Natural Inhibitors Targeting the Localization of Lipoprotein System in Vibrio parahaemolyticus

The localization of lipoprotein (Lol) system is responsible for the transport of lipoproteins in the outer membrane (OM) of Vibrio parahaemolyticus. LolB catalyzes the last step in the Lol system, where lipoproteins are inserted into the OM. If the function of LolB is impeded, growth of V. parahaemolyticus is inhibited, due to lack of an intact OM barrier for protection against the external environment. Additionally, it becomes progressively harder to generate antimicrobial resistance (AMR). In this study, LolB was employed as the receptor for a high-throughput virtual screening from a natural compounds database. Compounds with higher glide score were selected for an inhibition assay against V. parahaemolyticus. It was found that procyanidin, stevioside, troxerutin and rutin had both exciting binding affinity with LolB in the micromolar range and preferable antibacterial activity in a concentration-dependent manner. The inhibition rates of 100 ppm were 87.89%, 86.2%, 91.39% and 83.71%, respectively. The bacteriostatic mechanisms of the four active compounds were explored further via fluorescence spectroscopy and molecular docking, illustrating that each molecule formed a stable complex with LolB via hydrogen bonds and pi–pi stacking interactions. Additionally, the critical sites for interaction with V. parahaemolyticus LolB, Tyr108 and Gln68, were also illustrated. This paper demonstrates the inhibition of LolB, thus, leading to antibacterial activity, and identifies LolB as a promising drug target for the first time. These compounds could be the basis for potential antibacterial agents against V. parahaemolyticus.


Introduction
Vibrio parahaemolyticus is an important foodborne pathogen widely distributed in aquatic products, and marine and estuarine environments [1]. It not only infects the Penaeus vannamei juvenile, leading to acute hepatopancreatic necrosis disease (AHPND) and serious economic losses [2,3], but also causes serious foodborne diseases, such as diarrhea and sepsis [4], which pose a great threat to human health and food safety. Meanwhile, the antimicrobial resistance (AMR) of bacteria, including V. parahaemolyticus, is severely increasing, aggravating the negative effect of bacteria on global health and the economy. If we do not suppress their development, drug-resistant superbugs will claim 10 million lives a year and cost the global economy a cumulative $100 trillion by 2050 [5].
Virtual screening (VS) is the use of in silico techniques to screen active compounds, based on the compound database [16]. Using the molecular docking operation between compounds and drug targets, VS quickly selected active compounds with potential to be drugs from dozens to millions of molecules, greatly reducing the number of compounds for experimental screening, shortening the research cycle and decreasing the cost of drug development [17,18]. Therefore, VS has become one of the most promising tools for drug development.
The homology model of V. parahaemolyticus LolB protein used Escherichia coli (strain K12) LolB protein (PDB: 1iwm) [19] with 35% identity as template, where residues 36-211 of V. parahaemolyticus LolB were modeled successfully ( Figure 1A). The model was aligned with E. coli LolB crystal structure and full-length V. parahaemolyticus LolB predicted by Alphafold, which showed high structural similarity ( Figure 1B,C). In the established LolB model, as shown in the Ramachandran plot ( Figure S1), 89.8% of residues were located in the completely conformation-allowed region, 10.2% in the conformation-allowed region, and no residue in conformational-disallowed region, indicating that these residues had good conformational space. The LolB model was of ideal quality for subsequent virtual screening.
Based on the screening of 39,000 molecules in the Cayman/2D structure database, 70 compounds that bind to the LolB protein were screened ( Figure S2), and Table 1 lists the top 10 compounds with good glide scores to the target protein. With respect to the glide score, the lower score represents higher affinity with the receptor. The screened top 10 compounds had glide scores of −12.023~−7.781, lower than −6, demonstrating superior binding capability targeting V. parahaemolyticus LolB.

Inhibition Effect of the Top 10 Compounds on V. parahaemolyticus
The listed top 10 compounds were applied to analyze their antibacterial effects on V. parahaemolyticus RIMD 2210633 by measuring the inhibition rates under a concentration gradient of 100-10 ppm ( Figure 2). The results showed that these compounds had concentration-dependent bactericidal performances, among which procyanidin, stevioside, troxerutin and rutin presented relatively ideal inhibitory effects on V. parahaemolyticus, with inhibition rates of 100 ppm of 87.89%, 86.2%, 91.39% and 83.71%, respectively.   The listed top 10 compounds were applied to analyze their antibacterial effects on V. parahaemolyticus RIMD 2210633 by measuring the inhibition rates under a concentration gradient of 100-10 ppm ( Figure 2). The results showed that these compounds had concentration-dependent bactericidal performances, among which procyanidin, stevioside, troxerutin and rutin presented relatively ideal inhibitory effects on V. parahaemolyticus, with inhibition rates of 100 ppm of 87.89%, 86.2%, 91.39% and 83.71%, respectively.

Fluorescence Spectroscopy of Active Compounds with LolB
LolB protein produces endogenous fluorescence under UV irradiation since it contains three aromatic amino acid residues, namely, tryptophan, tyrosine, and phenylalanine. When protein reacts with substances, the fluorescence intensity of fluorophore decreases, known as fluorescence quenching [20]. Therefore, fluorescence spectroscopy is widely applied for studying the interaction between proteins and inhibitors. In order to verify whether the reason why procyanidin, stevioside, troxerutin and rutin inhibited the activity of V. parahaemolyticus was the targeting effect on LolB protein, the fluorescence spectra of the interactions between the four active compounds and the isolated LolB protein were further measured, respectively.

Inhibition Effect of the Top 10 Compounds on V. parahaemolyticus
The listed top 10 compounds were applied to analyze their antibacterial effects on V. parahaemolyticus RIMD 2210633 by measuring the inhibition rates under a concentration gradient of 100-10 ppm (Figure 2). The results showed that these compounds had concentration-dependent bactericidal performances, among which procyanidin, stevioside, troxerutin and rutin presented relatively ideal inhibitory effects on V. parahaemolyticus, with inhibition rates of 100 ppm of 87.89%, 86.2%, 91.39% and 83.71%, respectively.  As shown in Figure 3, the fluorescence emission peak emerged at 334 nm when the excitation wavelength was 280 nm, and the fluorescence intensity of LolB protein without quencher was 2166 a. u. The addition of procyanidin, stevioside, troxerutin and rutin significantly decreased the fluorescence intensity of LolB protein in a concentration-dependent manner from~2000 a. u. at 0 µM to~500 a. u. at 25 µM of active compounds. This suggested that the four molecules interacted with the LolB protein, resulting in fluorescence quenching of the LolB protein. Furthermore, with the increase of the concentration of active compounds, a blue shift of the fluorescence emission peak was observed in all fluorescence spectra (Figure 3), which implied that the interactions between LolB and the compounds possibly changed the microenvironment around LolB amino acid residues, so that the hydrophobicity of LolB increased and the polarity decreased. Together, fluorescence quenching and blue shift demonstrated that procyanidin, stevioside, troxerutin and rutin bound to LolB.

Fluorescence Spectroscopy of Active Compounds with LolB
LolB protein produces endogenous fluorescence under UV irradiation since it contains three aromatic amino acid residues, namely, tryptophan, tyrosine, and phenylalanine. When protein reacts with substances, the fluorescence intensity of fluorophore decreases, known as fluorescence quenching [20]. Therefore, fluorescence spectroscopy is widely applied for studying the interaction between proteins and inhibitors. In order to verify whether the reason why procyanidin, stevioside, troxerutin and rutin inhibited the activity of V. parahaemolyticus was the targeting effect on LolB protein, the fluorescence spectra of the interactions between the four active compounds and the isolated LolB protein were further measured, respectively.
As shown in Figure 3, the fluorescence emission peak emerged at 334 nm when the excitation wavelength was 280 nm, and the fluorescence intensity of LolB protein without quencher was 2166 a. u. The addition of procyanidin, stevioside, troxerutin and rutin significantly decreased the fluorescence intensity of LolB protein in a concentration-dependent manner from ~2000 a. u. at 0 μM to ~500 a. u. at 25 μM of active compounds. This suggested that the four molecules interacted with the LolB protein, resulting in fluorescence quenching of the LolB protein. Furthermore, with the increase of the concentration of active compounds, a blue shift of the fluorescence emission peak was observed in all fluorescence spectra (Figure 3), which implied that the interactions between LolB and the compounds possibly changed the microenvironment around LolB amino acid residues, so that the hydrophobicity of LolB increased and the polarity decreased. Together, fluorescence quenching and blue shift demonstrated that procyanidin, stevioside, troxerutin and rutin bound to LolB.

Fluorescence Quenching Mechanism of Active Compounds on LolB
To explore the interaction mechanism between the inhibitors and the LolB protein, the Stern-Volmer equation was used to determine the type of fluorescence quenching. Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorophore and quencher, or static, resulting from the formation of a ground-state complex between the fluorophore and quencher [20]. Figure 4A shows the Stern-Volmer plots of the interactions between the different compounds and LolB, which presented a good linear relationship. The K q values of procyanidin, stevioside, troxerutin and rutin inhibiting LolB were 8.3054 × 10 12 L/mol·s, 6.1570 × 10 12 L/mol·s, 14.2317 × 10 12 L/mol·s and 8.7014 × 10 12 L/mol·s, respectively ( Table 2). These values were much greater than the maximum dynamic quenching rate (2.0 × 10 10 L/mol·s) [21], confirming that these molecules combined with LolB to form complexes, producing static quenching. According to the One Site-Specific Binding model, the binding constant K d was analyzed to determine the affinity of inhibitors with LolB ( Figure 4B). The four K d values were in the micromolar range, especially that of procyanidin, reaching 14.68 ± 4.65 µM, indicative of strong binding effects with LolB protein and stable complex formations.
To explore the interaction mechanism between the inhibitors and the LolB protein, the Stern-Volmer equation was used to determine the type of fluorescence quenching. Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorophore and quencher, or static, resulting from the formation of a ground-state complex between the fluorophore and quencher [20]. Figure 4A shows the Stern-Volmer plots of the interactions between the different compounds and LolB, which presented a good linear relationship. The Kq values of procyanidin, stevioside, troxerutin and rutin inhibiting LolB were 8.3054 × 10 12 L/mol‧s, 6.1570 × 10 12 L/mol‧s, 14.2317 × 10 12 L/mol‧s and 8.7014 × 10 12 L/mol‧s, respectively (Table 2). These values were much greater than the maximum dynamic quenching rate (2.0 × 10 10 L/mol·s) [21], confirming that these molecules combined with LolB to form complexes, producing static quenching. According to the One Site-Specific Binding model, the binding constant Kd was analyzed to determine the affinity of inhibitors with LolB ( Figure 4B). The four Kd values were in the micromolar range, especially that of procyanidin, reaching 14.68 ± 4.65 μM, indicative of strong binding effects with LolB protein and stable complex formations.

Analysis of binding sites between active compounds and LolB
To investigate the key interaction sites between the four natural compounds and the LolB protein, the molecular docking was analyzed through Schrodinger's Maestro. It can be observed, from the 3D diagram of molecular docking, that these natural compounds were bound in the entrance area of the hydrophobic cavity of the critical transport lipoprotein LolB through many main chain contacts. Specifically, the hydroxyl groups and

Analysis of Binding Sites between Active Compounds and LolB
To investigate the key interaction sites between the four natural compounds and the LolB protein, the molecular docking was analyzed through Schrodinger's Maestro. Figures 5-8 describe the interactions between the individual compounds and the LolB protein, showing the docking region and the interaction between ligand and protein. It can be observed, from the 3D diagram of molecular docking, that these natural compounds were bound in the entrance area of the hydrophobic cavity of the critical transport lipoprotein LolB through many main chain contacts. Specifically, the hydroxyl groups and ether bonds of procyanidin formed hydrogen bonds with Tyr108 and Gln68 of the LolB protein, 6 of 17 respectively, and the two benzene rings of procyanidin formed pi-pi stacking interactions with Tyr108 ( Figure 5). The hydroxyl groups of stevioside formed 10 hydrogen bonds with Thr126, Asp65, Arg67, Gln68 and Ser69 ( Figure 6). The hydroxyl groups and ether bond of troxerutin interacted with Tyr123, Asp65, Ala93, Asp109 and Tyr108 of LolB protein (H bonds), and the pi-pi stacking interaction with Tyr108 was also observed (Figure 7). The hydroxyl group of rutin formed hydrogen bonds with Arg67, Gln68, Ser69, Thr126 and Tyr108, and the same pi-pi stacking interaction existed in the complex of rutin and LolB ( Figure 8). Consistent with the results of fluorescence spectra (Figure 3), the major acting force, pi-pi stacking interactions and hydrogen bonds, enhanced the hydrophobicity of the LolB protein, then, altered the environment of the amino acids at the entrance of the hydrophobic cavity, affecting the structure and function of OM transport lipoprotein LolB. Given that fluorescence quenching is commonly observed on binding ligands to binding sites, including tryptophan, tyrosine, and phenylalanine [20], the interaction of four inhibitors with LolB might indicate that procyanidin, that formed two pi-pi interactions with Tyr108, had the highest affinity, especially compared to stevioside, the compound with the highest glide score that had no pi-pi stacking. ether bonds of procyanidin formed hydrogen bonds with Tyr108 and Gln68 of the LolB protein, respectively, and the two benzene rings of procyanidin formed pi-pi stacking interactions with Tyr108 ( Figure 5). The hydroxyl groups of stevioside formed 10 hydrogen bonds with Thr126, Asp65, Arg67, Gln68 and Ser69 ( Figure 6). The hydroxyl groups and ether bond of troxerutin interacted with Tyr123, Asp65, Ala93, Asp109 and Tyr108 of LolB protein (H bonds), and the pi-pi stacking interaction with Tyr108 was also observed ( Figure 7). The hydroxyl group of rutin formed hydrogen bonds with Arg67, Gln68, Ser69, Thr126 and Tyr108, and the same pi-pi stacking interaction existed in the complex of rutin and LolB (Figure 8). Consistent with the results of fluorescence spectra (Figure 3), the major acting force, pi-pi stacking interactions and hydrogen bonds, enhanced the hydrophobicity of the LolB protein, then, altered the environment of the amino acids at the entrance of the hydrophobic cavity, affecting the structure and function of OM transport lipoprotein LolB. Given that fluorescence quenching is commonly observed on binding ligands to binding sites, including tryptophan, tyrosine, and phenylalanine [20], the interaction of four inhibitors with LolB might indicate that procyanidin, that formed two pi-pi interactions with Tyr108, had the highest affinity, especially compared to stevioside, the compound with the highest glide score that had no pi-pi stacking.

Discussion
Nowadays, the AMR of V. parahaemolyticus should not be underestimated [22,23]. Since an intact OM is required, both for viability and for resistance against antibiotics, in Gram-negative bacteria, therapeutics targeting OM biogenesis have the potential to kill the bacteria outright and sensitize them to antibiotics that are otherwise unable to penetrate an intact OM [15]. Here, we paid attention to the Lol system, the essential undertaker in transporting OM lipoproteins, and found antibacterial compounds targeting the LolB protein through VS, namely, procyanidin, stevioside, troxerutin and rutin.
OM biogenesis is involved in specific transport machines, called the Lol, BAM, and Lpt pathways, responsible for transferring lipoproteins, outer membrane proteins and lipopolysaccharide, respectively [24]. The attractiveness of OM biogenesis processes as antibacterial targets is driven, in particular, by their essentiality, conservation, and extra-cytoplasmic localization [25]. In the Lol system, previous research confirmed LolA and LolCD2E as antibacterial targets [26]. MAC13243, discovered with cell-based small-molecule screening, had a unique mechanism and promising activity against multidrug-resistant Pseudomonas aeruginosa and inhibited the function of the LolA protein ( Figure 9) [27]. Moreover, the degradation products of MAC13243 resulted in thiourea compounds, which shared a similar cellular mechanism interacting with LolA ( Figure 9) [28]. The first reported inhibitors of the LolCD2E complex were the pyridineimidazoles, shown to inhibit the LolA-dependent release of the outer membrane lipoprotein Lpp from E. coli spheroplasts [29]. A similar phenomenon was also observed with the presence of the pyrrolopy-

Discussion
Nowadays, the AMR of V. parahaemolyticus should not be underestimated [22,23]. Since an intact OM is required, both for viability and for resistance against antibiotics, in Gram-negative bacteria, therapeutics targeting OM biogenesis have the potential to kill the bacteria outright and sensitize them to antibiotics that are otherwise unable to penetrate an intact OM [15]. Here, we paid attention to the Lol system, the essential undertaker in transporting OM lipoproteins, and found antibacterial compounds targeting the LolB protein through VS, namely, procyanidin, stevioside, troxerutin and rutin.
OM biogenesis is involved in specific transport machines, called the Lol, BAM, and Lpt pathways, responsible for transferring lipoproteins, outer membrane proteins and lipopolysaccharide, respectively [24]. The attractiveness of OM biogenesis processes as antibacterial targets is driven, in particular, by their essentiality, conservation, and extracytoplasmic localization [25]. In the Lol system, previous research confirmed LolA and LolCD 2 E as antibacterial targets [26]. MAC13243, discovered with cell-based small-molecule screening, had a unique mechanism and promising activity against multidrug-resistant Pseudomonas aeruginosa and inhibited the function of the LolA protein ( Figure 9) [27]. Moreover, the degradation products of MAC13243 resulted in thiourea compounds, which shared a similar cellular mechanism interacting with LolA ( Figure 9) [28]. The first reported inhibitors of the LolCD 2 E complex were the pyridineimidazoles, shown to inhibit the LolA-dependent release of the outer membrane lipoprotein Lpp from E. coli spheroplasts [29]. A similar phenomenon was also observed with the presence of the pyrrolopyrimidinedione compound G0507, another LolCD 2 E inhibitor, which was identified in a phenotypic screen for inhibitors of E. coli lacking the tripartite efflux pump component, TolC [25]. In this paper, we screened natural inhibitors, procyanidin, stevioside, troxerutin and rutin, in inhibiting V. parahaemolyticus (Figure 2), and verified their binding with LolB through in vitro assays (Figure 3), confirming that LolB is a promising drug target. To our knowledge, this is the first demonstration of inhibition of LolB leading to antibacterial activity. phenotypic screen for inhibitors of E. coli lacking the tripartite efflux pump component, TolC [25]. In this paper, we screened natural inhibitors, procyanidin, stevioside, troxerutin and rutin, in inhibiting V. parahaemolyticus (Figure 2), and verified their binding with LolB through in vitro assays (Figure 3), confirming that LolB is a promising drug target. To our knowledge, this is the first demonstration of inhibition of LolB leading to antibacterial activity. Concerning accessibility, safety and applicability [30], we selected natural compounds as the database, then obtained four LolB inhibitors inhibiting V. parahaemolyticus (Table 1 and Figure 2). Firstly, procyanidin is a class of flavonoids from plants like grape Concerning accessibility, safety and applicability [30], we selected natural compounds as the database, then obtained four LolB inhibitors inhibiting V. parahaemolyticus (Table 1 and Figure 2). Firstly, procyanidin is a class of flavonoids from plants like grape seeds [31]. It was demonstrated that procyanidin had clear and well-defined beneficial effects against several pathologies, including cardiovascular heart disease, obesity, where it prevents weight gain and adipose tissue mass increase, and diabetes and insulin resistance [32,33]. Procyanidin also possesses the potency of free radical scavenging, antioxidant activity and cancer chemoprevention [34,35]. Recently, the potential role of procyanidin as a therapeutic agent against SARS-CoV-2 was discovered [35], indicating its promising utility. As the inhibitor of LolB protein with the best glide score in VS (Table 1), stevioside is a natural glycoside extracted from the leaves of Stevia rebaudiana [36]. It became well-known for its intense sweetness (250-300 times sweeter than sucrose) and is used as a safe, non-caloric, sweetener in food industries [37]. It is suited for both diabetics, hypertensive and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet [36]. Troxerutin, also known as vitamin P4, is a naturally occurring flavonoid which is isolated from tea, coffee and cereal grains, as well as from vegetables. It has a variety of valuable pharmacological and therapeutic activities, such as antioxidant, anti-inflammatory, anti-diabetic, anti-tumor, antihyperlipidemic, and nephroprotective activities [38,39]. Furthermore, clinical trials revealed the efficacy of troxerutin for management of phlebocholosis and hemorrhoidal diseases [39]. The last active compound, rutin, also known as vitamin P, or rutoside, is one of the most common dietary polyphenols found in vegetables, fruits, and other plants. It is metabolized by the mammalian gut microbiota and absorbed from the intestines, and becomes bioavailable in the form of conjugated metabolites [40,41]. Rutin showed a wide range of pharmacological applications, including antimicrobial, antifungal, anti-inflammatory, anticancer, antidiabetic, anti-hypertension and anti-hypercholesterolemia, as well as potential antiviral activity against SARS-CoV-2, due to its significant antioxidant and nontoxic properties [42,43]. Taking its numerous benefits into account, rutin can be widely used in functional foods, dietary supplements, and pharmaceuticals. The weaknesses of low aqueous solubility, poor stability and limited membrane permeability need to be resolved in practical application [41,44]. Overall, procyanidin, stevioside, troxerutin and rutin are natural products with low toxicity and have a broad spectrum of pharmacological and therapeutic benefits, compared with synthetic compounds, exhibiting tremendous prospects for development. Admittedly, however, the four compounds are moderate antibacterial inhibitors, which need to be in relatively high concentrations (100 ppm) for inhibition of V. parahaemolyticus, and it is possible that they poorly penetrate the OM of Gram-negative bacteria. Future work will focus on enhancing their inhibition effects on V. parahaemolyticus and other Gram-negative bacteria, by means of structural optimization.
When resolving the E. coli LolB structure, researchers obtained the crystal structure of the complex of LolB with PEGMME2000 at the hydrophobic cavity (PDB 1IWN) [19]. In our study, molecular docking showed the complex of V. parahaemolyticus LolB with inhibitor at the entrance of the hydrophobic cavity ( Figure 10A and Figure S2), where LolB interacted with LolA in the putative mouth-to-mouth lipoprotein transfer model [45]. Upon binding, the "mouth" of LolB is blocked by the inhibitor, the acyl chain of the lipoprotein is, therefore, unable to enter the hydrophobic cavity of LolB, and the trafficking of lipoprotein and the growth of bacterial cells is possibly prevented. The docking results suggested that the four compounds interacted with LolB through both main chain contacts and side chain contacts ( Figure 10A). Some active sites involving backbone contacts, Arg67, Ser69 and Ala93, were located in β-sheet, and Tyr123 and Thr126 positions at the α-helix, that is speculated to undergo conformational changes to give access of the lipoprotein substance to the LolB hydrophobic cavity ( Figure 10A) [46]. Therefore, the interaction of stevioside, troxerutin and rutin with Tyr123 and Thr126 likely hindered conformational changes of the α-helix, locked the close state of LolB, and affected LolB functioning. Furthermore, given that the LolB protein has no large structural difference among various microorganisms ( Figure 1B), it is quite possible that these backbone contacts also exist between inhibitors and other LolB proteins from different bacteria. and 8). Sequence alignment revealed that the corresponding amino acids of Gln68 and Tyr108 were Val and Asn, respectively, in E. coli LolB ( Figure 10B). The two residues were specific to V. parahaemolyticus, perhaps to procyanidin, interacting with the side chains of Tyr108 and Gln68 only, and could not bind E. coli LolB, and, thus, had no better effect on other microbial infections. Moreover, the side chain carboxyl groups in Asp65 and Asp84 were also involved in the formation of hydroxyl bonds, but the residues were not homologous with E. coli either. (Figures 6, 7 and 10). In summary, inhibitors targeting V. parahaemolyticus LolB, in the future, could be designed and optimized, based on the identified critical residues, Tyr108 and Gln68. We hope this study can provide strong technical support for the accurate control of V. parahaemolyticus, and contribute new strategies for in-depth research in the control of microbial safety. This targeted sterilization technology, aimed at the OM, paves the way for resolving AMR, reducing the risk of bacterial infection and safeguarding human health.

Homology Modeling
The sequence of LolB of V. parahaemolyticus RIMD 2210633 (protein id: BAC59004.1) from NCBI GenBank [47] was submitted to SWISS-MODEL (https://beta.swissmodel.expasy.org/, accessed on 1 December 2020) to construct the 3D structure of the protein [48,49]. The most homologous sequence was selected as the template for homology modeling. The effectiveness of the obtained model of V. parahaemolyticus LolB, constructed Through analyzing the side chain interactions of the LolB protein with inhibitors, it could be concluded that the residue Tyr108 of V. parahaemolyticus LolB was one of the most important active sites, of which the benzene ring and phenolic hydroxyl group of the side chain played a key role for the interaction of procyanidin, troxerutin and rutin with receptor LolB (Figures 5, 7 and 8). Additionally, Gln68 was another important amino acid residue in the V. parahaemolyticus LolB protein, since the carboxamido group of Gln68 formed at least two hydroxyl bonds with procyanidin, stevioside, and rutin ( Figures 5, 6 and 8). Sequence alignment revealed that the corresponding amino acids of Gln68 and Tyr108 were Val and Asn, respectively, in E. coli LolB ( Figure 10B). The two residues were specific to V. parahaemolyticus, perhaps to procyanidin, interacting with the side chains of Tyr108 and Gln68 only, and could not bind E. coli LolB, and, thus, had no better effect on other microbial infections. Moreover, the side chain carboxyl groups in Asp65 and Asp84 were also involved in the formation of hydroxyl bonds, but the residues were not homologous with E. coli either. (Figures 6, 7 and 10). In summary, inhibitors targeting V. parahaemolyticus LolB, in the future, could be designed and optimized, based on the identified critical residues, Tyr108 and Gln68.
We hope this study can provide strong technical support for the accurate control of V. parahaemolyticus, and contribute new strategies for in-depth research in the control of microbial safety. This targeted sterilization technology, aimed at the OM, paves the way for resolving AMR, reducing the risk of bacterial infection and safeguarding human health.

High-Throughput Virtual Screening
The active site of V. parahaemolyticus LolB was determined by homologous protein alignment, or prediction through the sitemap module of the Schrodinger Suite (Schrödinger, Inc., New York, NY, USA). After structure preparation (e.g., structure optimization of protein and ligands, receptor grid generation, etc.), 39,000 molecules from the Cayman/2D structure database were used to screen active compounds binding V. parahaemolyticus LolB at the large and deep hydrophobic cavity by structure-based virtual screening (provided by APExBIO Technology LLC, Houston, TX, USA). The top 10 compound glide scores were selected and purchased for further research.

Determination of Inhibition on V. parahaemolyticus
V. parahaemolyticus RIMD 2210633 strain was a gift from Dr. Craig and Dr. David of the Centre for Environment, Fisheries and Aquaculture Science. The antibacterial effect of the screened compounds was determined by broth microdilution method [51]. Briefly, a single colony of V. parahaemolyticus, on Thiosulfate citrate bile salts sucrose (TCBS) agar (Beijing Land Bridge Technology Co., Ltd., Beijing, China), was selected to inoculate into Tryptic Soy Broth (TSB) medium (Beijing Land Bridge Technology Co., Ltd., Beijing, China) containing 3% NaCl, overnight at 37 • C with shaking. The cells were centrifuged at 3000 rpm for 10 min and resuspended in Mueller-Hinton Broth (MHB) medium (Beijing Land Bridge Technology Co., Ltd., Beijing, China), 20 µL of which was dispensed into 96-well plates containing serially diluted compounds (purchased from Shanghai Macklin Biochemical Co., Ltd., Topscience Co., Ltd., Shanghai Acmec Biochemical Co., Ltd., or Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) ranging from 100-10 ppm. Polymyxin B was used as control. Plates were incubated statically at 37 • C for 16 h. The OD 600 of each well was read on Synergy2 Microplate Reader (Agilent Technologies, Inc., Santa Clara, CA, USA). Experiments were repeated three times in triplicate. The inhibition rate of each compound was calculated using the following equation [52]: where OD blank is the OD value of medium before incubation, OD compound is the OD value of medium with compound before incubation, OD contro l is the OD value of inoculum and OD treated is the OD value of inoculum with compound. The DNA sequence of LolB from V. parahaemolyticus RIMD 2210633 was obtained from NCBI GenBank [47]. Residues 26-212 of LolB were codon optimized using E. coli Codon Usage Analyzer 2.1 (http://faculty.ucr.edu/~mmaduro/codonusage/usage.htm, accessed on 1 September 2020) [53]. A single plasmid, pET28b, containing the codon-optimized gene, was chemically synthesized (GENEWIZ, Suzhou, China) with an N-terminal His 6 tag and NdeI and HindIII restriction sites.

Fluorescence Spectroscopy
Samples of purified LolB protein were mixed with 0 µM, 5 µM, 10 µM, 15 µM, 20 µM and 25 µM specific compounds dissolved in Tris-HCl buffer, respectively. After 30 min, the fluorescence spectra of the mixtures were measured with a F-7100 fluorescence spectrophotometer (Hitachi High-Tech Corporation, Tokyo, Japan). The excitation wavelength was set at 280 nm, the range of 300-400 nm was selected for emission scanning, and the width of the excitation slit and the emission slit was 5 nm.

Identification of Fluorescence Quenching Mechanism
Based on the data of fluorescence spectra, the type of fluorescence quenching of reaction between compounds and LolB protein was identified by the Stern-Volmer equation [54]: F 0 /F = 1 + K q τ 0 [Q] = 1 + K sv [Q] (2) In the equation, F 0 and F denote the fluorescence intensities before and after the addition of the compound, respectively. K q and K sv are the biomacromolecule quenching constant and the Stern-Volmer quenching constant, respectively. The value τ 0 is the average lifetime of the biomacromolecule without the quencher (10 −8 s) and [Q] represents the concentration of the compound.
The equilibrium binding constant K d was calculated using nonlinear regression with 'One Site-Specific Binding' model [55]: Y = B max * X/(K d + X) In the equation, X is the ligand concentration, Y is the fluorescence intensity, B max denotes the maximum specific binding.

Analysis of Molecular Docking
The docking results of the top 10 compounds on the V. parahaemolyticus LolB protein were visualized by Maestro (Schrödinger, CA, USA) [56]. Ligand-receptor protein interactions were shown in both two-dimensional and three-dimensional plots. In order to clarify the inhibitory mechanism, specific compounds were docked with LolB protein and the distribution of binding sites were analyzed, respectively.

Conclusions
The development of new antibacterial compounds targeting the Lol system of V. parahaemolyticus helps to construct efficient sterilization technology, and could potentially solve the problem of AMR. The present study was carried out to discover active compounds targeting the LolB protein to kill V. parahaemolyticus. The present data indicated that procyanidin, stevioside, troxerutin and rutin screened via VS had both exciting binding affinity with LolB and preferable antibacterial activity towards V. parahaemolyticus at concentrations of 100 ppm. Fluorescence spectroscopy and molecular docking demonstrated that these active compounds formed stable complexes with LolB through hydrogen bonds and pi-pi stacking interactions, and Tyr108 and Gln68 were the critical active sites in the LolB protein. Our study corroborated LolB as being a promising drug target, elucidated the active sites in LolB, and provided natural antibacterial agents for the targeted elimination of V. parahaemolyticus.