Antibacterial protein helveticin NX371 fused with penetrating peptide response to Vibrio parahaemolyticus in salmon

Antibacterial protein helveticin NX371 fused with penetrating peptide response to Vibrio parahaemolyticus in salmon | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antibacterial protein helveticin NX371 fused with penetrating peptide response to Vibrio parahaemolyticus in salmon Qing Sun, Yong Hao, Qing Li, Libang Zhou, Xiaoyu Zhu, Zhaoxin Lu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7359643/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Vibrio parahaemolyticus is a prevalent foodborne pathogen associated with seafood and is a significant causative agent of foodborne illness. There is a market demand for enhancing food safety and quality through the use of bacteriocins derived from lactic acid bacteria to control V. parahaemolyticus infections. This study focuses on enhancing the efficacy of the bacteriocin Helveticin NX371, derived from Lactobacillus helveticus , against V. parahaemolyticus . This was achieved by modifying the surface charge and employing the fusion and expression of transmembrane peptides. Among the tested combinations, NX371 + OP4 significantly reduced the minimum inhibitory concentration (MIC) against V. parahaemolyticus from 180 µg/mL to 35 µg/mL, representing a decrease of 80.5%. In salmon artificially contaminated with V. parahaemolyticus , NX371 + OP4 demonstrated a potent bactericidal effect, achieving a mortality rate of 93.3%. Furthermore, throughout a 12-day storage period, the concentration of V. parahaemolyticus remained below 10 ^ 5 CFU/g. This study offers an effective strategy for preventing V. parahaemolyticus contamination in seafood and enhancing its shelf life. Vibrio parahaemolyticus antimicrobial agents helveticin gram-negative bacteria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction V. parahaemolyticus is a significant foodborne pathogen commonly found in offshore waters and seabed sediments, particularly in seafood like fish, shrimp, crab, and shellfish 1 , 2 . It primarily causes acute gastroenteritis, with symptoms such as abdominal pain, diarrhea, nausea, vomiting, and mild fever 3 , 4 . In severe cases, it can lead to dehydration, shock, coma, or even death. In mainland China, V. parahaemolyticus frequently causes foodborne diseases, particularly from May to October due to increased seafood consumption 5 . Data from the National Foodborne Disease Monitoring Network shows it is a leading cause of outbreaks, ranking first among microbial foodborne diseases from 2011 to 2018. From 2010 to 2020, mainland China reported 1,772 V. parahaemolyticus foodborne outbreaks, resulting in 27,212 illnesses and 5,944 hospitalizations 5 , 6 . Bacteriocins from lactic acid bacteria are effective biopreservatives that inhibit pathogens like V. parahaemolyticus 7 , 8 . They also enhance aquaculture animal health by improving water quality and boosting immune responses 9 . Lactic acid bacteria like Lactobacillus plantarum and Lactobacillus pentosus can inhibit V. parahaemolyticus , lowering infection risk 8 . Bacteriocins also minimize reliance on chemical preservatives, aligning with consumer demands for safer, healthier food 10 , 11 . Research on using lactic acid bacteria bacteriocins in aquaculture is growing, with studies indicating that their inclusion in shrimp feed can enhance growth and disease resistance 12 , 13 , particularly against V. parahaemolyticus . This offers new solutions for aquaculture and supports the commercial use of these bacteriocins 14 , 15 . Helveticin, a natural antibacterial, has broad application potential. Lactocin 27 from Lactobacillus helveticus LP27 targets only Lactobacillus acidophilus and L. helveticus 16 . Helveticin J from L. helveticus 481 is effective against related lactic acid bacteria 17 and L. helveticus 34.9 produces a large bacteriocin (35 KDa) that is active across a wide pH range 18 , 19 . Helveticin-M, a type III bacteriocin from Lactobacillus crispatus , effectively targets Staphylococcus aureus , S. saprophytes , and Enterobacter cloacae 20 . Meanwhile, L. helveticus M14-1 produces a bacteriocin with strong activity against Listeria monocytogenes 21 . We previously isolated helveticin NX371 from Lactobacillus helveticus NX2-6 22, 23 , with a molecular weight of 37Kda 24 . It effectively inhibits various lactic acid and Gram-positive bacteria like S. aureus but is less effective against Gram-negative bacteria such as Escherichia coli , Salmonella , and V. parahaemolyticus 25 , 26 , which are key food poisoning agents. Thus, creating a more potent helveticin could better control food-borne pathogen infections and minimize the use of preservatives. Results 1. Helveticin NX371 exhibits limited inhibitory activity against Gram-negative bacteria. Helveticin NX371, a bacteriocin derived from L. helveticus and classified as a class III lactic acid bacteria bacteriocin, is characterized as a heat-labile macro-molecular antimicrobial protein. It demonstrates significant inhibitory effects on Gram-positive bacteria, including S. aureus , L. monocytogenes , and Micrococcus luteus , with a minimum inhibitory concentration (MIC) ranging from 67 to 125 µg/mL. Nevertheless, the inhibitory efficacy of helveticin NX371 against Gram-negative bacteria, including prevalent foodborne pathogens such as Salmonella typhimurium , E. coli , and V. parahaemolyticus , is limited, with a minimum inhibitory concentration (MIC) ranging from 175 to 242 µg/mL (Fig. 1 ). The robust outer membrane structure of Gram-negative bacteria confers resistance to various food preservatives and pharmaceutical antibiotics. Consequently, enhancing the inhibitory activity of helveticin NX371 against Gram-negative bacteria is a critical focus of this research. This study focused on V. parahaemolyticus , a common seafood pathogen, to explore how helveticin NX371 acts against Gram-negative bacteria. It was found that helveticin NX371's ability to hydrolyze peptidoglycan showed no significant difference between S. aureus and V. parahaemolyticus ( p = 0.075) (Fig. 2 ). Adding 90 µg/mL of helveticin NX371 (MIC = 183 µg/mL) increased V. parahaemolyticus permeability by 9.4%. At 180 µg/mL and 360 µg/mL, permeability rose by 38.46% and 464%, respectively. This suggests that helveticin NX371 enhances outer membrane permeability to exert antibacterial effects. The robust outer membrane of V. parahaemolyticus acts as a barrier, preventing helveticin NX371 from accessing the periplasmic space to hydrolyze peptidoglycan or compromise the cell membrane. Consequently, enhancing the efficacy of helveticin NX371 against V. parahaemolyticus necessitates augmenting its capacity to disrupt the outer membrane. 2. helveticin NX371 protein structure prediction and property analysis Alphafold 2 predicted that helveticin NX371 closely resembles the propeller domain of the host recognition protein Gp45 from the S. aureus phage phi11. Gp45 is crucial for the phage's infection process, targeting GlcNAc residues on the bacterial cell wall. Experiments confirmed that helveticin NX371 can recognize and hydrolyze the peptidoglycan of S. aureus . Additionally, an analysis of the hydrophobicity and charge distribution of helveticin NX371 was conducted, revealing that helveticin NX371 exhibits significant hydrophobicity and possesses an extensive hydrophobic surface area (Fig. 3 ). Upon heterologous expression in E. coli , helveticin NX371 was found to exist exclusively as inclusion bodies, further corroborating its pronounced hydrophobic nature. The charge distribution analysis indicated that positive and negative charges are predominantly localized at the termini of the β-folded structure (Fig. 3 ). The protein contained 42 negatively charged residues and 27 positively charged residues, yet its isoelectric point (pI) was determined to be 5.71. Consequently, under neutral pH conditions, the protein exhibited a net positive charge. 3.Design of helveticin NX371 to enhance its effectiveness against outer membrane permeabilization To enhance helveticin NX371's outer membrane permeability against V. parahaemolyticus : (1) Adjust its surface charge to concentrate positive charges at one end with some hydrophobicity, and (2) fuse it with a membrane-penetrating peptide effective against negative bacteria. Consequently, we engineered mutations at one terminus of the protein, substituting the negatively charged glutamic acid and aspartic acid residues with positively charged histidine and lysine (designated as M1 and M2, respectively), with the aim of enhancing its efficacy in disrupting the outer membrane of V. parahaemolyticus (Fig. 4 ). Furthermore, previous research led to the identification of an outer membrane permeability peptide, PlnA (OP4), from L. plantarum . This peptide was subjected to targeted mutations and modifications, resulting in the mutant OP4, which exhibits a significantly enhanced capacity to increase outer membrane permeability. Based on this approach, we substituted the H122-H136 segment of NX371 with OP4 to generate mutant M3, and appended the OP4 segment following N90 to create mutant M4 (refer to Fig. 4 ). In the structural representations, positively charged amino acids are depicted in blue, while negatively charged amino acids are illustrated in red for the wild type, mutant 1, and mutant 2. The membrane-penetrating peptide OP4 is shown in purple for mutants 3 and 4. First, the base sequence for the M1-M4 mutants was synthesized based on their amino acid sequence, inserted into the pNC-GST vector, and transformed into E. coli BL21 (DE3). Figure 26 confirms the expected size of positive clones. However, after IPTG induction, the four mutants failed to express in E. coli , possibly due to higher toxicity compared to the wild-type NX371. Then A double-end blocking strategy was used to reduce recombinant protein toxicity. GST and Sumo tags were added to both ends of the helveticin NX371 mutant to reduce toxicity. These mutants were purified using Ni-NTA affinity chromatography, and the blocking fusion proteins were removed with TEV, yielding soluble M1-M4 mutants. Helveticin NX371 showed lysozyme-like peptidoglycan hydrolysis activity against Gram-positive bacteria. M1 and M2 mutants had reduced hydrolysis activity on S. aureus and V. parahaemolyticus , while M3 and M4 mutants showed no significant change in activity (Fig. 5 a). We used the liquid microdilution method to determine the MIC of M1-M4 mutants against V. parahaemolyticus . The MIC of NX371 against V. parahaemolyticus was 180 µg/mL, while M1-M4 mutants had MICs of 230, 240, 170, and 170 µg/mL, respectively. M1 and M2 showed significantly reduced inhibitory activity against V. parahaemolyticus ( p = 0.025 and 0.007), while M3 and M4 did not show a statistically significant difference compared to the wild type. Increasing the positive charge on the surface of helveticin NX371 does not enhance, but rather inhibits, its antibacterial activity against V. parahaemolyticus . Thus, its effectiveness is not necessarily linked to the surface charge distribution. Based on M3-M4 results, incorporating the membrane-penetrating peptide OP4 into the H122-H136 segment's random coil doesn't replicate OP4's membrane-penetrating effect. While it enhances antibacterial action against V. parahaemolyticus , the improvement isn't statistically significant (Fig. 5 b). 4. Fusion expression of helveticin NX371 and OP4 and its antibacterial activity To evaluate the influence of the membrane-penetrating peptide on the antibacterial efficacy of helveticin NX371, both separate (NX371 + OP4) and fused (NX371-OP4) peptide configurations were examined. The separate peptide OP4 significantly enhanced the antibacterial activity of NX371 against V. parahaemolyticus , reducing the minimum inhibitory concentration (MIC) from 180 µg/mL to 35 µg/mL ( p = 0.0005). In contrast, the MIC for the C-terminally fused peptide, NX371-OP4, against V. parahaemolyticus was established at 100 µg/mL. This value did not significantly differ from that of the wild-type helveticin NX371 ( p = 0.44) (see Fig. 6 ). These findings indicate that the fusion-mode membrane-penetrating peptide OP4 does not substantially enhance the permeability of the outer membrane of V. parahaemolyticus . 5、Effects of Helveticin NX371 on the Permeability of Inner and Outer Membranes. The NPN assay results indicated that the effects of helveticin NX371 at 1/2 MIC (18 µg/mL) and the combination of NX371 (18 µg/mL) with OP4 on the outer membrane permeability of V. parahaemolyticus were minimal. A significant increase in outer membrane permeability was observed only when the concentration of helveticin NX371 reached 180 µg/mL (Fig. 7 a). These findings suggest that the presence of OP4 enables NX371 to enhance outer membrane permeability at a reduced concentration. The results demonstrated that both helveticin NX371 and NX371 + OP4 significantly enhanced cell membrane permeability, resulting in the leakage of intracellular β-galactosidase. This leakage was observed to increase proportionally with the concentration. In comparison, the control, consisting of 18 µg/mL OP4, also significantly increased the permeability of both the outer and inner membranes; however, its effect was less pronounced than that of 18 µg/mL NX371 + OP4. This suggests that helveticin NX371 effectively damages the inner membrane following its penetration through the outer membrane of V. parahaemolyticus (Fig. 7 a). This observation aligns with prior research findings. The OP4 transmembrane peptide interacts with the lipopolysaccharide of V. parahaemolyticus , disrupting the outer membrane structure and enhancing its permeability. Consequently, helveticin NX371 can more effectively penetrate the periplasmic space, facilitating its role in hydrolyzing peptidoglycan and inducing membrane damage(Fig. 7 b). 6、Fresh-keeping experiment in salmon Colony counts over time revealed that the untreated salmon fillet contained about 10^4 to 10^ 5 CFU/g of V. parahaemolyticus , indicating contamination in the purchased salmon sashimi. This highlights the common issue of V. parahaemolyticus in salmon fillets. The bacterial count in the control group rose with storage time, surpassing 10^ 5 CFU/g by the third day. The positive control group, contaminated with V. parahaemolyticus , saw colony counts rise above 10^ 6 CFU/g by the third day, nearing 10^ 7 CFU/g. In contrast, the treated group, initially inoculated with nearly 10^ 6 CFU/g, showed a reduction to 10^ 4 CFU/g on day 0, demonstrating that NX371 + OP4 effectively kills V. parahaemolyticus with a 93.3% mortality rate. During the 12-day storage period, the concentration of V. parahaemolyticus remained below 10^ 5 CFU/g. The application of NX371 + OP4 demonstrated a substantial bactericidal and inhibitory effect on V. parahaemolyticus , thereby significantly prolonging the shelf life of salmon fillets, as illustrated in Fig. 8 . Discussion Chemical and biological preservatives are key in preventing foodborne pathogens. Chemical preservatives, like nitrites and potassium sorbate, are synthetic and have raised health concerns among consumers 7 , 13 . Conversely, biological preservatives come from natural sources, such as plant, animal, and microbial metabolites 27 – 29 . For instance, antimicrobial peptides synthesized by lactic acid bacteria, including Nisin and Diplococcin, have been extensively utilized in food preservation and have demonstrated significant antibacterial efficacy 30 , 31 . Recently, as consumer concern for food safety and health grows, the use of natural preservatives has risen. Despite their potential, biological preservatives face industrial challenges, particularly regarding effectiveness and stability 31 . Further research is needed to enhance their application and ensure their safety and efficacy. Lactic acid bacteria produce bacteriocins, natural antimicrobial agents used in food preservation. These bacteriocins, particularly from Lactobacillus , inhibit harmful bacteria like Salmonella , E. coli , and S. aureus , protecting food from contamination. They work by disrupting bacterial cell wall synthesis, energy metabolism, and membrane structure 8 , 32 , 33 . Previous research demonstrated that helveticin NX371, isolated and identified from L. helveticus NX2-6, exhibited significant inhibitory effects on Gram-positive bacteria, including S. aureus 24 . The present study explored various strategies to enhance its inhibitory activity against Gram-negative bacteria, with a particular focus on V. parahaemolyticus . By employing a combination of fusion expression with the membrane-penetrating peptide OP4 and subsequent OP4 release via TEV protease treatment, the minimum inhibitory concentration (MIC) of NX371 against V. parahaemolyticus was reduced by 80.5%, achieving a concentration of 35 µg/mL. Furthermore, the preservation and freshness experiment conducted on salmon demonstrated a significant reduction in the number of viable bacteria, thereby extending the shelf life of the product. The outer membrane of Gram-negative bacteria, made of lipopolysaccharide (LPS), phospholipids, and proteins, is highly hydrophobic and acts as a crucial barrier against environmental stress. Transmembrane peptides can alter the structure and function of these bacteria by interacting with their outer membrane 34 , 35 . For instance, bee venom peptides can rapidly bind to the LPS region, affecting the membrane's permeability and capacitance without fully compromising its integrity. Antimicrobial peptides like polymyxin B and E alter membrane structure by interacting with LPS, leading to antibacterial effects. Some peptides can penetrate the outer membrane's phospholipid bilayer, disrupt surface potential, form pores, and cause cell lysis. In this experiment, OP4 can be incorporated into the outer membrane via electrostatic and hydrophobic interactions, thereby disrupting the membrane structure by altering the arrangement of lipopolysaccharides (LPS) and phospholipids, and consequently enhancing the membrane's permeability 36 . When combined with NX371, which exhibits lysozyme-like activity, OP4 significantly facilitates the translocation of NX371 across the outer membrane. This synergistic interaction enhances NX371's efficacy in hydrolyzing the peptidoglycan of Vibrio parahaemolyticus and inducing membrane damage. The design and development of novel, highly effective, broad-spectrum antibacterial agents derived from bacteriocins of lactic acid bacteria hold significant potential to address the inherent limitations of bacteriocins, including their narrow antibacterial spectrum and limited stability. This advancement is of considerable importance for food preservation, the control of foodborne pathogens, and the enhancement of food quality. Methods 1. Materials and strains Auto induction medium, LB medium, brain heart infusion medium, and TCBS medium were sourced from Qingdao Haibo Biotechnology Co., Ltd. V. parahaemolyticus and S. aureus peptidoglycan, Tev protease, NPN, and ONPG were obtained from Sigma. High-fidelity DNA polymerase and an infusion seamless cloning kit were acquired from Takara Biotechnology Co., Ltd. Table 1 strains used in this study strains Application Description References E. coli DH5α Plasmid construction and subcloning F − Φ80 lacZ ΔM15 Δ( lacZYA - argF ) U169 rec A1 end A1 hsd R17(rk − , mk + ) pho A sup E44 thi -1 gyr A96 rel A1 λ − Beijing Zoman Biotechnology Co., Ltd. E. coli BL21(DE3) Protein expression F − ompT hsdSB(rB − mB − )gal dcm(DE3) Beijing Zoman Biotechnology Co., Ltd. E. coli BL21(DE3)- NX371 Expression of NX371with GST tag E. coli BL21(DE3) with pNC-GST-NX371 This study E. coli BL21(DE3)- M1 Expression of NX371 mutant M1 with GST tag E. coli BL21(DE3) with pNC-GST-M1 This study E. coli BL21(DE3)- M2 Expression of NX371 mutant M2 with GST tag E. coli BL21(DE3) with pNC-GST-M2 This study E. coli BL21(DE3)- M3 Expression of NX371 mutant M3 with GST tag E. coli BL21(DE3) with pNC-GST-M3 This study E. coli BL21(DE3)- M4 Expression of NX371 mutant M4 with GST tag E. coli BL21(DE3) with pNC-GST-M4 This study E. coli BL21(DE3)- M1s Expression of NX371 mutant M1 with GST tag and Sumo tag E. coli BL21(DE3) with pNC-GST-M1-Sumo This study E. coli BL21(DE3)- M2s Expression of NX371 mutant M2 with GST tag and Sumo tag E. coli BL21(DE3) with pNC-GST-M2-Sumo This study E. coli BL21(DE3)- M3s Expression of NX371 mutant M3 with GST tag and Sumo tag E. coli BL21(DE3) with pNC-GST-M3-Sumo This study E. coli BL21(DE3)- M4s Expression of NX371 mutant M4 with GST tag and Sumo tag E. coli BL21(DE3) with pNC-GST-M4-Sumo This study E. coli BL21(DE3)- NX371-OP4 Expression of NX371 fused with OP4 peptide E. coli BL21(DE3) with pNC-GST-NX371-OP4-Sumo This study 2.Determination of Minimum Inhibitory Concentration (MIC) MIC is the lowest concentration of helveticin NX371 and its mutants that prevents visible microbial growth in vitro. This is assessed by preparing serial dilutions of the agent in a liquid growth medium with 10^6 cfu/mL of Vibrio parahaemolyticus or other strains, incubating for 12 hours, and then checking for turbidity. 3.Determination of Peptidoglycan Hydrolysis To assess peptidoglycan hydrolysis, prepare a dilution of V. parahaemolyticus or S. aureus peptidoglycan (Sigma) using 1 mL of 60 mM MES buffer (pH 6.0, 180 mM NaCl) to achieve an optical density at 600 nm (OD600) of 1.0. Conduct the experiment in triplicate by utilizing three tubes for each reaction condition. Introduce 1 mg of helveticin NX371 or its mutants into each tube, followed by incubation at 37°C for 30 minutes. Subsequently, measure the OD600 and plot the absorbance values to evaluate peptidoglycan hydrolysis. 4.Determination of outer Permeability N-phenyl-1-naphthylamine (NPN) was utilized to evaluate changes in the permeability of the outer membrane of V. parahaemolyticus . NPN, a hydrophobic fluorescent dye, integrates into the cell membrane, resulting in a significant fluorescent signal. Helveticin NX371 and the NX371 + OP4 were added to the V. parahaemolyticus suspension at concentrations equivalent to 1/2 the minimum inhibitory concentration (MIC) and the MIC, respectively. Fluorescence measurements were then conducted using a fluorescence microplate reader following an incubation period of 5 minutes. 5. Determination of Intimal Permeability The compound o-Nitrophenyl β-D-galactopyranoside (ONPG) undergoes hydrolysis by the enzyme β-galactosidase from Vibrio parahaemolyticus, yielding the yellow compound o-nitrophenol (ONP). This chromatic transition serves as an indicator of β-galactosidase leakage, implying modifications in membrane permeability. To assess this phenomenon, cells should be cultured to an optical density at 600 nm (OD 600) of 0.5–0.6, followed by centrifugation and washing with a solution of 10 mM sodium phosphate (pH 7.4) containing 100 mM NaCl. The cell suspension should then be adjusted to an OD 600 of 0.5. Subsequently, 1.5 mM ONPG is added to the mixture, along with protein samples at varying concentrations. The reaction mixture is incubated at 37°C for 30 minutes to facilitate the hydrolysis of ONPG, resulting in the production of ONP. 6. Protein expression and purification The sequence-verified expression vectors (Table 1 ) were introduced into BL21(DE3) Escherichia coli cells. A single colony was cultured in 50 mL of Auto Induction Medium supplemented with 50 µg/mL kanamycin and incubated at 25°C for 24 hours. The cells were harvested by centrifugation at 5000×g. The resulting cell pellet was resuspended in 10 mL of lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM EDTA) and lysed using an ultrasonic crusher. The lysate was clarified by centrifugation at 14,000×g for 10 minutes. Subsequently, proteins were purified from each supernatant via Ni-NTA chromatography using a 5 mL HisTrap FF column (Thermofisher). The imidazole was eliminated through ultrafiltration employing a 3 kDa ultrafiltration membrane. The purified protein was subsequently stored at − 80°C in a solution containing 50% glycerol. 7. Fresh-keeping experiment in salmon Salmon slices were artificially inoculated with a concentration of 10^6 CFU/g of V. parahaemolyticus . The experimental group underwent immersion in a solution containing 250 µg/mL of NX371 + OP4 for a duration of 10 minutes. Following immersion, excess surface moisture was removed using a paper towel. Subsequently, the salmon slices were allowed to air dry for 10 minutes on a sterile clean bench, which was covered with plastic wrap. Thereafter, the slices were placed into sterile low-density polyethylene bags and stored under refrigeration at 4°C. This procedure was designed to simulate cold chain conditions in order to evaluate the storage parameters of raw salmon slices. Take 1 gram of salmon at three-day intervals and incorporate 9 milliliters of saline solution. Homogenize the tissue thoroughly, subsequently applying gradient dilutions onto Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) agar plates. Incubate the plates at 37°C for a duration of 12 hours, and subsequently enumerate the colonies of V. parahaemolyticus present on the plates. Declarations Availability of data and material All data generated or analyzed during this study are included in this published article. Competing interests The authors declare no conflict of interest. Acknowledgements The authors would like to thank professor Fengxia Lyu and Xiaomei Bie of Nanjing Agricultural University, professor Yingjian Lu of Nanjing University of Finance and Economics for helpful discussions on topics related to this work. We would like to thank the anonymous editors and reviewers for their helpful remarks. Funding Declaration The authors would like to thank the financial support of the National Natural Science Foundation of China (No.32101910 and 32072182). Author Contributions Fanqiang Meng conceived the study, designed the experiments, analyzed and interpreted the data and wrote the manuscript. Qing Sun and Yong Hao performed the experimental work. Qing Sun and Qing Li analyzed the data and elaborated tables and figures. Libang Zhou contributed to data interpretation. Xiaoyu Zhu and Zhaoxin Lu designed the experiments and reviewed the manuscript. All authors discussed the results and approved the submitted manuscript. References Martinez-Urtaza J, Baker-Austin C (2020) Vibrio parahaemolyticus. Trends Microbiol 28(10):867–868 He P, Zhang J, Zhou Y, Hou S, Tao X, Wang A, Yang Z, Bai Z, Wu X (2022) Genomic characteristics of a notable emerging serotype O10:K4 of Vibrio parahaemolyticus from food-borne cluster events in Guangzhou, China. J Infect 85(6):702–769 Matsuda S, Okada R, Tandhavanant S, Hiyoshi H, Gotoh K, Iida T, Kodama T (2019) Export of a Vibrio parahaemolyticus toxin by the Sec and type III secretion machineries in tandem. Nat Microbiol 4(5):781–788 Ndraha N, Wong HC, Hsiao HI (2020) Managing the risk of Vibrio parahaemolyticus infections associated with oyster consumption: A review. Compr Rev Food Sci Food Saf 19(3):1187–1217 Yang C, Li Y, Jiang M, Wang L, Jiang Y, Hu L, Shi X, He L, Cai R, Wu S, Qiu Y, Lu L, Zuo L, Chen Q, Wu Y, Martinez-Urtaza J, Wan C, Yang R, Cui Y, Hu Q (2022) Outbreak dynamics of foodborne pathogen Vibrio parahaemolyticus over a seventeen year period implies hidden reservoirs. Nat Microbiol 7(8):1221–1229 Liu Y, Yan L, Xia Y (2022) Assessment of maternal services in China based on WHO's comprehensive evaluation model. BMC Health Serv Res 22(1):1571 Soltani S, Hammami R, Cotter PD, Rebuffat S, Ben Said L, Gaudreau H, Bedard F, Biron E, Drider D, Fliss I (2021) Bacteriocins as a new generation of antimicrobials: toxicity aspects and regulations. FEMS Microbiol Rev 45 (1) Hernández-González JC, Martínez-Tapia A, Lazcano-Hernández G, García-Pérez BE, Castrejón-Jiménez NS (2021) Bacteriocins from Lactic Acid Bacteria. A Powerful Alternative as Antimicrobials, Probiotics, and Immunomodulators in Veterinary Medicine. Animals 11(4):979 Zimina M, Babich O, Prosekov A, Sukhikh S, Ivanova S, Shevchenko M, Noskova S (2020) Overview of Global Trends in Classification, Methods of Preparation and Application of Bacteriocins. Antibiotics-Basel 9 (9) Kaya HI, Ozel B, Simsek O (2019) A Natural Way of Food Preservation: Bacteriocins and Their Applications . ; p 633–659 Pang X, Song X, Chen M, Tian S, Lu Z, Sun J, Li X, Lu Y, Yuk HG (2022) Combating biofilms of foodborne pathogens with bacteriocins by lactic acid bacteria in the food industry. Compr Rev Food Sci Food Saf 21(2):1657–1676 Sugrue I, Ross RP, Hill C (2024) Bacteriocin diversity, function, discovery and application as antimicrobials. Nat Rev Microbiol And HC, Hoover DG (2003) Bacteriocins and their Food Applications. Compr Rev Food Sci Food Saf 2(3):82–100 Abdulhussain Kareem R, Razavi SH (2020) Plantaricin bacteriocins: As safe alternative antimicrobial peptides in food preservation—A review. 40 (1), e12735 Chikindas ML, Weeks R, Drider D, Chistyakov VA, Dicks LMT (2018) Functions and emerging applications of bacteriocins. Curr Opin Biotech 49:23–28 Upreti GC, Hinsdill RD (1975) Production and mode of action of lactocin 27: bacteriocin from a homofermentative Lactobacillus. Antimicrob Agents Chemother 7(2):139–145 Joerger MC, Klaenhammer TR (1990) Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J. J Bacteriol 172(11):6339–6347 Angelescu IR, Grosu-Tudor SS, Cojoc LR, Maria GM, Chiritoiu GN, Munteanu CVA, Zamfir M (2022) Isolation, characterization, and mode of action of a class III bacteriocin produced by Lactobacillus helveticus 34.9. World J Microbiol Biotechnol 38(12):220 Chelladhurai K, Ayyash M, Turner MS, Kamal-Eldin A (2023) Lactobacillus helveticus : Health effects, current applications, and future trends in dairy fermentation. Trends Food Sci Technol 136:159–168 Sun Z, Wang X, Zhang X, Wu H, Wang D (2018) Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane. J Industrial Microbiol Biotechnol Lett 45(3):1–15 Zhao J, Zeng X, Xi Y, Li J (2024) Recent advances in the applications of Lactobacillus helveticus in the fermentation of plant-based beverages: A review. Trends Food Sci Technol 147:104427 Tang C, Cao G, Zhao W, Bie X, Lu F, Lu Z, Lu Y (2021) Lactobacillus acidophilus NX2-6 Improved High-Fat Diet-Induced Glucose Metabolism Disorder Independent of Promotion of Insulin Secretion in Mice. J Agric Food Chem 69(51):15598–15610 Tang C, Meng F, Pang X, Chen M, Zhou L, Lu Z, Lu Y (2020) Protective effects of Lactobacillus acidophilus NX2-6 against oleic acid-induced steatosis, mitochondrial dysfunction, endoplasmic reticulum stress and inflammatory responses. J Funct Foods 74:104206 Meng F, Zhu X, Zhao H, Nie T, Lu F, Lu Z, Lu Y (2020) A class Ⅲ bacteriocin with broad-spectrum antibacterial activity from Lactobacillus acidophilus NX2-6 and its preservation in milk and cheese. Food Control 107597 Wuyundalai; Lu FX, Sun HG, Cao GQ, Lu YP, Lu ZX (2010) The antibacterial properties and strain identification of Lactobacillus acidophilus NX2-6 screened from Chigee. Milchwissenschaft 65(2):144–148 Zhang Q, Lu Y, Liu X, Bie X, Lv F, Lu Z (2014) Preservative effect of food-based fermentate from Lactobacillus acidophilus NX2-6 on chilled pork patties. J Food Prot 77(3):459–465 Chen C, Chen L, Mao C, Jin L, Wu S, Zheng Y, Cui Z, Li Z, Zhang Y, Zhu S, Jiang H, Liu X (2024) Natural Extracts for Antibacterial Applications. Small 20 (9), e2306553 Kenny O, Smyth TJ, Walsh D, Kelleher CT, Hewage CM, Brunton NP (2014) Investigating the potential of under-utilised plants from the Asteraceae family as a source of natural antimicrobial and antioxidant extracts. Food Chem 161:79–86 Ye L, Zhang J, Xiao W, Liu S (2020) Efficacy and mechanism of actions of natural antimicrobial drugs. Pharmacol Ther 216:107671 Onaizi SA, Leong SS (2011) Tethering antimicrobial peptides: current status and potential challenges. Biotechnol Adv 29(1):67–74 Carratalá JV, Serna N, Villaverde A, Vázquez E, Ferrer-Miralles N (2020) Nanostructured antimicrobial peptides: The last push towards clinics. Biotechnol Adv 44:107603 Meng F, Zhao H, Nie T, Lu F, Zhang C, Lu Y, Lu Z (2021) Acetate improves the competitive advantage of Lactobacillus by activating the synthesis of bacteriocins. Appl Environ Microb Abdulhussain Kareem R, Razavi SH (2019) Plantaricin bacteriocins: As safe alternative antimicrobial peptides in food preservation-A review. J Food Saf Guha S, Ghimire J, Wu E, Wimley WC (2019) Mechanistic Landscape of Membrane-Permeabilizing Peptides. Chem Rev 119(9):6040–6085 Kabelka I, Vácha R (2021) Advances in Molecular Understanding of α-Helical Membrane-Active Peptides. Acc Chem Res 54(9):2196–2204 Meng F, Liu Y, Nie T, Tang C, Lyu F, Bie X, Lu Y, Zhao M, Lu Z, Plantaricin A (2022) Derived from Lactiplantibacillus plantarum , Reduces the Intrinsic Resistance of Gram-Negative Bacteria to Hydrophobic Antibiotics. Appl Environ Microbiol 88 (10), e0037122 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7359643","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":505251809,"identity":"2743ec71-67cb-4028-be09-9fa302476bf2","order_by":0,"name":"Qing Sun","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Sun","suffix":""},{"id":505251810,"identity":"afae8ed8-3415-4fe0-9d53-adb5627420ef","order_by":1,"name":"Yong Hao","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Hao","suffix":""},{"id":505251812,"identity":"3109faca-1eb0-45f8-ace6-96229bd58a62","order_by":2,"name":"Qing Li","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Li","suffix":""},{"id":505251813,"identity":"cc31ee44-3183-4010-9480-a4cfef40aa43","order_by":3,"name":"Libang Zhou","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Libang","middleName":"","lastName":"Zhou","suffix":""},{"id":505251815,"identity":"64abb119-031e-4412-b62f-1cb9aa617372","order_by":4,"name":"Xiaoyu Zhu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Zhu","suffix":""},{"id":505251816,"identity":"e0c9a819-e7e9-4075-8fae-ba3fc92585cf","order_by":5,"name":"Zhaoxin Lu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhaoxin","middleName":"","lastName":"Lu","suffix":""},{"id":505251817,"identity":"d1410d54-5776-42dc-8d7a-6ca453fe7775","order_by":6,"name":"Fanqiang Meng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYDAC5gOMDxh4wEwDIrWwJTAbkKyFTQLKJFKLbhvztuoCGbvEBvbmbRIMNXcIazE7xlZ2ewZPcmIDz7EyCYZjz4jQcr/H7DYPD3Nig0SOmQRjw2FibOExK+bhqU9skH9DghZmHp7DQFt4iNbCVizNw3PcuI0nrdgi4RhRWpg3fubtqZbtZz+88caHGiK0MICig7EHGD8gZgJRGsAx+INIpaNgFIyCUTAyAQDuqzKOH4hbcwAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Fanqiang","middleName":"","lastName":"Meng","suffix":""}],"badges":[],"createdAt":"2025-08-13 00:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7359643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7359643/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89924133,"identity":"e035bbb9-d7c2-41fe-9fd5-3025b107065d","added_by":"auto","created_at":"2025-08-26 13:15:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33849,"visible":true,"origin":"","legend":"\u003cp\u003eMIC of helveticin NX371\u003c/p\u003e\n\u003cp\u003eThe bacterial strains \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eM. luteus\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003ewere diluted in Luria-Bertani (LB) medium to a concentration of 10^6 colony-forming units per milliliter (CFU/mL). Subsequently, helveticin NX371 was introduced at concentrations of 50, 100, 150, 200, 250, and 300 μg/mL, and the cultures were incubated for an additional 12 hours. Meanwhile, \u003cem\u003eL. monocytogenes\u003c/em\u003e and \u003cem\u003eS. typhimurium\u003c/em\u003e were cultured in brain heart infusion (BHI) medium, whereas \u003cem\u003eV. parahaemolyticus\u003c/em\u003e was cultured in LB medium supplemented with 3% NaCl.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/009f2831e3e784ed403a16d1.png"},{"id":89926009,"identity":"f848de66-5719-4cf9-b20f-2d1f21f1ba50","added_by":"auto","created_at":"2025-08-26 13:31:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29701,"visible":true,"origin":"","legend":"\u003cp\u003ePeptidoglycan hydrolytic activity and outer membrane permeability of helveticin NX371\u003c/p\u003e\n\u003cp\u003ea. The peptidoglycan hydrolytic activity of helveticin NX371 at a concentration of 1 mg/mL was assessed at 37 ºC for a duration of 30 minutes. b. The outer membrane permeability of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e was evaluated following treatment with helveticin NX371.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/4aba2c917ccf77ebadd28ad7.png"},{"id":89924136,"identity":"c4c26d92-b521-46e7-9a17-3065f355d2c5","added_by":"auto","created_at":"2025-08-26 13:15:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":827080,"visible":true,"origin":"","legend":"\u003cp\u003eprotein structure and properties of helveticin NX371\u003c/p\u003e\n\u003cp\u003ea) Predicted structure of helveticin NX371; b) Homology comparison between helveticin NX371 (depicted in green) and Gp45 (depicted in yellow, PDB: 5EFV); c) Distribution of hydrophobic amino acids in helveticin NX371; d) Hydrophobic surface representation of helveticin NX371; e) Charge distribution analysis of helveticin NX371.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/65b07479bbcb4c713f73b4fc.png"},{"id":89926010,"identity":"4824eb1a-fdab-4b54-bc08-b79402939b8f","added_by":"auto","created_at":"2025-08-26 13:31:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":571268,"visible":true,"origin":"","legend":"\u003cp\u003eschematic of designed NX371\u003c/p\u003e\n\u003cp\u003eIn the structural representations, positively charged amino acids are depicted in blue, while negatively charged amino acids are illustrated in red for the wild type, mutant 1, and mutant 2. The membrane-penetrating peptide OP4 is shown in purple for mutants 3 and 4.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/015796526e316f20e673f2be.png"},{"id":89924145,"identity":"994aada0-31bf-4236-a1df-1725fa07b26d","added_by":"auto","created_at":"2025-08-26 13:15:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28662,"visible":true,"origin":"","legend":"\u003cp\u003ePeptidoglycan hydrolytic activity and antibacterial activity of helveticin NX371 mutants\u003c/p\u003e\n\u003cp\u003ea. The peptidoglycan hydrolytic activity of helveticin NX371 and its mutants at a concentration of 1 mg/mL was assessed at 37 ºC for a duration of 30 minutes. b. The MIC of helveticin NX371 and its mutants for \u003cem\u003eV. parahaemolyticus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/ebd14c93e3da5416ff71caf9.png"},{"id":89925093,"identity":"632ffb45-3e19-477c-bcdc-293d5d27f772","added_by":"auto","created_at":"2025-08-26 13:23:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":229293,"visible":true,"origin":"","legend":"\u003cp\u003eFusion expression of helveticin NX371 and OP4 and its antibacterial activity\u003c/p\u003e\n\u003cp\u003ea. Fusion expression of helveticin NX371 and OP4 with GST tag and Sumo tag. b, The MIC of helveticin NX371 fused NX371-OP4 and separated NX371+OP4 for \u003cem\u003eV. parahaemolyticus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/58232951dcfa5b9761c007b6.png"},{"id":89924139,"identity":"c805ab3b-6e1a-4518-ba3b-18f748e90dba","added_by":"auto","created_at":"2025-08-26 13:15:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":47862,"visible":true,"origin":"","legend":"\u003cp\u003ePeptidoglycan hydrolytic activity and outer membrane permeability of helveticin NX371 mutants\u003c/p\u003e\n\u003cp\u003ea. The outer membrane permeability of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e was evaluated following treatment with helveticin NX371, NX371 and OP4 mixture, peptide OP4. b. The inner membrane permeability of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e treatment with helveticin NX371, NX371 and OP4 mixture, peptide OP4.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/53c22237d2c0f17793bec883.png"},{"id":89924143,"identity":"6956c8aa-b520-482f-8e21-b033b3d8205e","added_by":"auto","created_at":"2025-08-26 13:15:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":37672,"visible":true,"origin":"","legend":"\u003cp\u003evisual colony of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSalmon homogenate was diluted by 0.9% saline, and coated on TCBS plates, cultured at 37ºC for 12 hours, count the number of monoclones on the plate.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/ac6eef64db68d91e163a7f87.png"},{"id":90821012,"identity":"3c9d06d0-8304-45cf-bbca-084f3a77d163","added_by":"auto","created_at":"2025-09-08 14:17:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2526015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7359643/v1/62c511dc-26eb-4ee7-8c75-2bdcd68606b0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antibacterial protein helveticin NX371 fused with penetrating peptide response to Vibrio parahaemolyticus in salmon","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eV. parahaemolyticus\u003c/em\u003e is a significant foodborne pathogen commonly found in offshore waters and seabed sediments, particularly in seafood like fish, shrimp, crab, and shellfish\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It primarily causes acute gastroenteritis, with symptoms such as abdominal pain, diarrhea, nausea, vomiting, and mild fever\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In severe cases, it can lead to dehydration, shock, coma, or even death. In mainland China, \u003cem\u003eV. parahaemolyticus\u003c/em\u003e frequently causes foodborne diseases, particularly from May to October due to increased seafood consumption\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Data from the National Foodborne Disease Monitoring Network shows it is a leading cause of outbreaks, ranking first among microbial foodborne diseases from 2011 to 2018. From 2010 to 2020, mainland China reported 1,772 \u003cem\u003eV. parahaemolyticus\u003c/em\u003e foodborne outbreaks, resulting in 27,212 illnesses and 5,944 hospitalizations\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBacteriocins from lactic acid bacteria are effective biopreservatives that inhibit pathogens like \u003cem\u003eV. parahaemolyticus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. They also enhance aquaculture animal health by improving water quality and boosting immune responses\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Lactic acid bacteria like \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and \u003cem\u003eLactobacillus pentosus\u003c/em\u003e can inhibit \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, lowering infection risk\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Bacteriocins also minimize reliance on chemical preservatives, aligning with consumer demands for safer, healthier food\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Research on using lactic acid bacteria bacteriocins in aquaculture is growing, with studies indicating that their inclusion in shrimp feed can enhance growth and disease resistance\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, particularly against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. This offers new solutions for aquaculture and supports the commercial use of these bacteriocins\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHelveticin, a natural antibacterial, has broad application potential. Lactocin 27 from \u003cem\u003eLactobacillus helveticus\u003c/em\u003e LP27 targets only \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e and \u003cem\u003eL. helveticus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Helveticin J from \u003cem\u003eL. helveticus\u003c/em\u003e 481 is effective against related lactic acid bacteria\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eL. helveticus\u003c/em\u003e 34.9 produces a large bacteriocin (35 KDa) that is active across a wide pH range\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Helveticin-M, a type III bacteriocin from \u003cem\u003eLactobacillus crispatus\u003c/em\u003e, effectively targets \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eS. saprophytes\u003c/em\u003e, and \u003cem\u003eEnterobacter cloacae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Meanwhile, \u003cem\u003eL. helveticus\u003c/em\u003e M14-1 produces a bacteriocin with strong activity against \u003cem\u003eListeria monocytogenes\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe previously isolated helveticin NX371 from Lactobacillus helveticus NX2-6\u003csup\u003e22, 23\u003c/sup\u003e, with a molecular weight of 37Kda\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. It effectively inhibits various lactic acid and Gram-positive bacteria like \u003cem\u003eS. aureus\u003c/em\u003e but is less effective against Gram-negative bacteria such as \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, which are key food poisoning agents. Thus, creating a more potent helveticin could better control food-borne pathogen infections and minimize the use of preservatives.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003e1. Helveticin NX371 exhibits limited inhibitory activity against Gram-negative bacteria.\u003c/h3\u003e\n\u003cp\u003eHelveticin NX371, a bacteriocin derived from \u003cem\u003eL. helveticus\u003c/em\u003e and classified as a class III lactic acid bacteria bacteriocin, is characterized as a heat-labile macro-molecular antimicrobial protein. It demonstrates significant inhibitory effects on Gram-positive bacteria, including \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eL. monocytogenes\u003c/em\u003e, and \u003cem\u003eMicrococcus luteus\u003c/em\u003e, with a minimum inhibitory concentration (MIC) ranging from 67 to 125 \u0026micro;g/mL. Nevertheless, the inhibitory efficacy of helveticin NX371 against Gram-negative bacteria, including prevalent foodborne pathogens such as \u003cem\u003eSalmonella typhimurium\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, is limited, with a minimum inhibitory concentration (MIC) ranging from 175 to 242 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The robust outer membrane structure of Gram-negative bacteria confers resistance to various food preservatives and pharmaceutical antibiotics. Consequently, enhancing the inhibitory activity of helveticin NX371 against Gram-negative bacteria is a critical focus of this research.\u003c/p\u003e\n\u003cp\u003eThis study focused on \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, a common seafood pathogen, to explore how helveticin NX371 acts against Gram-negative bacteria. It was found that helveticin NX371's ability to hydrolyze peptidoglycan showed no significant difference between \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.075) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Adding 90 \u0026micro;g/mL of helveticin NX371 (MIC\u0026thinsp;=\u0026thinsp;183 \u0026micro;g/mL) increased \u003cem\u003eV. parahaemolyticus\u003c/em\u003e permeability by 9.4%. At 180 \u0026micro;g/mL and 360 \u0026micro;g/mL, permeability rose by 38.46% and 464%, respectively. This suggests that helveticin NX371 enhances outer membrane permeability to exert antibacterial effects. The robust outer membrane of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e acts as a barrier, preventing helveticin NX371 from accessing the periplasmic space to hydrolyze peptidoglycan or compromise the cell membrane. Consequently, enhancing the efficacy of helveticin NX371 against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e necessitates augmenting its capacity to disrupt the outer membrane.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e2. helveticin NX371 protein structure prediction and property analysis\u003c/h3\u003e\n\u003cp\u003eAlphafold 2 predicted that helveticin NX371 closely resembles the propeller domain of the host recognition protein Gp45 from the \u003cem\u003eS. aureus\u003c/em\u003e phage phi11. Gp45 is crucial for the phage's infection process, targeting GlcNAc residues on the bacterial cell wall. Experiments confirmed that helveticin NX371 can recognize and hydrolyze the peptidoglycan of \u003cem\u003eS. aureus\u003c/em\u003e. Additionally, an analysis of the hydrophobicity and charge distribution of helveticin NX371 was conducted, revealing that helveticin NX371 exhibits significant hydrophobicity and possesses an extensive hydrophobic surface area (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Upon heterologous expression in \u003cem\u003eE. coli\u003c/em\u003e, helveticin NX371 was found to exist exclusively as inclusion bodies, further corroborating its pronounced hydrophobic nature. The charge distribution analysis indicated that positive and negative charges are predominantly localized at the termini of the \u0026beta;-folded structure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The protein contained 42 negatively charged residues and 27 positively charged residues, yet its isoelectric point (pI) was determined to be 5.71. Consequently, under neutral pH conditions, the protein exhibited a net positive charge.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e3.Design of helveticin NX371 to enhance its effectiveness against outer membrane permeabilization\u003c/h3\u003e\n\u003cp\u003eTo enhance helveticin NX371's outer membrane permeability against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e: (1) Adjust its surface charge to concentrate positive charges at one end with some hydrophobicity, and (2) fuse it with a membrane-penetrating peptide effective against negative bacteria. Consequently, we engineered mutations at one terminus of the protein, substituting the negatively charged glutamic acid and aspartic acid residues with positively charged histidine and lysine (designated as M1 and M2, respectively), with the aim of enhancing its efficacy in disrupting the outer membrane of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, previous research led to the identification of an outer membrane permeability peptide, PlnA (OP4), from \u003cem\u003eL. plantarum\u003c/em\u003e. This peptide was subjected to targeted mutations and modifications, resulting in the mutant OP4, which exhibits a significantly enhanced capacity to increase outer membrane permeability. Based on this approach, we substituted the H122-H136 segment of NX371 with OP4 to generate mutant M3, and appended the OP4 segment following N90 to create mutant M4 (refer to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the structural representations, positively charged amino acids are depicted in blue, while negatively charged amino acids are illustrated in red for the wild type, mutant 1, and mutant 2. The membrane-penetrating peptide OP4 is shown in purple for mutants 3 and 4.\u003c/p\u003e\n\u003cp\u003eFirst, the base sequence for the M1-M4 mutants was synthesized based on their amino acid sequence, inserted into the pNC-GST vector, and transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3). Figure\u0026nbsp;26 confirms the expected size of positive clones. However, after IPTG induction, the four mutants failed to express in \u003cem\u003eE. coli\u003c/em\u003e, possibly due to higher toxicity compared to the wild-type NX371. Then A double-end blocking strategy was used to reduce recombinant protein toxicity. GST and Sumo tags were added to both ends of the helveticin NX371 mutant to reduce toxicity. These mutants were purified using Ni-NTA affinity chromatography, and the blocking fusion proteins were removed with TEV, yielding soluble M1-M4 mutants.\u003c/p\u003e\n\u003cp\u003eHelveticin NX371 showed lysozyme-like peptidoglycan hydrolysis activity against Gram-positive bacteria. M1 and M2 mutants had reduced hydrolysis activity on \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, while M3 and M4 mutants showed no significant change in activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). We used the liquid microdilution method to determine the MIC of M1-M4 mutants against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. The MIC of NX371 against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e was 180 \u0026micro;g/mL, while M1-M4 mutants had MICs of 230, 240, 170, and 170 \u0026micro;g/mL, respectively. M1 and M2 showed significantly reduced inhibitory activity against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025 and 0.007), while M3 and M4 did not show a statistically significant difference compared to the wild type. Increasing the positive charge on the surface of helveticin NX371 does not enhance, but rather inhibits, its antibacterial activity against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. Thus, its effectiveness is not necessarily linked to the surface charge distribution. Based on M3-M4 results, incorporating the membrane-penetrating peptide OP4 into the H122-H136 segment's random coil doesn't replicate OP4's membrane-penetrating effect. While it enhances antibacterial action against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, the improvement isn't statistically significant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\n\u003ch3\u003e4. Fusion expression of helveticin NX371 and OP4 and its antibacterial activity\u003c/h3\u003e\n\u003cp\u003eTo evaluate the influence of the membrane-penetrating peptide on the antibacterial efficacy of helveticin NX371, both separate (NX371\u0026thinsp;+\u0026thinsp;OP4) and fused (NX371-OP4) peptide configurations were examined. The separate peptide OP4 significantly enhanced the antibacterial activity of NX371 against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, reducing the minimum inhibitory concentration (MIC) from 180 \u0026micro;g/mL to 35 \u0026micro;g/mL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005). In contrast, the MIC for the C-terminally fused peptide, NX371-OP4, against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e was established at 100 \u0026micro;g/mL. This value did not significantly differ from that of the wild-type helveticin NX371 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.44) (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). These findings indicate that the fusion-mode membrane-penetrating peptide OP4 does not substantially enhance the permeability of the outer membrane of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e5、Effects of Helveticin NX371 on the Permeability of Inner and Outer Membranes.\u003c/h3\u003e\n\u003cp\u003eThe NPN assay results indicated that the effects of helveticin NX371 at 1/2 MIC (18 \u0026micro;g/mL) and the combination of NX371 (18 \u0026micro;g/mL) with OP4 on the outer membrane permeability of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e were minimal. A significant increase in outer membrane permeability was observed only when the concentration of helveticin NX371 reached 180 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea). These findings suggest that the presence of OP4 enables NX371 to enhance outer membrane permeability at a reduced concentration.\u003c/p\u003e\n\u003cp\u003eThe results demonstrated that both helveticin NX371 and NX371\u0026thinsp;+\u0026thinsp;OP4 significantly enhanced cell membrane permeability, resulting in the leakage of intracellular \u0026beta;-galactosidase. This leakage was observed to increase proportionally with the concentration. In comparison, the control, consisting of 18 \u0026micro;g/mL OP4, also significantly increased the permeability of both the outer and inner membranes; however, its effect was less pronounced than that of 18 \u0026micro;g/mL NX371\u0026thinsp;+\u0026thinsp;OP4. This suggests that helveticin NX371 effectively damages the inner membrane following its penetration through the outer membrane of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea). This observation aligns with prior research findings. The OP4 transmembrane peptide interacts with the lipopolysaccharide of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, disrupting the outer membrane structure and enhancing its permeability. Consequently, helveticin NX371 can more effectively penetrate the periplasmic space, facilitating its role in hydrolyzing peptidoglycan and inducing membrane damage(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e6、Fresh-keeping experiment in salmon\u003c/h3\u003e\n\u003cp\u003eColony counts over time revealed that the untreated salmon fillet contained about 10^4 to 10^\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e CFU/g of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, indicating contamination in the purchased salmon sashimi. This highlights the common issue of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e in salmon fillets. The bacterial count in the control group rose with storage time, surpassing 10^\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e CFU/g by the third day. The positive control group, contaminated with \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, saw colony counts rise above 10^\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e CFU/g by the third day, nearing 10^\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e CFU/g. In contrast, the treated group, initially inoculated with nearly 10^\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e CFU/g, showed a reduction to 10^\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e CFU/g on day 0, demonstrating that NX371\u0026thinsp;+\u0026thinsp;OP4 effectively kills \u003cem\u003eV. parahaemolyticus\u003c/em\u003e with a 93.3% mortality rate. During the 12-day storage period, the concentration of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e remained below 10^\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e CFU/g. The application of NX371\u0026thinsp;+\u0026thinsp;OP4 demonstrated a substantial bactericidal and inhibitory effect on \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, thereby significantly prolonging the shelf life of salmon fillets, as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eChemical and biological preservatives are key in preventing foodborne pathogens. Chemical preservatives, like nitrites and potassium sorbate, are synthetic and have raised health concerns among consumers\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Conversely, biological preservatives come from natural sources, such as plant, animal, and microbial metabolites\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e–\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. For instance, antimicrobial peptides synthesized by lactic acid bacteria, including Nisin and Diplococcin, have been extensively utilized in food preservation and have demonstrated significant antibacterial efficacy\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Recently, as consumer concern for food safety and health grows, the use of natural preservatives has risen. Despite their potential, biological preservatives face industrial challenges, particularly regarding effectiveness and stability\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Further research is needed to enhance their application and ensure their safety and efficacy.\u003c/p\u003e\u003cp\u003eLactic acid bacteria produce bacteriocins, natural antimicrobial agents used in food preservation. These bacteriocins, particularly from \u003cem\u003eLactobacillus\u003c/em\u003e, inhibit harmful bacteria like \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e, protecting food from contamination. They work by disrupting bacterial cell wall synthesis, energy metabolism, and membrane structure\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Previous research demonstrated that helveticin NX371, isolated and identified from \u003cem\u003eL. helveticus\u003c/em\u003e NX2-6, exhibited significant inhibitory effects on Gram-positive bacteria, including \u003cem\u003eS. aureus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The present study explored various strategies to enhance its inhibitory activity against Gram-negative bacteria, with a particular focus on \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. By employing a combination of fusion expression with the membrane-penetrating peptide OP4 and subsequent OP4 release via TEV protease treatment, the minimum inhibitory concentration (MIC) of NX371 against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e was reduced by 80.5%, achieving a concentration of 35 µg/mL. Furthermore, the preservation and freshness experiment conducted on salmon demonstrated a significant reduction in the number of viable bacteria, thereby extending the shelf life of the product.\u003c/p\u003e\u003cp\u003eThe outer membrane of Gram-negative bacteria, made of lipopolysaccharide (LPS), phospholipids, and proteins, is highly hydrophobic and acts as a crucial barrier against environmental stress. Transmembrane peptides can alter the structure and function of these bacteria by interacting with their outer membrane\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. For instance, bee venom peptides can rapidly bind to the LPS region, affecting the membrane's permeability and capacitance without fully compromising its integrity. Antimicrobial peptides like polymyxin B and E alter membrane structure by interacting with LPS, leading to antibacterial effects. Some peptides can penetrate the outer membrane's phospholipid bilayer, disrupt surface potential, form pores, and cause cell lysis. In this experiment, OP4 can be incorporated into the outer membrane via electrostatic and hydrophobic interactions, thereby disrupting the membrane structure by altering the arrangement of lipopolysaccharides (LPS) and phospholipids, and consequently enhancing the membrane's permeability\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. When combined with NX371, which exhibits lysozyme-like activity, OP4 significantly facilitates the translocation of NX371 across the outer membrane. This synergistic interaction enhances NX371's efficacy in hydrolyzing the peptidoglycan of Vibrio parahaemolyticus and inducing membrane damage.\u003c/p\u003e\u003cp\u003eThe design and development of novel, highly effective, broad-spectrum antibacterial agents derived from bacteriocins of lactic acid bacteria hold significant potential to address the inherent limitations of bacteriocins, including their narrow antibacterial spectrum and limited stability. This advancement is of considerable importance for food preservation, the control of foodborne pathogens, and the enhancement of food quality.\u003c/p\u003e\n\n\n\n\n\n\n\n\n\n\n\n\n\n"},{"header":"Methods","content":"\u003ch3\u003e1. Materials and strains\u003c/h3\u003e\u003cp\u003eAuto induction medium, LB medium, brain heart infusion medium, and TCBS medium were sourced from Qingdao Haibo Biotechnology Co., Ltd. \u003cem\u003eV. parahaemolyticus\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e peptidoglycan, Tev protease, NPN, and ONPG were obtained from Sigma. High-fidelity DNA polymerase and an infusion seamless cloning kit were acquired from Takara Biotechnology Co., Ltd.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003estrains used in this study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003estrains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eApplication\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReferences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e DH5α\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePlasmid construction and subcloning\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003csup\u003e−\u003c/sup\u003eΦ80\u003cem\u003elacZ\u003c/em\u003eΔM15 Δ(\u003cem\u003elacZYA\u003c/em\u003e-\u003cem\u003eargF\u003c/em\u003e) U169 \u003cem\u003erec\u003c/em\u003eA1 \u003cem\u003eend\u003c/em\u003eA1 \u003cem\u003ehsd\u003c/em\u003eR17(rk\u003csup\u003e−\u003c/sup\u003e, mk\u003csup\u003e+\u003c/sup\u003e) \u003cem\u003epho\u003c/em\u003eA \u003cem\u003esup\u003c/em\u003eE44 \u003cem\u003ethi\u003c/em\u003e-1 \u003cem\u003egyr\u003c/em\u003eA96 \u003cem\u003erel\u003c/em\u003eA1 λ\u003csup\u003e−\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBeijing Zoman Biotechnology Co., Ltd.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProtein expression\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003csup\u003e−\u003c/sup\u003eompT hsdSB(rB\u003csup\u003e−\u003c/sup\u003emB\u003csup\u003e−\u003c/sup\u003e)gal dcm(DE3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBeijing Zoman Biotechnology Co., Ltd.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- NX371\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371with GST tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-NX371\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- M1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 mutant M1 with GST tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-M1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- M2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 mutant M2 with GST tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-M2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- M3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 mutant M3 with GST tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-M3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- M4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 mutant M4 with GST tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-M4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- M1s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 mutant M1 with GST tag and Sumo tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-M1-Sumo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- M2s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 mutant M2 with GST tag and Sumo tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-M2-Sumo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- M3s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 mutant M3 with GST tag and Sumo tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-M3-Sumo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- M4s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 mutant M4 with GST tag and Sumo tag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-M4-Sumo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)- NX371-OP4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExpression of NX371 fused with OP4 peptide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with pNC-GST-NX371-OP4-Sumo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003ch3\u003e2.Determination of Minimum Inhibitory Concentration (MIC)\u003c/h3\u003e\u003cp\u003eMIC is the lowest concentration of helveticin NX371 and its mutants that prevents visible microbial growth in vitro. This is assessed by preparing serial dilutions of the agent in a liquid growth medium with 10^6 cfu/mL of Vibrio parahaemolyticus or other strains, incubating for 12 hours, and then checking for turbidity.\u003c/p\u003e\u003ch3\u003e3.Determination of Peptidoglycan Hydrolysis\u003c/h3\u003e\u003cp\u003eTo assess peptidoglycan hydrolysis, prepare a dilution of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e or \u003cem\u003eS. aureus\u003c/em\u003e peptidoglycan (Sigma) using 1 mL of 60 mM MES buffer (pH 6.0, 180 mM NaCl) to achieve an optical density at 600 nm (OD600) of 1.0. Conduct the experiment in triplicate by utilizing three tubes for each reaction condition. Introduce 1 mg of helveticin NX371 or its mutants into each tube, followed by incubation at 37°C for 30 minutes. Subsequently, measure the OD600 and plot the absorbance values to evaluate peptidoglycan hydrolysis.\u003c/p\u003e\u003ch3\u003e4.Determination of outer Permeability\u003c/h3\u003e\u003cp\u003eN-phenyl-1-naphthylamine (NPN) was utilized to evaluate changes in the permeability of the outer membrane of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. NPN, a hydrophobic fluorescent dye, integrates into the cell membrane, resulting in a significant fluorescent signal. Helveticin NX371 and the NX371 + OP4 were added to the \u003cem\u003eV. parahaemolyticus\u003c/em\u003e suspension at concentrations equivalent to 1/2 the minimum inhibitory concentration (MIC) and the MIC, respectively. Fluorescence measurements were then conducted using a fluorescence microplate reader following an incubation period of 5 minutes.\u003c/p\u003e\u003ch3\u003e5. Determination of Intimal Permeability\u003c/h3\u003e\u003cp\u003eThe compound o-Nitrophenyl β-D-galactopyranoside (ONPG) undergoes hydrolysis by the enzyme β-galactosidase from Vibrio parahaemolyticus, yielding the yellow compound o-nitrophenol (ONP). This chromatic transition serves as an indicator of β-galactosidase leakage, implying modifications in membrane permeability. To assess this phenomenon, cells should be cultured to an optical density at 600 nm (OD 600) of 0.5–0.6, followed by centrifugation and washing with a solution of 10 mM sodium phosphate (pH 7.4) containing 100 mM NaCl. The cell suspension should then be adjusted to an OD 600 of 0.5. Subsequently, 1.5 mM ONPG is added to the mixture, along with protein samples at varying concentrations. The reaction mixture is incubated at 37°C for 30 minutes to facilitate the hydrolysis of ONPG, resulting in the production of ONP.\u003c/p\u003e\u003ch3\u003e6. Protein expression and purification\u003c/h3\u003e\u003cp\u003eThe sequence-verified expression vectors (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were introduced into BL21(DE3) \u003cem\u003eEscherichia coli\u003c/em\u003e cells. A single colony was cultured in 50 mL of Auto Induction Medium supplemented with 50 µg/mL kanamycin and incubated at 25°C for 24 hours. The cells were harvested by centrifugation at 5000×g. The resulting cell pellet was resuspended in 10 mL of lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM EDTA) and lysed using an ultrasonic crusher. The lysate was clarified by centrifugation at 14,000×g for 10 minutes. Subsequently, proteins were purified from each supernatant via Ni-NTA chromatography using a 5 mL HisTrap FF column (Thermofisher). The imidazole was eliminated through ultrafiltration employing a 3 kDa ultrafiltration membrane. The purified protein was subsequently stored at − 80°C in a solution containing 50% glycerol.\u003c/p\u003e\u003ch3\u003e7. Fresh-keeping experiment in salmon\u003c/h3\u003e\u003cp\u003eSalmon slices were artificially inoculated with a concentration of 10^6 CFU/g of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. The experimental group underwent immersion in a solution containing 250 µg/mL of NX371 + OP4 for a duration of 10 minutes. Following immersion, excess surface moisture was removed using a paper towel. Subsequently, the salmon slices were allowed to air dry for 10 minutes on a sterile clean bench, which was covered with plastic wrap. Thereafter, the slices were placed into sterile low-density polyethylene bags and stored under refrigeration at 4°C. This procedure was designed to simulate cold chain conditions in order to evaluate the storage parameters of raw salmon slices. Take 1 gram of salmon at three-day intervals and incorporate 9 milliliters of saline solution. Homogenize the tissue thoroughly, subsequently applying gradient dilutions onto Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) agar plates. Incubate the plates at 37°C for a duration of 12 hours, and subsequently enumerate the colonies of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e present on the plates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank professor Fengxia Lyu and Xiaomei Bie of Nanjing Agricultural University, professor Yingjian Lu of Nanjing University of Finance and Economics for helpful discussions on topics related to this work. We would like to thank the anonymous editors and reviewers for their helpful remarks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Funding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the financial support of the National Natural Science Foundation of China (No.32101910 and 32072182).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFanqiang Meng conceived the study, designed the experiments, analyzed and interpreted the data and wrote the manuscript. Qing Sun and Yong Hao performed the experimental work. Qing Sun and Qing Li analyzed the data and elaborated tables and figures. Libang Zhou contributed to data interpretation. Xiaoyu Zhu and Zhaoxin Lu designed the experiments and reviewed the manuscript. All authors discussed the results and approved the submitted manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMartinez-Urtaza J, Baker-Austin C (2020) Vibrio parahaemolyticus. Trends Microbiol 28(10):867\u0026ndash;868\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe P, Zhang J, Zhou Y, Hou S, Tao X, Wang A, Yang Z, Bai Z, Wu X (2022) Genomic characteristics of a notable emerging serotype O10:K4 of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e from food-borne cluster events in Guangzhou, China. J Infect 85(6):702\u0026ndash;769\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatsuda S, Okada R, Tandhavanant S, Hiyoshi H, Gotoh K, Iida T, Kodama T (2019) Export of a \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e toxin by the Sec and type III secretion machineries in tandem. Nat Microbiol 4(5):781\u0026ndash;788\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNdraha N, Wong HC, Hsiao HI (2020) Managing the risk of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e infections associated with oyster consumption: A review. Compr Rev Food Sci Food Saf 19(3):1187\u0026ndash;1217\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang C, Li Y, Jiang M, Wang L, Jiang Y, Hu L, Shi X, He L, Cai R, Wu S, Qiu Y, Lu L, Zuo L, Chen Q, Wu Y, Martinez-Urtaza J, Wan C, Yang R, Cui Y, Hu Q (2022) Outbreak dynamics of foodborne pathogen \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e over a seventeen year period implies hidden reservoirs. Nat Microbiol 7(8):1221\u0026ndash;1229\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu Y, Yan L, Xia Y (2022) Assessment of maternal services in China based on WHO's comprehensive evaluation model. BMC Health Serv Res 22(1):1571\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoltani S, Hammami R, Cotter PD, Rebuffat S, Ben Said L, Gaudreau H, Bedard F, Biron E, Drider D, Fliss I (2021) Bacteriocins as a new generation of antimicrobials: toxicity aspects and regulations. FEMS Microbiol Rev \u003cem\u003e45\u003c/em\u003e (1)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHern\u0026aacute;ndez-Gonz\u0026aacute;lez JC, Mart\u0026iacute;nez-Tapia A, Lazcano-Hern\u0026aacute;ndez G, Garc\u0026iacute;a-P\u0026eacute;rez BE, Castrej\u0026oacute;n-Jim\u0026eacute;nez NS (2021) Bacteriocins from Lactic Acid Bacteria. A Powerful Alternative as Antimicrobials, Probiotics, and Immunomodulators in Veterinary Medicine. Animals 11(4):979\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZimina M, Babich O, Prosekov A, Sukhikh S, Ivanova S, Shevchenko M, Noskova S (2020) Overview of Global Trends in Classification, Methods of Preparation and Application of Bacteriocins. Antibiotics-Basel \u003cem\u003e9\u003c/em\u003e (9)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaya HI, Ozel B, Simsek O (2019) \u003cem\u003eA Natural Way of Food Preservation: Bacteriocins and Their Applications\u003c/em\u003e. ; p 633\u0026ndash;659\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePang X, Song X, Chen M, Tian S, Lu Z, Sun J, Li X, Lu Y, Yuk HG (2022) Combating biofilms of foodborne pathogens with bacteriocins by lactic acid bacteria in the food industry. Compr Rev Food Sci Food Saf 21(2):1657\u0026ndash;1676\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSugrue I, Ross RP, Hill C (2024) Bacteriocin diversity, function, discovery and application as antimicrobials. Nat Rev Microbiol\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnd HC, Hoover DG (2003) Bacteriocins and their Food Applications. Compr Rev Food Sci Food Saf 2(3):82\u0026ndash;100\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbdulhussain Kareem R, Razavi SH (2020) Plantaricin bacteriocins: As safe alternative antimicrobial peptides in food preservation\u0026mdash;A review. \u003cem\u003e40\u003c/em\u003e (1), e12735\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChikindas ML, Weeks R, Drider D, Chistyakov VA, Dicks LMT (2018) Functions and emerging applications of bacteriocins. Curr Opin Biotech 49:23\u0026ndash;28\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUpreti GC, Hinsdill RD (1975) Production and mode of action of lactocin 27: bacteriocin from a homofermentative Lactobacillus. Antimicrob Agents Chemother 7(2):139\u0026ndash;145\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoerger MC, Klaenhammer TR (1990) Cloning, expression, and nucleotide sequence of the \u003cem\u003eLactobacillus helveticus\u003c/em\u003e 481 gene encoding the bacteriocin helveticin J. J Bacteriol 172(11):6339\u0026ndash;6347\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAngelescu IR, Grosu-Tudor SS, Cojoc LR, Maria GM, Chiritoiu GN, Munteanu CVA, Zamfir M (2022) Isolation, characterization, and mode of action of a class III bacteriocin produced by \u003cem\u003eLactobacillus helveticus\u003c/em\u003e 34.9. World J Microbiol Biotechnol 38(12):220\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChelladhurai K, Ayyash M, Turner MS, Kamal-Eldin A (2023) \u003cem\u003eLactobacillus helveticus\u003c/em\u003e: Health effects, current applications, and future trends in dairy fermentation. Trends Food Sci Technol 136:159\u0026ndash;168\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun Z, Wang X, Zhang X, Wu H, Wang D (2018) Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane. J Industrial Microbiol Biotechnol Lett 45(3):1\u0026ndash;15\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao J, Zeng X, Xi Y, Li J (2024) Recent advances in the applications of \u003cem\u003eLactobacillus helveticus\u003c/em\u003e in the fermentation of plant-based beverages: A review. Trends Food Sci Technol 147:104427\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang C, Cao G, Zhao W, Bie X, Lu F, Lu Z, Lu Y (2021) \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NX2-6 Improved High-Fat Diet-Induced Glucose Metabolism Disorder Independent of Promotion of Insulin Secretion in Mice. J Agric Food Chem 69(51):15598\u0026ndash;15610\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang C, Meng F, Pang X, Chen M, Zhou L, Lu Z, Lu Y (2020) Protective effects of \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NX2-6 against oleic acid-induced steatosis, mitochondrial dysfunction, endoplasmic reticulum stress and inflammatory responses. J Funct Foods 74:104206\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng F, Zhu X, Zhao H, Nie T, Lu F, Lu Z, Lu Y (2020) A class Ⅲ bacteriocin with broad-spectrum antibacterial activity from \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NX2-6 and its preservation in milk and cheese. Food Control 107597\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWuyundalai; Lu FX, Sun HG, Cao GQ, Lu YP, Lu ZX (2010) The antibacterial properties and strain identification of \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NX2-6 screened from Chigee. Milchwissenschaft 65(2):144\u0026ndash;148\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Q, Lu Y, Liu X, Bie X, Lv F, Lu Z (2014) Preservative effect of food-based fermentate from \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NX2-6 on chilled pork patties. J Food Prot 77(3):459\u0026ndash;465\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen C, Chen L, Mao C, Jin L, Wu S, Zheng Y, Cui Z, Li Z, Zhang Y, Zhu S, Jiang H, Liu X (2024) Natural Extracts for Antibacterial Applications. Small 20 (9), e2306553\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKenny O, Smyth TJ, Walsh D, Kelleher CT, Hewage CM, Brunton NP (2014) Investigating the potential of under-utilised plants from the \u003cem\u003eAsteraceae\u003c/em\u003e family as a source of natural antimicrobial and antioxidant extracts. Food Chem 161:79\u0026ndash;86\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYe L, Zhang J, Xiao W, Liu S (2020) Efficacy and mechanism of actions of natural antimicrobial drugs. Pharmacol Ther 216:107671\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOnaizi SA, Leong SS (2011) Tethering antimicrobial peptides: current status and potential challenges. Biotechnol Adv 29(1):67\u0026ndash;74\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarratal\u0026aacute; JV, Serna N, Villaverde A, V\u0026aacute;zquez E, Ferrer-Miralles N (2020) Nanostructured antimicrobial peptides: The last push towards clinics. Biotechnol Adv 44:107603\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng F, Zhao H, Nie T, Lu F, Zhang C, Lu Y, Lu Z (2021) Acetate improves the competitive advantage of \u003cem\u003eLactobacillus\u003c/em\u003e by activating the synthesis of bacteriocins. Appl Environ Microb\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbdulhussain Kareem R, Razavi SH (2019) Plantaricin bacteriocins: As safe alternative antimicrobial peptides in food preservation-A review. J Food Saf\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuha S, Ghimire J, Wu E, Wimley WC (2019) Mechanistic Landscape of Membrane-Permeabilizing Peptides. Chem Rev 119(9):6040\u0026ndash;6085\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKabelka I, V\u0026aacute;cha R (2021) Advances in Molecular Understanding of α-Helical Membrane-Active Peptides. Acc Chem Res 54(9):2196\u0026ndash;2204\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng F, Liu Y, Nie T, Tang C, Lyu F, Bie X, Lu Y, Zhao M, Lu Z, Plantaricin A (2022) Derived from \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, Reduces the Intrinsic Resistance of Gram-Negative Bacteria to Hydrophobic Antibiotics. Appl Environ Microbiol 88 (10), e0037122\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vibrio parahaemolyticus, antimicrobial agents, helveticin, gram-negative bacteria","lastPublishedDoi":"10.21203/rs.3.rs-7359643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7359643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e is a prevalent foodborne pathogen associated with seafood and is a significant causative agent of foodborne illness. There is a market demand for enhancing food safety and quality through the use of bacteriocins derived from lactic acid bacteria to control \u003cem\u003eV. parahaemolyticus\u003c/em\u003e infections. This study focuses on enhancing the efficacy of the bacteriocin Helveticin NX371, derived from \u003cem\u003eLactobacillus helveticus\u003c/em\u003e, against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. This was achieved by modifying the surface charge and employing the fusion and expression of transmembrane peptides. Among the tested combinations, NX371\u0026thinsp;+\u0026thinsp;OP4 significantly reduced the minimum inhibitory concentration (MIC) against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e from 180 \u0026micro;g/mL to 35 \u0026micro;g/mL, representing a decrease of 80.5%. In salmon artificially contaminated with \u003cem\u003eV. parahaemolyticus\u003c/em\u003e, NX371\u0026thinsp;+\u0026thinsp;OP4 demonstrated a potent bactericidal effect, achieving a mortality rate of 93.3%. Furthermore, throughout a 12-day storage period, the concentration of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e remained below 10\u003csup\u003e^\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e CFU/g. This study offers an effective strategy for preventing \u003cem\u003eV. parahaemolyticus\u003c/em\u003e contamination in seafood and enhancing its shelf life.\u003c/p\u003e","manuscriptTitle":"Antibacterial protein helveticin NX371 fused with penetrating peptide response to Vibrio parahaemolyticus in salmon","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-26 13:15:40","doi":"10.21203/rs.3.rs-7359643/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"047a52f6-e0db-4145-a94e-142094660010","owner":[],"postedDate":"August 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T14:08:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-26 13:15:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7359643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7359643","identity":"rs-7359643","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}