mendes et al., 2012). We screened targeted compounds that can inhibit VHH-mediated hemolysis and found that the polyphenolic compound RSV significantly inhibited the hemolytic activity of VHH. In this research, V. harveyi and RSV were used as experimental materials to reveal the mechanism by which RSV inhibits the virulence of V. harveyi and to evaluate the therapeutic effect.
2.2. Preparation of the VHH protein The vhh gene fragment was obtained by PCR (primer sequences are shown in Table 1) and inserted in the pET28a(+) vector, which was then transformed into BL21(DE3) cells, and the vhh-expressing strain pET28a(+)-vhh-BL21(DE3) was obtained. The strain was induced with IPTG and lysed by sonication to obtain inclusion bodies. After a series of purification, renaturation and concentration steps, the recombinant VHH protein was finally obtained. 2.3. Homology modeling and docking In this study, the three-dimensional structure of VHH (GenBank: AAG25957.1) was modeled using the homology model of Modeller 9.18 (Webb and Sali, 2014), and the molecular model of RSV (CAS: 501–360) was constructed using the Gaussian 03 program. The interaction between RSV and VHH receptors was analyzed by molecular docking analysis. Briefly, Modeller 9.18 was first used to search for proteins that are homologous to VHH in the Protein Data Bank (PDB) by superimposing the structure of homologous proteins to determine their structurally conserved regions (SCRs) and LOOP. Second, the model protein was compared with the homologous protein to acquire the SCR and LOOP of the model protein, and the structure of other parts of the model protein was predicted simultaneously. Finally, the flexible parts of VHH and RSV were determined using AutoDock Tools software, and the docking of the protein receptors and ligands was performed. Binding energy was calculated, and the binding sites were classified, with locking into several of the most likely binding sites eventually being observed.
2. Materials and methods 2.1. Materials The V. harveyi strain (NO. 2HWH020) used in the experiment was isolated from cultured fugu in the early stage of the disease at Dalian Tianzheng Industrial Co., Ltd. and showed strong virulence. Stock cultures were maintained at −80 °C in a suspension of nutrient broth (NB, Qingdao Hope Biotech) with 15‰ salinity and 20% glycerol. When required, the isolate was grown on nutrient agar (NA, Qingdao
2.4. Site-directed mutagenesis The L247A and Y368A mutations were introduced into pET28a 2
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corresponding well and cocultured with the cells. After 12 h of incubation, the supernatants from each well were collected for cell viability analysis using the Cell Counting Kit (CCK, TransGen Biotech) according to the manufacturer's directions. To determine the effects of RSV on VHH-triggered cell damage, the cells were cultured in the same manner described above. 2 μL of purified protein (10 μg/mL) was mixed with RSV at different concentrations in advance, and the mixture was placed at 28 °C for 30 min, after which it was added to the correct wells. At the indicated time points, cell viability was detected via the CCK assay at OD450nm.
(+)-vhh by PCR using the Fast Mutagenesis System (TransGen Biotech). The primers used to induce the point mutations are listed in Table 1. The DMT enzyme was added to the PCR product, followed by incubation at 37 °C for 1 h to digest the methylated plasmid template. A 2 μL aliquot of the digestion products was transformed into 50 μL of DMT competent cells, and all mutations were verified by DNA sequencing. The preparation and purification of the mutant proteins were identical to the procedure described in sections “2. vhh gene cloning and prokaryotic expression” and “3. Protein affinity”. 2.5. Hemolysis assay
2.9. Tissue distribution of RSV in T. rubripes The effect of RSV on hemolysis induced by VHH or its mutants was evaluated as described previously with slight modifications (Guo et al., 2020). Briefly, 2 μL of purified proteins (40 ng/mL) was mixed with different doses of RSV and 0.01 M PBS (pH = 7.4) in a total volume of 485 μL. The mixture was incubated at 28 °C for 30 min before the addition of defibrinated erythrocytes at a total volume of 3%. After 4 h, the reaction mixture was centrifuged (1290 g, 1 min) to remove the erythrocytes. Hemolytic activity was determined by measuring the OD543nm of the cell-free supernatant. Samples treated with 1% Triton X100 (Solarbio) were set as the 100% lysis controls. Hemolysis was expressed as the ratio of the OD543nm of each sample to the complete lysis controls.
To explore the distribution of RSV in the fugu liver and muscle, we first drew the standard curve of RSV. The RSV stock solution was diluted with PBS to produce a series of standard solutions (100, 150, and 200 μg/mL). Each RSV standard solution and acetonitrile (Solarbio) were mixed evenly at a ratio of 1:2, followed by centrifugation at 20700 g for 10 min. The supernatant was filtered through a 0.22 μm filtration membrane and injected into a high-performance liquid chromatography (HPLC) system equipped with a C18 column (4.6 mm × 250 mm, 5 μm; Thermo Scientific) and a UV detector set at 303 nm. The mobile phase of 30% acetonitrile and 70% acidified water (0.2%(v/v) phosphate) was run at a flow rate of 1 mL/min with an operating temperature of 25 °C. The area under the time concentration curve (AUC) of each sample was recorded, and the standard curve of the AUC-concentration was drawn. Next, the T. rubripes were administered intramuscularly with 50 μL of RSV at a dose of 100 mg/kg. The T. rubripes were sacrificed at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 8 and 12 h after drug administration. Their livers and muscles were promptly removed, and physiological saline was added to prepare a homogenate. HPLC was carried out as described above. The AUC was taken into the standard line, and the corresponding drug concentration was calculated.
2.6. Minimum inhibitory concentration assay The minimum inhibitory concentration (MIC) of RSV against V. harveyi was determined by the broth dilution method recommended by NCCLS (Bruck, 1980). A single colony of pure V. harveyi was selected and inoculated in fresh NB with 15‰ salinity, then cultured at 28 °C until reaching an OD600nm = 0.1. RSV stock solution was added to the culture medium to a final concentration of 2048 μg/mL, which was then continuously diluted to 1 μg/mL via a two-fold dilution method. Then, the culture was continued for 12 h. Thereafter, the effect of RSV on the growth of V. harveyi was evaluated by measuring the OD600nm value. For the positive control, no RSV was added, and for the negative control, no bacteria were included.
2.10. Animal experiments To assess the toxicity and treatment effects of RSV in T. rubripes fry, we conducted animal infection experiments. Briefly, 120 fry were evenly distributed in 12 tanks sterilized with potassium permanganate and divided into 4 groups: V. harveyi (infection group), RSV + V. harveyi (drug treatment group), RSV alone (drug toxicity group) and no treatment (control group). Infection was carried out by immersion. T. rubripes were immersed for 8 h in filtered sea water inoculated with a fresh bacterial suspension at a final concentration of 5 × 107 CFU/mL, and the challenged fish were transferred to a new site. The fry were administered RSV intramuscularly at 100 mg/kg postinfection (50 μL per fish), and the same doses were administered at 12-h intervals. The mortality of the fish was monitored for 7 d consecutively. The gills and kidneys were prepared for histopathological analysis. Briefly, the samples were fixed in 10% formalin for 24 h, then washed overnight with running water, dehydrated in graded ethanol solutions and fixed in paraffin wax. The tissues were cut to a thickness of 5 μm and stained with hematoxylin and eosin. The histologic sections were observed under a light microscope, and digital images of the histological features were obtained using a Leica microscope imaging system (Leica DM4000, Germany).
2.7. qRT-PCR V. harveyi was cultured in NB (15‰ salinity) with or without various doses of RSV (final concentrations of 16, 8, 4, 2, and 1 μg/mL) to the exponential growth phase (OD600nm = 1.0). The bacterial cells were then collected (4 °C, 1290 g, 5 min), and total RNA was extracted using the RNA Fast 200 kit (Fastagen Biotech). The concentration of the purified RNA was determined using a NanoDrop 2000c UV/Vis spectrophotometer (Thermo Scientific). The quality of the purified RNA was determined by electrophoresis on a 1% agarose gel. The primer pairs used in qRT-PCR are listed in Table 1. RNA was reverse transcribed into cDNA using the Prime Script RT reagent kit (Takara). PCR was performed using a StepOnePlus Real-Time PCR instrument (Applied Biosystems) in a 20 μL volume containing Mon Amp ChemoHS qPCR Mix (Monad) as recommended by the manufacturer. All samples were analyzed in triplicate, and the housekeeping gene toxR (Pang et al., 2006; Torky et al., 2016) was used as an endogenous control. In this study, the expression of the target gene relative to toxR was used to determine the differences in transcription levels between samples.
2.11. Statistical analysis
2.8. Cytotoxicity test and cell damage inhibition test
Statistical analysis and p value calculation were conducted using SPSS 19.0 statistical software. All experimental group data were compared with the corresponding control group data by t-tests. A p value of less than 0.05 was considered to indicate a significant difference, and the difference was considered extremely significant when the p value was less than 0.01. Three parallel plots were generated for all samples
For the detection of RSV cytotoxicity, after digestion, striped-fin loach epithelial cells that had grown to a suitable density were inoculated into 96-well plates at 2 × 104 cells per well in FBS-free medium overnight. RSV with gradient concentrations was added to the 3
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we successfully mutated these residues into Ala residues and performed a hemolysis assay to verify their function and investigated the effect of RSV on the hemolytic activity of wild-type VHH and its mutants. When there was no RSV in the mixed system, the hemolytic activities of WTVHH, L247A and Y368A were almost the same; with an increase in the RSV concentration, the hemolytic activities of all three showed decreases to different degrees, among which the inhibitory effect on VHH was the most obvious. When the concentration was 0.2 μg/mL, the inhibitory effect of RSV on VHH hemolytic activity was extremely significant (p < .01) and continued to increase. At a concentration of 8 μg/mL, the inhibitory effect reached a maximum. Thereafter, the hemoglobin release rate rebounded slightly. Compared with VHH, RSV showed weak inhibition of L247A and Y368A, as the inclusion of 8 μg/ mL of this compound in the reactions resulted in a hemolysis inhibition rate of less than 50% (Fig. 2). These results suggested that Leu247 and Tyr368 were indeed the key sites of RSV binding to VHH.
and were plotted using the mean and standard deviation. 3. Results 3.1. Cloning of the vhh gene and preparation of the protein The vhh gene (GenBank No. MN296414) was amplified by using V. harveyi DNA as a template and vhh-F/R as a primer. The fragment size was 1257 bp, and the sequence was 99% consistent with that of the vhhA gene (GenBank No. AF293430) according to BLAST alignment. pET28a(+)-vhh-BL21 (DE3) was induced by IPTG, sonicated and purified to obtain a VHH recombinant protein with a molecular weight of 47.38 kDa, and the protein existed in the form of inclusion bodies. After renaturation and concentration, the protein could be used in the following experiments. 3.2. Leu247 and Tyr368 are the key sites for the binding of RSV to VHH
3.4. RSV has no effect on the growth of V. harveyi and inhibits vhh transcription by promoting luxS transcription
Modeller 9.18 was used to search the full homologous sequence template of the VHH protein, and active site analysis of the modeled structure was performed. We found that PDB: 6JL2 (Wan et al., 2019) showed the highest degree of matching with the target protein, and the similarity between the amino acid sequences of the catalytic center and VHH was greater than 80%. When this structure was used as a template, the catalytic triad model of Asp390-His393-Ser153 could be obtained with an appropriate location. Therefore, it was used as the template to model the 3D structure of the target protein, and the RSV ligand was molecularly docked with the 3D structure of VHH. The docking energy of RSV and the VHH protein was −6.0 Kcal/ mol. RSV was integrated into the cavity composed of the triplet catalytic center and the oxygen anion hole. In this structure, the metaphenolic hydroxyl group formed a hydrogen bond with Tyr368, and the p-hydroxybenzene ring formed a π-alkyl interaction with Leu247 (Fig. 1). We speculate that RSV inhibited the hemolytic activity of VHH, possibly by acting directly on VHH and inactivating its biological activity.
As shown in Fig. 3A, when the concentration was 32 μg/mL, RSV showed an inhibitory effect on the growth of V. harveyi, and the antibacterial effect was enhanced with an increasing drug concentration. The MIC of RSV was 64 μg/mL, and a concentration of 128 μg/mL was completely bacteriostatic, indicating that RSV has an antibacterial effect at higher concentrations in vitro. To eliminate the interference caused by RSV inhibiting the growth of V. harveyi, we selected the concentration range of 0–16 μg/mL for qRT-PCR experiments to explore the influence of RSV on the transcription level of vhh. The mRNA level of vhh was investigated by qRT-PCR. Previous studies have shown that one of the signal molecules of the V. harveyi density sensing system, AI-2, whose precursor is synthesized by LuxS, plays a negative role in the transcription of vhh (Bai and Zhang, 2010). Therefore, we also detected the transcription of luxS after the RSV treatment of V. harveyi. As the dose of RSV was increased, the transcription level of luxS was increased, and vhh transcription was downregulated in a dose-dependent manner. When V. harveyi was cocultured with RSV at 1, 2, 4, 8 or 16 μg/mL, the transcription level of vhh was reduced 1.15, 1.42, 1.75, 2.29 or 2.67 times, respectively, compared with that in V. harveyi without RSV treatment (Fig. 3B).
3.3. Effect of RSV on the hemolytic activity of the WT-VHH protein and mutant proteins The results of molecular docking showed that Leu247 and Tyr368 played an important role in the binding of VHH to RSV. Subsequently,
Fig. 1. 3D and 2D interaction diagrams of RSV and VHH. The light green stick is RSV, the yellow stick is the amino acid residue of the catalytic center of triplet and oxygen anion hole, and the dark green stick is the amino acid residue involved in the binding. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 4
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Fig. 2. Effect of RSV on hemolytic activity of WT-VHH protein and mutant proteins. The inhibition of RSV on VHH hemolytic activity increased with the increase of RSV concentration, but the effect on the two mutated proteins was not so obvious. N, negative control; P, positive control.
in the liver. In addition, the half-lives of the elimination (t1/2) of RSV in the muscle and liver were 1.25 h and 1.5 h, respectively, and RSV was completely metabolized 4 h after injection. The concentration of RSV in tissues exceeded the minimum effective concentration for the inhibition of hemolysis and cell damage induced by VHH in vitro, so it was feasible to conduct therapeutic experiments at this dose. No deaths occurred during the entire experiment. The infected T. rubripes were accompanied by clinical symptoms such as swimming slowly and loss of appetite. After dissection, it could be observed that the gills, liver and kidneys presented different degrees of bleeding and swelling, and showed severe enteritis. All of the above symptoms were alleviated after treatment with RSV. Since the gill is the first site where the bacteria invade the host and the kidney is an important immune organ of fish, we obtained paraffin sections of these two tissues to further study the pathological changes before and after treatment. In the infected group, edema occurred in the gill filaments, and the gill lamella was expanded, congested and deformed. The renal interstitium became loose, and the original normal structure was destroyed. The glomeruli were atrophied, and the basilar membrane of the renal tubules exhibited edema. In the RSV-treated group, this pathology was discernibly alleviated, with only mild edema and renal interstitial hemorrhage being observed. In the drug toxicity group, no obvious lesions were observed in the gill, and the epithelial cells of the renal tubule showed mild granular degeneration (Fig. 4C). Overall, RSV can alleviate the histopathological damage caused by V. harveyi and has the potential to be used as a new anti-infection drug.
3.5. RSV has a protective effect against VHH-mediated cell damage Tetrazolium salt in CCK solution can be reduced to orange formazan by dehydrogenase, and the color depth shows a good linear relationship with the number of living cells. Therefore, we evaluated the drug toxicity of RSV and its inhibitory effect on VHH-mediated cell damage by measuring the light absorption value at 450 nm. As shown in Fig. 3C, RSV at 32 μg/mL exerted a certain degree of cytotoxicity (p < .05), resulting in 47% cell death. Prior to the application of this concentration, there was some cell death, but the level was not significant, so RSV could be considered safe. When 0.5 μg/mL RSV was added to the VHHcell coculture system, a significant protective effect was observed (p < .05), and the cell survival rate reached a maximum when the concentration was 2 μg/mL, which was 87.34% (Fig. 3D). Although the survival rate decreased as the concentration of RSV increased thereafter, it was still higher than the overall level in the RSV-free group, indicating that RSV could effectively alleviate the cell damage caused by VHH.
3.6. RSV protects T. rubripes from lesions caused by V. harveyi infection The standard curve of the concentration-AUC of RSV was drawn (Fig. 4A) and indicated that the concentration of RSV showed a good linear relationship with the AUC within the range of 0–200 μg/mL. After injection into the T. rubripes, RSV was rapidly distributed in the muscles and liver. From the drug concentration-time in the tissue curve (Fig. 4B), when the drug was administered at a concentration of 100 mg/kg, RSV reached a peak concentration (Cmax) of 64 mg/kg in the muscle at 1 h after injection. However, the peak time (tmax) in the liver occurred 0.25 h earlier, and the Cmax was only 42 mg/kg, indicating that the bioavailability of RSV in muscle was higher than that
4. Discussion As one of the core species of Vibrio, V. harveyi is a conditionally pathogenic bacteria (Austin and Zhang, 2010). When the host organism 5
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Fig. 3. RSV inhibits vhh transcription and VHH-mediated cell damage. A, RSV did not inhibit the extracellular growth of V. harveyi. At low concentrations, RSV had no bacteriostatic effect. However, with the increase in the concentration, the bacteriostatic effect was enhanced, and it was completely bacteriostatic at 128 μg/mL. The OD600nm value was used to determine the concentration of V. harveyi suspension after cultured for 12 h. B, The effects of different concentrations of RSV on gene expression in V. harveyi. With the increase in RSV concentration, the transcription levels of luxS was up-regulated and that was down-regulated of vhh. C, Cytotoxicity of RSV. D, RSV inhibited VHH cell damage. The inhibitory effect was greatest at RSV concentration of 2 μg/mL, although there was a decrease later, the overall survival rate was higher than that of the control group.
et al., 2007). Although these toxins are generally referred to as hemolysins because of their capacity to lyse erythrocytes, they exhibit two different modes of action, involving either a pore-forming protein or a phospholipase enzyme, and VHH belongs to the second group (Arachchige et al., 2012; Rowe and Welch, 1994). Studies by Bai et al. (Fangfang et al., 2010) have shown that VHH has an amphiphilic structure and can induce tubular protrusion formation in the erythrocyte membrane in a short time. After coculture with erythrocytes, VHH was localized by the immunogold staining technique, and it was found that the gold particles were mainly deposited on the membrane, indicating that VHH relies on its phospholipase activity to destroy the cell membrane. However, a small amount of gold particles was deposited inside the cells, suggesting that the cell membrane was not the only target and that VHH may present enzyme activity other than phospholipase activity (Fangfang et al., 2010). VHH belongs to the GDSL hydrolytic enzyme family and is characterized by flexible active sites that bind to multiple substrates by altering conformation (Akoh et al., 2004). Therefore, VHH may also exhibit a flexible exchange site that can be combined with a variety of targets. In addition to dissolving cell membranes, VHH activates caspase-3 and induces apoptosis (Fangfang et al., 2010). In the past, the widespread and frequent use of antibiotics in aquaculture has increased the selective pressure exerted on microorganisms, encouraged the natural emergence of bacterial resistance, and enabled horizontal transfer of resistant genes across organisms through mobile genetic factors such as plasmids, transposons, phages, and integrons (You et al., 2016; Zhu et al., 2006). Reports of multidrug
is immune suppressed or otherwise physiologically stressed, large-scale outbreaks will occur, with the frequency of infection often attributable to intensive culture and adverse environmental conditions (Defoirdt et al., 2007). With the contributions of phospholipase C, hemolysin, lipopolysaccharide, adhesion factor and other virulence factors (Zhang et al., 2007), V. harveyi can cause serious infections in important economic species such as shellfish (Wang et al., 2017), shrimp (Ting et al., 2015), T. rubripes and orange-spotted grouper (Hai et al., 2017). Comparative genomic analysis has revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by deletion and gene acquisitions through duplication or horizontal transfer (Ruwandeepika et al., 2010b). To cope with its complex environment, V. harveyi is very likely to obtain virulence genes from other Vibrio species in the aquatic environment through horizontal gene transfer, thereby increasing the virulence of bacteria to specific hosts and effectively improving their ability to infect aquatic organisms (Ruwandeepika et al., 2010b). In this context, some V. harveyi strains have been transformed from secondary pathogens to a primary pathogens through increases in their virulence (Zhou et al., 2012). In addition, through long-term evolution, V. harveyi has attained strong resistance to the killing effect of host serum by interfering with the P38 MAPK pathway, thereby blocking the phagocytosis of blood cells (Li et al., 2011). V. harveyi is becoming more virulent and harmful, but researchers have unfortunately not yet sufficiently elucidated its pathogenesis. Hemolysin is arguably the most widely distributed exotoxin among pathogenic vibrios and is involved in disease pathogenesis (Boguang 6
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Fig. 4. RSV protects T. rubripes from lesions caused by V. harveyi infection. A, The standard curve of RSV. B, Tissue distribution of RSV in T. rubripes. After inoculation at the concentration of 100 mg/kg, RSV spread rapidly in T. rubripes, reached Cmax in muscle and liver about 1 h, and was completely metabolized at 4 h. C, RSVtreated T. rubripes exhibited less pathological abnormalities in gills and kidneys. After treatment of RSV, pathology was discernibly alleviated, with only mild edema and renal interstitial hemorrhage.
role of the two residues in the binding process. Furthermore, RSV downregulated the transcription of vhh and inhibited the toxicity of VHH to fin epithelial cells in a concentration range that did not affect the growth of V. harveyi (i.e., drug resistance was not easy to achieve). Finally, RSV showed a satisfactory therapeutic effect on tissue lesions caused by V. harveyi infection. RSV was inoculated into the body and spread rapidly, reaching the Cmax in the muscles and liver within approximately 1 h. After RSV treatment, the gills and kidneys of the infected fish showed significant improvement. From the pathological sections, we could see that the organ tissues of the drug toxicity group showed mild kidney injury, similar to the situation reported previously (Cottart et al., 2010; Crowell et al., 2004), which may be due to the toxicity caused by drug accumulation. In the process of the experiment, RSV accumulated to a high dose, resulting in toxic side effects; in practical applications, the dose could be reduced so that the toxicity is weakened or may not even manifest. In future studies, researchers could improve the functional groups of RSV by chemical modification and reduce its toxicity. In summary, our results have demonstrated the effectiveness of this natural compound in inhibiting the cytotoxicity of VHH, which should pave the way for future structural modification of RSV or the generation of RSV derivatives to obtain molecules showing higher potency against VHH and a protective effect, leading to the development of effective and clinically useful VHH-specific therapeutics.
resistance in V. harveyi are common and can be traced back to the last century. In the case described by Karunsagar et al., mass mortality in black tiger shrimp larvae was caused by V. harveyi with multiple resistance to chloramphenicol, erythromycin, cotrimoxazole and streptomycin (Karunasagar et al., 1994). In response to this situation, a variety of new treatments have emerged. Anti-virulence factor therapy is considered one of the promising effective strategies for meeting the challenge posed by widespread drug resistance among bacterial pathogens and could reduce the virulence of pathogens by interfering with the regulation of virulence factor expression or specifically inhibiting a virulence factor (Tom et al., 2011). Compared with vaccines, phage therapy, probiotics and other antibiotic replacement therapy, virulence factor inhibitors are generally small molecular compounds that exist naturally, which are widely available and inexpensive. In the process of use, it can be fed through mixed feeding. In addition, V. harveyi can also infect some invertebrates, for which virulence factor inhibitors are more feasible than others. Here, we provide evidence that RSV is one such agent with the potential to treat infections caused by V. harveyi by specifically antagonizing the activity of VHH. RSV effectively inhibited hemolysis induced by VHH. Molecular docking is increasingly considered as a tool for lead discovery, as the structures of an increasing number of proteins and nucleic acids become available (Shoichet et al., 2002). We obtained the binding sites of RSV and VHH through this technique and selected Leu247 and Tyr368 as the targets for site-directed mutagenesis. The results of the hemolysis assay showed that after the mutation of Leu247 and Tyr368 into Ala, the inhibitory effect of RSV on the hemolytic activity of the mutant protein decreased significantly, revealing the key
Ethical statement In this research, all experimental animals were used according to 7
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the ethical requirements, and experimental procedures were following the guidelines for the Care and Use of Laboratory Animals in China.
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Data availability statement The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Funding information National Key R&D Program of China (2017YFD0701701). Natural Science Foundation of Liaoning Province of China (2019MS-031). Declaration of Competing Interest No conflict of interest was existed. References Akoh, C.C., Lee, G.C., Liaw, Y.C., Huang, T.H., Shaw, J.F., 2004. GDSL family of serine esterases/lipases. Prog. Lipid Res. 43, 534–552. Arachchige, H., Ruwandeepika, D., Sanjeewa, T., Jayaweera, P., Bhowmick, P.P., Karunasagar, I., Bossier, P., Defoirdt, T., 2012. Pathogenesis, virulence factors and virulence regulation of vibrios belonging to the Harveyi clade. Rev. Aquac. 4, 59–74. Austin, B., Zhang, X., 2010. Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Lett. Appl. Microbiol. 43, 119–124. Bai, F.-F., Zhang, X.-H., 2010. Studies on the effect of quorum sensing systems of Vibrio harveyi on the activities of extracellular enzymes. Period. Ocean Univ. China 40, 91–95. Boguang, S., Xiao-Hua, Z., Xuexi, T., Shushan, W., Yingbin, Z., Jixiang, C., Brian, A., 2007. A single residue change in Vibrio harveyi hemolysin results in the loss of phospholipase and hemolytic activities and pathogenicity for turbot (Scophthalmus maximus). J. Bacteriol. 189, 2575. Bruck, E., 1980. National Committee for clinical laboratory standards. Pediatrics 65, 187. Cottart, C.H., Nivet-Antoine, V., Laguillier-Morizot, C., Beaudeux, J.L., 2010. Resveratrol bioavailability and toxicity in humans. Mol. Nutr. Food Res. 54, 7–16. Crowell, J.A., Korytko, P.J., Morrissey, R.L., Booth, T.D., Levine, B.S., 2004. Resveratrolassociated renal toxicity. Toxicol. Sci. 82, 614. Defoirdt, T., Boon, N., Sorgeloos, P., Verstraete, W., Bossier, P., 2007. Alternatives to antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as an example. Trends Biotechnol. 25, 472–479. Fangfang, B., Boguang, S., Woo, N.Y.S., Xiao-Hua, Z., 2010. Vibrio harveyi hemolysin induces ultrastructural changes and apoptosis in flounder (Paralichthys olivaceus) cells. Biochem. Biophys. Res. Commun. 395, 70–75. Frémont, L., 2000. Biological effects of resveratrol. Life Sci. 66, 663–673. Granzotto, A., Zatta, P., 2014. Resveratrol and Alzheimer’s disease: message in a bottle on red wine and cognition. Front. Aging Neurosci. 6, 95. Guo, Y., Xing, Z., He, Z., Zhao, X., Ye, S., 2020. Honokiol inhibits Vibrio harveyi hemolysin virulence by reducing its haemolytic activity. Aquac. Res. 51 (1), 206–214. Hai, T.N., Nguyen, T.T.T., Tsai, M.A., Ya-Zhen, E., Wang, P.C., Chen, S.C., 2017. A formalin-inactivated vaccine provides good protection against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides). Fish Shellfish Immunol. 65, 118–126. Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas, C.F., Beecher, C.W., Fong, H.H., Farnsworth, N.R., Kinghorn, A.D., Mehta, R.G., 1997. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275, 218–220. Karunasagar, I., Pai, R., Malathi, G.R., Karunasagar, I., 1994. Mass mortality of Penaeus monodon larvae due to antibiotic-resistant Vibrio harveyi infection. Aquaculture 128, 203–209. Koizumi, K., Hiratsuka, S., 2009. Fatty acid compositions in muscles of wild and cultured ocellate puffer Takifugu rubripes. Fish. Sci. 75, 1323. Kyoungmi, W., Kihong, K., Sooil, P., 2010. Vibrio harveyi associated with deep skin ulcer in river puffer, Takifugu obscures, exhibited in a public aquarium. Aquac. Res. 40,
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