Characterization of a secondary hydroxy-acyltransferase for lipid A in Vibrio parahaemolyticus.

Lipid A plays a crucial role in Vibrio parahaemolyticus. Previously we have reported the diversity of secondary acylation of lipid A in V. parahaemolyticus and four V. parahaemolyticus genes VP_RS08405, VP_RS01045, VP_RS12170, and VP_RS00880 exhibiting homology to the secondary acyltransferases in Escherichia coli. In this study, the gene VP_RS12170 was identified as a specific lipid A secondary hydroxy-acyltransferase responsible for transferring a 3-hydroxymyristate to the 2'-position of lipid A. Four E. coli mutant strains WHL00, WHM00, WH300, and WH001 were constructed, and they would synthesize lipid A with different structures due to the absence of genes encoding lipid A secondary acyltransferases or Kdo transferase. Then V. parahaemolyticus VP_RS12170 was overexpressed in W3110, WHL00, WHM00, WH300, and WH001, and lipid A was isolated from these strains and analyzed by using thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. The detailed structural changes of lipid A in these mutant strains with and without VP_RS12170 overexpression were compared and conclude that VP_RS12170 can specifically transfer a 3-hydroxymyristate to the 2'-position of lipid A. This study also demonstrated that the function of VP_RS12170 is Kdo-dependent and its favorite substrate is Kdo-lipid IVA. These findings give us better understanding the biosynthetic pathway and the structural diversity of V. parahaemolyticus lipid A.

Antibacterial and antibiofilm activity of halogenated phenylboronic acids against Vibrio parahaemolyticus and Vibrio harveyi.

Vibrios are associated with live seafood because they are part of the indigenous marine microflora. In Asia, foodborne infections caused by Vibrio spp. are common. In recent years, V. parahaemolyticus has become the leading cause of all reported food poisoning outbreaks. Therefore, the halogenated acid and its 33 derivatives were investigated for their antibacterial efficacy against V. parahaemolyticus. The compounds 3,5-diiodo-2-methoxyphenylboronic acid (DIMPBA) and 2-fluoro-5-iodophenylboronic acid (FIPBA) exhibited antibacterial and antibiofilm activity. DIMPBA and FIPBA had minimum inhibitory concentrations of 100 µg/mL for the planktonic cell growth and prevented biofilm formation in a dose-dependent manner. Both iodo-boric acids could diminish the several virulence factors influencing the motility, agglutination of fimbria, hydrophobicity, and indole synthesis. Consequently, these two active halogenated acids hampered the proliferation of the planktonic and biofilm cells. Moreover, these compounds have the potential to effectively inhibit the presence of biofilm formation on the surface of both squid and shrimp models.

Antibacterial and antibiofilm activities of iodinated hydrocarbons against Vibrio parahaemolyticus and Staphylococcus aureus.

Food-related illnesses have become a growing public concern due to their considerable socioeconomic and medical impacts. Vibrio parahaemolyticus and Staphylococcus aureus have been implicated as causative organisms of food-related infections and poisoning, and both can form biofilms which confer antibiotic resistance. Hence, the need for continuous search for compounds with antibiofilm and antivirulence properties. In this study, 22 iodinated hydrocarbons were screened for their antibiofilm activity, and of these, iodopropynyl butylcarbamate (IPBC) was found to effectively control biofilm formation of both pathogens with a MIC of 50 µg/mL which was bactericidal to V. parahaemolyticus and S. aureus. Microscopic studies confirmed IPBC inhibits biofilm formation of both bacteria and also disrupted their mixed biofilm formation. Furthermore, IPBC suppressed virulence activities such as motility and hemolytic activity of V. parahaemolyticus and the cell surface hydrophobicity of S. aureus. It exhibited a preservative potential against both pathogens in a shrimp model. IPBC disrupted the cell membrane of S. aureus and V. parahaemolyticus and differentially affected gene expressions related to biofilm formation and virulence. Additionally, it displayed broad-spectrum antibiofilm activities against other clinically relevant pathogens. These findings indicate IPBC offers a potential means of controlling infections mediated by Vibrio and Staphylococcus biofilms.

Isolation and characterization of an antimicrobial Bacillus subtilis strain O-741 against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine bacterium that can infect and cause the death of aquatic organisms. V. parahaemolyticus can also cause human foodborne infection via contaminated seafood, with clinical syndromes which include diarrhea, abdominal cramps, nausea and so on. Since controlling V. parahaemolyticus is important for aquaculture and human health, various strategies have been explored. This study investigates the application of antagonistic microorganisms to inhibit the growth of V. parahaemolyticus. We screened aquaculture environment samples and identified a Bacillus subtilis strain O-741 with potent antimicrobial activities. This strain showed a broad spectrum of antagonistic activities against V. parahaemolyticus and other Vibrio species. Application of the O-741 bacterium significantly increased the survival of Artemia nauplii which were infected with V. parahaemolyticus. Furthermore, the cell-free supernatant (CFS) of O-741 bacterium exhibited inhibitory ability against V. parahaemolyticus, and its activity was stable to heat, acidity, UV, enzymes, and organic solvents. Next, the O-741 CFS was extracted by ethyl acetate, and analyzed by ultra-performance liquid chromatography-mass-mass spectrometry (UPLC-MS/MS), and the functional faction was identified as an amicoumacin A compound. The organic extracts of CFS containing amicoumacin A had bactericidal effects on V. parahaemolyticus, and the treated V. parahaemolyticus cells showed disruption of the cell membrane and formation of cell cavities. These findings indicate that B. subtilis strain O-741 can inhibit the V. parahaemolyticus in vitro and in vivo, and has potential for use as a biocontrol agent for preventing V. parahaemolyticus infection.

The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.

Characterization of a novel Type-I Crustin (carcininPm2) from black tiger shrimp Penaeus monodon.

Carcinins are type-I crustins from crustaceans and play an important role in innate immune system. In this study, type-I crustins, carcininPm1 and carcininPm2, from the hemocytes of Penaeus monodon were identified. Comparison of their amino acid sequences and the phylogenetic tree revealed that they were closely related to the other crustacean carcinin proteins, but were clustered into different groups of the carcinin proteins. The full-length amino acids of carcininPm1 and carcininPm2 were 92 and 111 residues, respectively. CarcininPm1 and carcininPm2 were expressed mainly in hemocytes and intestine compared to the other tissues. The expression of carcininPm1 and carcininPm2 were dramatically increased in early time of bacterial challenged shrimp hemocytes. In contrast, the carcininPm1 and carcininPm2 were expressed in response to late state of YHV-infected shrimp hemocytes where the copy number of virus was high. The recombinant carcininPm2 (rcarcininPm2) but not its WAP domain (rcarcininPm2_WAP) exhibited antimicrobial activity against Vibrio harveyi and Vibrio parahaemolyticus AHPND but not other bacteria tested. The rcarcininPm2 was able to prolong the survival rate of VH-treated post larval shrimp from about 102 h to 156 h. These studies indicated that the carcininPm2 possessed the potential and challenges as antibacterial in innate immunity of shrimp.

Vibrio Species and Cyanobacteria: Understanding Their Association in Local Shrimp Farm Using Canonical Correspondence Analysis (CCA).

In aquatic environments, Vibrio and cyanobacteria establish varying relationships influenced by environmental factors. To investigate their association, this study spanned 5 months at a local shrimp farm, covering the shrimp larvae stocking cycle until harvesting. A total of 32 samples were collected from pond A (n = 6), pond B (n = 6), effluent (n = 10), and influent (n = 10). Vibrio species and cyanobacteria density were observed, and canonical correspondence analysis (CCA) assessed their correlation. CCA revealed a minor correlation (p = 0.847, 0.255, 0.288, and 0.304) between Vibrio and cyanobacteria in pond A, pond B, effluent, and influent water, respectively. Notably, Vibrio showed a stronger correlation with pH (6.14-7.64), while cyanobacteria correlated with pH, salinity (17.4-24 ppt), and temperature (30.8-31.5 °C), with salinity as the most influential factor. This suggests that factors beyond cyanobacteria influence Vibrio survival. Future research could explore species-specific relationships, regional dynamics, and multidimensional landscapes to better understand Vibrio-cyanobacteria connections. Managing water parameters may prove more efficient in controlling vibriosis in shrimp farms than targeting cyanobacterial populations.

Synergistic effects of combined probiotics Bacillus pumilis D5 and Leuconostoc mesenteroide B4 on immune enhancement and disease resistance in Litopenaeus vannamei.

This study investigated the effects of two distinct probiotics, Leuconostoc mesenteroides B4 (B4) and Bacillus pumilus D5 (D5), along with their combination, on the diet of white shrimp (Litopenaeus vannamei) during an eight-week feeding trial. The diets tested included B4 + dextran at 107 CFU/g feed (the B4 group), D5 alone at 107 CFU/g feed (the D5 group), and a combination of B4 + dextran and D5 at 5 × 106 CFU/g feed each (the B4+dextran + D5 group). Relative to the control group, those administered probiotics exhibited moderate enhancements in growth. By the eighth week, the weight gain for the B4, D5, and B4+D5 groups was 696.50 ± 78.15%, 718.53 ± 130.73%, and 693.05 ± 93.79%, respectively, outperforming the control group's 691.66 ± 31.10% gain. The feed conversion ratio was most efficient in the B4 group (2.16 ± 0.06), closely followed by B4+D5 (2.21 ± 0.03) and D5 (2.22 ± 0.06), with the control group having the highest ratio (2.27 ± 0.03). While phenoloxidase activity was somewhat elevated in the B4 and D5 groups, no significant differences were noted in respiratory burst activity or total hemocyte count across all groups. Challenge tests at weeks 4 and 8 showed that the B4 + D5 combination offered superior protection against AHPND-causing Vibrio parahaemolyticus. The 4-week cumulative survival rate was highest in shrimp treated with B4 + dextran + D5 (56.25%), followed by B4 + dextran (31.25%), control (18.75%), and lowest in D5 (12.5%). By week 8, the B4 + dextran + D5 (43.75%) and B4 + dextran (37.5%) groups significantly outperformed the control group (6.25%, p < 0.05), with no significant difference observed between the D5 group (37.5%) and the control group at day 56. Analysis of the shrimp's foregut microbiota revealed an increase in unique OTUs in the B4 and B4 + D5 groups. Compared to the control, Proteobacteria abundance was reduced in all probiotic groups. Potential pathogens like Vibrio, Bacteroides, Neisseria, Botrytis, Clostridioides, and Deltaentomopoxvirus were detected in the control but were reduced or absent in probiotic groups. Beneficial microbes such as Methanobrevibacter and Dictyostelium in the B4+D5 group, and Sugiyamaella in the B4 group, showed significant increases. Probiotics also led to higher transcript levels of nitric oxide synthase in the hemocytes, and lysozyme and transglutaminase in the midgut, along with lysozyme and α2-macroglobulin in the foregut. Notably, the combined B4 + D5 probiotics synergistically enhanced the expression of superoxide dismutase and prophenoloxidase in the foregut, indicating an improved immune response. In summary, this study demonstrates that the probiotics evaluated, especially when used in combination, significantly boost the expression of specific immune-related genes, enhance the bacterial diversity and richness of the intestine, and thus prevent the colonization and proliferation of Vibrio spp. in L. vannamei.

Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei.

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

In vitro and in vivo evaluation of the efficacies of commercial probiotics and disinfectant against acute hepatopancreatic necrosis disease and luminescent vibriosis in Litopenaeus vannamei.

The bioactivities of two commercially available probiotics and one chemical disinfectant were tested against strains of Vibrio parahaemolyticus (VPAHPND) and V. harveyi. This study aimed to determine shrimp pathogenic Vibrios' in vitro and in vivo sensitivities to commercial probiotics and a chemical disinfectant. The probiotics and disinfectant were tested first in vitro, followed by the in vivo trials. Results showed that upon administration of probiotics either through diet or adding into the tank water, the survivability of shrimp was increased during challenge with VPAHPND and V. harveyi. Also, the disinfectant was tested against the same pathogens and showed positive bactericidal effects at 2500 ppm and 5000 ppm. The present findings suggest that adding probiotics to the rearing water or the shrimp feeds effectively prevents infection by lowering the load of pathogenic bacteria. In comparison, the effectiveness of the disinfectant (PUR) depends on its appropriate concentration and timing of application. It is not only limited to rearing water but is also applicable for decontaminating pond liners, tanks, and other paraphernalia.

Morinda citrifolia fruit extract enhances the resistance of Penaeus vannamei to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a gram-negative facultative anaerobic bacterium implicated as the causative agent of several shrimp diseases. As part of the effort to provide biocontrol and cost-effective treatments, this research was designed to elucidate the effect of Morinda citrifolia fruit extract on the immunity of Penaeus vannamei postlarvae (PL) to V. parahaemolyticus. The methanol extract of M. citrifolia was vacuum evaporated, and the bioactive compounds were detected using gas chromatography‒mass spectrometry (GC‒MS). Thereafter, P. vannamei PL diets were supplemented with M. citrifolia at different concentrations (0, 10, 20, 30, 40, and 50 mg/g) and administered for 30 days before 24 h of exposure to the bacterium V. parahaemolyticus. A total of 45 bioactive compounds were detected in the methanol extract of M. citrifolia, with cyclononasiloxane and octadecamethyl being the most abundant. The survival of P. vannamei PLs fed the extract supplement was better than that of the control group (7.1-26.7% survival greater than that of the control group) following V. parahaemolyticus infection. Shrimp fed 50 mg/g M. citrifolia had the highest recorded survival. The activities of digestive and antioxidant enzymes as well as hepatopancreatic cells were significantly reduced, except for those of lipase and hepatopancreatic E-cells, which increased following challenge with V. parahaemolyticus. Histological assessment of the hepatopancreas cells revealed reduced cell degeneration following the administration of the plant extracts (expecially those fed 50 mg/g M. citrifolia) compared to that in the control group. Therefore, the enhanced immunity against V. parahaemolyticus infection in P. vannamei could be associated with the improved hepatopancreas health associated with M. citrifolia fruit extract supplementation.

ZntA maintains zinc and cadmium homeostasis and promotes oxidative stress resistance and virulence in Vibrio parahaemolyticus.

Although metals are essential for life, they are toxic to bacteria in excessive amounts. Therefore, the maintenance of metal homeostasis is critical for bacterial physiology and pathogenesis. Vibrio parahaemolyticus is a significant food-borne pathogen that mainly causes acute gastroenteritis in humans and acute hepatopancreatic necrosis disease in shrimp. Herein, we report that ZntA functions as a zinc (Zn) and cadmium (Cd) homeostasis mechanism and contributes to oxidative stress resistance and virulence in V. parahaemolyticus. zntA is remarkably induced by Zn, copper, cobalt, nickel (Ni), and Cd, while ZntA promotes V. parahaemolyticus growth under excess Zn/Ni and Cd conditions via maintaining Zn and Cd homeostasis, respectively. The growth of ΔzntA was inhibited under iron (Fe)-restricted conditions, and the inhibition was associated with Zn homeostasis disturbance. Ferrous iron supplementation improved the growth of ΔzntA under excess Zn, Ni or Cd conditions. The resistance of ΔzntA to H2O2-induced oxidative stress also decreased, and its virulence was attenuated in zebrafish models. Quantitative real-time PCR, mutagenesis, and ß-galactosidase activity assays revealed that ZntR positively regulates zntA expression by binding to its promoter. Collectively, the ZntR-regulated ZntA is crucial for Zn and Cd homeostasis and contributes to oxidative stress resistance and virulence in V. parahaemolyticus.

Genome-wide analysis of ATP-binding cassette (ABC) transporter in Penaeus vannamei and identification of two ABC genes involved in immune defense against Vibrio parahaemolyticus by affecting NF-κB signaling pathway.

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5, PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates.

QsvR and OpaR coordinately regulate the transcription of cpsS and cpsR in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, has a strong capacity to form biofilms on surfaces, which is strictly regulated by the CpsS-CpsR-CpsQ regulatory cascade. OpaR, a master regulator of quorum sensing, is a global regulator that controls multiple cellular pathways including biofilm formation and virulence. QsvR is an AraC-type regulator that works coordinately with OpaR to control biofilm formation and virulence gene expression of V. parahaemolyticus. QsvR and OpaR activate cpsQ transcription. OpaR also activates cpsR transcription, but lacks the detailed regulatory mechanisms. Furthermore, it is still unknown whether QsvR regulates cpsR transcription, as well as whether QsvR and OpaR regulate cpsS transcription. In this study, the results of quantitative real-time PCR and LacZ fusion assays demonstrated that deletion of qsvR and/or opaR significantly decreased the expression levels of cpsS and cpsR compared to the wild-type strain. However, the results of two-plasmid lacZ reporter and electrophoretic mobility-shift assays showed that both QsvR and OpaR were unable to bind the regulatory DNA regions of cpsS and cpsR. Therefore, transcription of cpsS and cpsR was coordinately and indirectly activated by QsvR and OpaR. This work enriched our knowledge on the regulatory network of biofilm formation in V. parahaemolyticus.

Characterization of the antibacterial and opsonic functions of the antimicrobial peptide LvCrustinVI from Litopenaeus vannamei.

Microbial drug resistance is becoming increasingly severe due to antibiotic abuse. The development and utilization of antimicrobial peptides is one of the important ways to solve this difficult problem. Crustins are a family of antimicrobial peptides that play important roles in the innate immune system of crustaceans. Several types of crustins exist in shrimp and their activities vary greatly. In the present study, we studied the immune function of one newly identified crustin and found that the type VI crustin encoding gene in Litopenaeus vannamei (LvCrustinVI) was mainly expressed in gills. Its expression was significantly up-regulated after Vibrio parahaemolyticus infection and knockdown of the gene promoted Vibrio proliferation in the hepatopancreas of shrimp, indicating that LvCrustinVI was involved in pathogens infection. The recombinant LvCrustinVI (rLvCrustinVI) showed strong inhibitory activities against both Gram-negative and Gram-positive bacteria, and exhibited binding activities with the bacteria and bacterial polysaccharides including Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVI showed a strong agglutination effect on V. parahaemolyticus and could significantly enhance the phagocytic ability of shrimp hemocytes against V. parahaemolyticus. In conclusion, LvCrustinVI played important roles as antimicrobial peptide and opsonin in the innate immune defense of L. vannamei. The study enriched our understanding of the functional activity of Crustin and provides an important basis for the development and utilization of antimicrobial peptides.

Assessing biofilm formation and resistance of vibrio parahaemolyticus on UV-aged microplastics in aquatic environments.

UV degradation of marine microplastics (MPs) could increase their vector potential for pathogenic bacteria and threaten human health. However, little is known about how the degree of UV aging affects interactions between MPs and pathogens and how various types of MPs differ in their impact on seafood safety. This study investigated five types of UV-aged MPs and their impact on Vibrio parahaemolyticus, a seafood pathogen. MPs exposed to UV for 60 days showed similar physicochemical changes such as surface cracking and hydrophobicity reduction. Regardless of the type, longer UV exposure of MPs resulted in more biofilm formation on the surface under the same conditions. V. parahaemolyticus types that formed biofilms on the MP surface showed 1.4- to 5.0-fold upregulation of virulence-related genes compared to those that did not form biofilms, independently of UV exposure. However, longer UV exposure increased resistance of V. parahaemolyticus on MPs to chlorine, heat, and human gastrointestinal environment. This study implies that the more UV degradation occurs on MPs, the more microbial biofilm formation is induced, which can significantly increase virulence and environmental resistance of bacteria regardless of the type of MP.

OmpU and OmpC are the key OMPs for Litopenaeus vannamei hemocyanin recognizes Vibrio parahaemolyticus.

Hemocyanin is a multifunctional protein present in arthropods and mollusks, responsible for oxygen transport and participating in multiple roles of immune defense including antibacterial activity. However, the molecular basis of how hemocyanin recognizes pathogens and exerts antibacterial activity remains poorly understood. In the present study, the pull-down assay was used to isolate Vibrio parahaemolyticus outer membrane proteins (OMPs) that bind to Litopenaeus vannamei hemocyanin. Two interacting OMPs bands were determined as OmpC and OmpU, and the heterogeneous interaction between hemocyanin and the two OMPs was further confirmed by far-Western blot. After construction of ompC and ompU deletion mutants, we found that the agglutinating activity and antibacterial activity of hemocyanin significantly decreased compared to the wild-type strain. After hemocyanin treatment, we identified four intracellular proteins of V. parahaemolyticus, including fructose-bisphosphate aldolase and ribosomal proteins could interact with rOmpC and rOmpU, respectively. Furthermore, we found that the mRNA levels of ompC, ompU, fbaA, rpsB and rpsC significantly decreased after hemocyanin treatment. These findings indicated that OmpC and OmpU are the key targets for L. vannamei hemocyanin recognize pathogens and exert its antibacterial activity.

The analysis of complete genome sequence and comparative genomics of Vibrio parahaemolyticus LF1113 in Hainan.

Vibrio parahaemolyticus is a Gram-negative, halophilic and polymorphic coccobacillus. It is world-widely distributed and has resulted in great economic losses since its first appearance. In this study, a pathogenic strain was isolated from diseased pearl gentian grouper and identified as V. parahaemolyticus based on the sequencing results of 16S rDNA gene. In order to gain a comprehensive understanding of this isolation, the whole genome sequencing was conducted. Phylogenetic analysis of the complete genomes of 16 Vibrio species showed that LF1113, ATCC17802, ATCC33787, 2210633, FORC 004, and 160807 were the most closely related. Animal experiments demonstrated that the isolated LF1113 strain was pathogenic in a fish model. This study is the first study to describe the complete genome sequence of a V. parahaemolyticus isolate, which infected pearl gentian grouper from an outbreak in a fish factory farm in Hainan. The results will expand our understanding of genetic characteristics, pathogenesis, diagnostics and disease prevention of V. parahaemolyticus, and lay the foundation for further study.

Characterization of a pseudohemocyanin gene (PtPhc1) and its immunity function in response to Vibrio parahaemolyticus infection in the swimming crab Portunus trituberculatus.

Pseudohemocyanin is a member of the hemocyanin superfamily, but little research is available on its function in immunology. In this study, a Portunus trituberculatus pseudohemocyanin gene, named PtPhc1, was obtained by gene cloning. The PtPhc1 cDNA was 2312 bp in length, encoding 684 amino acids while exhibiting a characteristic hemocyanin structural domain. Tissue expression analysis revealed ubiquitous expression of PtPhc1 across all tissues, with the highest level of expression observed in the hepatopancreas. The expression pattern of PtPhc1 in response to Vibrio parahaemolyticus infection was clarified using RT-qPCR in swimming crabs. Notably, the expression peaked at 24 h, and increased 1435-fold compared to the control group in the hepatopancreas. While the expression level reached the maximum value at 72 h, which was 3.24 times higher than that of the control group in hemocytes. Remarkably, the reduction in PtPhc1 expression led to a noteworthy 30% increase in the mortality rate of P. trituberculatus when exposed to V. parahaemolyticus. In addition, in vitro bacterial inhibition assays exhibited a dose-dependent suppression of bacterial proliferation by recombinant PtPhc1 protein, with a notable inhibition rate of 48.33% against V. parahaemolyticus at a concentration of 0.03 mg/mL. To the best of our knowledge, the results establish the function of pseudohaemocyanin in immunity for the first time, contributing to a deeper comprehension of innate immune regulatory mechanisms in aquatic organisms and advancing strategies for disease-resistant breeding.

Biological Function of Prophage-Related Gene Cluster ΔVpaChn25_RS25055~ΔVpaChn25_0714 of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is the primary foodborne pathogen known to cause gastrointestinal infections in humans. Nevertheless, the molecular mechanisms of V. parahaemolyticus pathogenicity are not fully understood. Prophages carry virulence and antibiotic resistance genes commonly found in Vibrio populations, and they facilitate the spread of virulence and the emergence of pathogenic Vibrio strains. In this study, we characterized three such genes, VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055, within the largest prophage gene cluster in V. parahaemolyticus CHN25. The deletion mutants ΔVpaChn25_RS25055, ΔVpaChn25_0713, ΔVpaChn25_0714, and ΔVpaChn25_RS25055-0713-0714 were derived with homologous recombination, and the complementary mutants ΔVpaChn25_0713-com, ΔVpaChn25_0714-com, ΔVpaChn25_RS25055-com, ΔVpaChn25_RS25055-0713-0714-com were also constructed. In the absence of the VpaChn25_RS25055, VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055-0713-0714 genes, the mutants showed significant reductions in low-temperature survivability and biofilm formation (p < 0.001). The ΔVpaChn25_0713, ΔVpaChn25_RS25055, and ΔVpaChn25_RS25055-0713-0714 mutants were also significantly defective in swimming motility (p < 0.001). In the Caco-2 model, the above four mutants attenuated the cytotoxic effects of V. parahaemolyticus CHN25 on human intestinal epithelial cells (p < 0.01), especially the ΔVpaChn25_RS25055 and ΔVpaChn25_RS25055-0713-0714 mutants. Transcriptomic analysis showed that 15, 14, 8, and 11 metabolic pathways were changed in the ΔVpaChn25_RS25055, ΔVpaChn25_0713, ΔVpaChn25_0714, and ΔVpaChn25_RS25055-0713-0714 mutants, respectively. We labeled the VpaChn25_RS25055 gene with superfolder green fluorescent protein (sfGFP) and found it localized at both poles of the bacteria cell. In addition, we analyzed the evolutionary origins of the above genes. In summary, the prophage genes VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055 enhance V. parahaemolyticus CHN25's survival in the environment and host. Our work improves the comprehension of the synergy between prophage-associated genes and the evolutionary process of V. parahaemolyticus.