ABSTRACT
Vibrio mimicus bacteria have caused sporadic cases and outbreaks of cholera-like diarrhea throughout the world, but the association of lineages with such events is unexplored. Genomic analyses revealed V. mimicus lineages carrying the virulence factors cholera toxin and toxin coregulated pilus, one of which has persisted for decades in China and the United States.
Subject(s)
Cholera Toxin , Genomic Islands , Vibrio mimicus , China/epidemiology , Humans , Vibrio mimicus/genetics , Vibrio mimicus/pathogenicity , United States/epidemiology , Cholera Toxin/genetics , Cholera/microbiology , Cholera/epidemiology , Phylogeny , Vibrio Infections/microbiology , Vibrio Infections/epidemiology , Virulence Factors/geneticsABSTRACT
Infection with Vibrio mimicus in the Siluriformes has demonstrated a rapid and high infectivity and mortality rate, distinct from other hosts. Our earlier investigations identified necrosis, an inflammatory storm, and tissue remodeling as crucial pathological responses in yellow catfish (Pelteobagrus fulvidraco) infected with V. mimicus. The objective of this study was to further elucidate the impact linking these pathological responses within the host during V. mimicus infection. Employing metabolomics and transcriptomics, we uncovered infection-induced dense vacuolization of perimysium; Several genes related to nucleosidase and peptidase activities were significantly upregulated in the skin and muscles of infected fish. Concurrently, the translation processes of host cells were impaired. Further investigation revealed that V. mimicus completes its infection process by enhancing its metabolism, including the utilization of oligopeptides and nucleotides. The high susceptibility of yellow catfish to V. mimicus infection was associated with the composition of its body surface, which provided a microenvironment rich in various nucleotides such as dIMP, dAMP, deoxyguanosine, and ADP, in addition to several amino acids and peptides. Some of these metabolites significantly boost V. mimicus growth and motility, thus influencing its biological functions. Furthermore, we uncovered an elevated expression of gangliosides on the surface of yellow catfish, aiding V. mimicus adhesion and increasing its infection risk. Notably, we observed that the skin and muscles of yellow catfish were deficient in over 25 polyunsaturated fatty acids, such as Eicosapentaenoic acid, 12-oxo-ETE, and 13-Oxo-ODE. These substances play a role in anti-inflammatory mechanisms, possibly contributing to the immune dysregulation observed in yellow catfish. In summary, our study reveals a host immune deviation phenomenon that promotes bacterial colonization by increasing nutrient supply. It underscores the crucial factors rendering yellow catfish highly susceptible to V. mimicus, indicating that host nutritional sources not only enable the establishment and maintenance of infection within the host but also aid bacterial survival under immune pressure, ultimately completing its lifecycle.
Subject(s)
Catfishes , Fish Diseases , Vibrio Infections , Vibrio mimicus , Animals , Catfishes/immunology , Catfishes/genetics , Fish Diseases/immunology , Fish Diseases/microbiology , Vibrio Infections/veterinary , Vibrio Infections/immunology , Vibrio mimicus/immunology , Disease Susceptibility/veterinary , Disease Susceptibility/immunology , Epidermis/immunology , Epidermis/microbiology , NutrientsABSTRACT
[This corrects the article DOI: 10.3389/fmicb.2023.1295065.].
ABSTRACT
Vibrio mimicus is a serious pathogen in aquatic animals, resulting in significant economic losses. The cAMP receptor protein (CRP) often acts as a central regulator in highly pathogenic pathogens. V. mimicus SCCF01 is a highly pathogenic strain isolated from yellow catfish; the crp gene deletion strain (Δcrp) was constructed by natural transformation to determine whether this deletion affects the virulence phenotypes. Their potential molecular connections were revealed by qRT-PCR analysis. Our results showed that the absence of the crp gene resulted in bacterial and colony morphological changes alongside decreases in bacterial growth, hemolytic activity, biofilm formation, enzymatic activity, motility, and cell adhesion. A cell cytotoxicity assay and animal experiments confirmed that crp contributes to V. mimicus pathogenicity, as the LD50 of the Δcrp strain was 73.1-fold lower compared to the WT strain. Moreover, qRT-PCR analysis revealed the inhibition of type II secretion system genes, flagellum genes, adhesion genes, and metalloproteinase genes in the deletion strain. This resulted in the virulence phenotype differences described above. Together, these data demonstrate that the crp gene plays a core regulatory role in V. mimicus virulence and pathogenicity.
ABSTRACT
Vibrio mimicus is a pathogenic bacterium that cause red body disease in Macrobrachium nipponense, leading to high mortality and financial loss. Based on previous studies, rpoS gene contribute to bacterial pathogenicity during infection, but the role of RpoS involved in the immune response of M. nipponense under V. mimicus infection remains unclear. In this study, the pathogen load and the RNA-seq of M. nipponense under wild-type and ΔrpoS strain V. mimicus infection were investigated. Over the entire infection period, the ΔrpoS strain pathogen load was always lower than that of the wild-type strain in the M. nipponense hemolymph, hepatopancreas, gill and muscle. Furthermore, the expression level of rpoS gene in the hepatopancreas was the highest at 24 hours post infection (hpi), then the samples of hepatopancreas tissue infected with the wild type and ΔrpoS strain at 24 hpi were selected for RNA-seq sequencing. The results revealed a significant change in the transcriptomes of the hepatopancreases infected with ΔrpoS strain. In contrast to the wild-type infected group, the ΔrpoS strain infected group exhibited differentially expressed genes (DEGs) enriched in 181 KEGG pathways at 24 hpi. Among these pathways, 8 immune system-related pathways were enriched, including ECM-receptor interaction, PI3K-Akt signaling pathway, Rap1 signaling pathway, Gap junction, and Focal adhesion, etc. Among these pathways, up-regulated genes related to Kazal-type serine protease inhibitors, S-antigen protein, copper zinc superoxide dismutase, tight junction protein, etc. were enriched. This study elucidates that rpoS can affect tissue bacterial load and immune-related pathways, thereby impacting the survival rate of M. nipponense under V. mimicus infection. These findings validate the potential of rpoS as a promising target for the development of a live attenuated vaccine against V. mimicus.
Subject(s)
Palaemonidae , Vibrio Infections , Vibrio mimicus , Animals , Palaemonidae/genetics , Phosphatidylinositol 3-Kinases/genetics , Transcriptome , Vibrio Infections/prevention & control , ImmunityABSTRACT
OBJECTIVES: South Asia remains home to foodborne diseases caused by the Vibrio species. We aimed to compile and update information on the epidemiology of vibriosis in South Asia. METHODS: For this systematic review and meta-analysis, we searched PubMed, Web of Science, EMBASE, and Google Scholar for studies related to vibriosis in South Asia published up to May 2023. A random-effects meta-analysis was used to estimate the pooled isolation rate of non-cholera-causing Vibrio species. RESULTS: In total, 38 studies were included. Seven of these were case reports and 22 were included in the meta-analysis. The reported vibriosis cases were caused by non-O1/non-O139 V. cholerae, V. parahaemolyticus, V. fluvialis, and V. vulnificus. The overall pooled isolation rate was 4.0% (95% confidence interval [CI] 3.0-5.0%) in patients with diarrhea. Heterogeneity was high (I2 = 98.0%). The isolation rate of non-O1/non-O139 V. cholerae, V. parahaemolyticus, and V. fluvialis were 9.0 (95% CI 7.0-10.0%), 1.0 (95% CI 1.0-2.0%), and 2.0 (95% CI: 1.0-3.0%), respectively. Regarding V. parahaemolyticus, O3:K6 was the most frequently isolated serotype. Cases peaked during summer. Several studies reported antibiotic-resistant strains and those harboring extended-spectrum beta-lactamases genes. CONCLUSIONS: This study demonstrates a high burden of infections caused by non-cholera-causing Vibrio species in South Asia.
Subject(s)
Vibrio Infections , Vibrio , Humans , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/pharmacology , Asia, Southern/epidemiology , Vibrio/isolation & purification , Vibrio/genetics , Vibrio/classification , Vibrio Infections/epidemiology , Vibrio Infections/microbiologyABSTRACT
Vibrio mimicus caused a seafood-associated outbreak in Florida, USA, in which 4 of 6 case-patients were hospitalized; 1 required intensive care for severe diarrhea. Strains were ctx-negative but carried genes for other virulence determinants (hemolysin, proteases, and types I-IV and VI secretion systems). Cholera toxin-negative bacterial strains can cause cholera-like disease.
Subject(s)
Cholera , Vibrio mimicus , Humans , Cholera/epidemiology , Florida/epidemiology , Vibrio mimicus/genetics , Disease Outbreaks , SeafoodABSTRACT
Vibrio mimicus (V. mimicus) is known to cause severe bacterial diseases with high mortality rates in fish, resulting in significant economic losses in the global aquaculture industry. Therefore, the objective of this study was to develop a safe and effective vaccine for protecting Carassius auratus (C. auratus) against V. mimicus infection. Recombinant Lactobacillus casei (L. casei) strains, Lc-pPG-612-OmpU and Lc-pPG-612-OmpU-CTB (surface-displayed), were constructed using a L. casei strain (ATCC 393) as an antigen delivery carrier and the cholera toxin B subunit (CTB) as an adjuvant. The two recombinant strains of L. casei were administered to C. auratus via oral immunization, and the protective efficacy of the oral vaccines was assessed. The results demonstrated that oral immunization with the two strains significantly increased the levels of nonspecific immune indicators in C. auratus, including alkaline phosphatase (AKP), lysozyme (LYS), acid phosphatase (ACP), complement 3 (C3), complement 4 (C4), lectin, and superoxide dismutase (SOD). Moreover, the experiment groups exhibited significant increases in specific immunoglobulin M (IgM) antibodies against OmpU, as well as the transcription of immune-related genes (ie., IL-1ß, TNF-α, IL-10, and TGF-ß), when compared to the control groups. Following infection of C. auratus with V. mimicus, the mortality rate of the recombinant L. casei-treated fish was observed to be lower compared to the control group. This finding suggests that recombinant L. casei demonstrates effective protection against V. mimicus infection in C. auratus. Furthermore, the addition of the immune adjuvant CTB was found to induce a more robust adaptive and innate immune response in C. auratus, resulting in reduced mortality after infection with V. mimicus.
Subject(s)
Carps , Lacticaseibacillus casei , Vibrio Infections , Vibrio mimicus , Animals , Goldfish , Bacterial Vaccines , Vibrio Infections/prevention & control , Vibrio Infections/veterinaryABSTRACT
Type II secretion systems (T2SS) are important molecular machines used by bacteria to transport a wide range of proteins across the outer membrane from the periplasm. Vibrio mimicus is an epidemic pathogen threats to both aquatic animals and human health. Our previous study demonstrates that T2SS deletion reduced virulence by 307.26 times in yellow catfish. However, the specific effects of T2SS-mediated extracellular protein secretion in V. mimicus, including its potential role in exotoxin secretion or other mechanisms, require further investigation. Through proteomics and phenotypic analyses, this study observed that the ΔT2SS strain exhibited significant self-aggregation and dynamic deficiency, with a notable negative correlation with subsequent biofilm formation. The proteomics analysis revealed 239 different abundances of extracellular proteins after T2SS deletion, including 19 proteins with higher abundance and 220 proteins with lower and even absent in the ΔT2SS strain. These extracellular proteins are involved in various pathways, such as metabolism, virulence factors expression, and enzymes. Among them, purine, pyruvate, and pyrimidine metabolism, and the Citrate cycle, were the primary pathways affected by T2SS. Our phenotypic analysis is consistent with these findings, suggesting that the decreased virulence of ΔT2SS strains is due to the effect of T2SS on these proteins, which negatively impacts growth, biofilm formation, auto-aggregation, and motility of V. mimicus. These results provide valuable insights for designing deletion targets for attenuated vaccines development against V. mimicus and expand our understanding of the biological functions of T2SS.
Subject(s)
Type II Secretion Systems , Animals , Humans , Type II Secretion Systems/genetics , Type II Secretion Systems/metabolism , Vaccines, Attenuated , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Virulence , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Introduction: The pathogenesis of Vibrio mimicus infection in yellow catfish (Pelteobagrus fulvidraco) remains poorly understood, particularly regarding the impact of infection with the pathogen on primary target organs such as the skin and muscle. Methods: In this study, we aim to analyze the pathological intricacies of the skin and muscle of yellow catfish after being infected with V. mimicus using a 1/10 LC50 seven-day post-infection model. Furthermore, we have utilized integrated bioinformatics to comprehensively elucidate the regulatory mechanisms and identify the key regulatory genes implicated in this phenomenon. Results: Our histopathological examination revealed significant pathological changes in the skin and muscle, characterized by necrosis and inflammation. Moreover, tissue remodeling occurred, with perimysium degeneration and lesion invasion into the muscle along the endomysium, accompanied by a transformation of type I collagen into a mixture of type I and type III collagens in the perimysium and muscle bundles. Our eukaryotic transcriptomic and 4D label-free analyses demonstrated a predominantly immune pathway response in both the skin and muscle, with downregulation observed in several cell signaling pathways that focused on focal adhesion-dominated cell signaling pathways. The upregulated genes included interleukins (IL)-1 and -6, chemokines, and matrix metallopeptidases (mmp)-9 and -13, while several genes were significantly downregulated, including col1a and col1a1a. Further analysis revealed that these pathways were differentially regulated, with mmp-9 and mmp-13 acting as the potential core regulators of cytokine and tissue remodeling pathways. Upregulation of NF-κB1 and FOSL-1 induced by IL-17C and Nox 1/2-based NADPH oxidase may have held matrix metallopeptidase and cytokine-related genes. Also, we confirmed these relevant regulatory pathways by qPCR and ELISA in expanded samples. Discussion: Our findings unequivocally illustrate the occurrence of a cytokine storm and tissue remodeling, mediated by interleukins, chemokines, and MMPs, in the surface of yellow catfish infected with V. mimicus. Additionally, we unveil the potential bidirectional regulatory role of MMP-9 and MMP-13. These results provide novel perspectives on the intricate immune response to V. mimicus infection in yellow catfish and highlight potential targets for developing therapies.
Subject(s)
Catfishes , Vibrio mimicus , Animals , Matrix Metalloproteinase 13 , Matrix Metalloproteinase 9 , Catfishes/genetics , Cytokine Release Syndrome , InterleukinsABSTRACT
Vibrio mimicus (V. mimicus) is a pathogenic bacterium that causes diseases in humans and various aquatic animals. A particularly efficient way to provide protection against V. mimicus is through vaccination. However, there are few commercial vaccines against V. mimics, especially oral vaccines. In our study, two surface-display recombinant Lactobacillus casei (L. casei) Lc-pPG-OmpK and Lc-pPG-OmpK-CTB were constructed using L. casei ATCC393 as an antigen delivery vector, outer membrane protein K (OmpK) of V. mimicus as an antigen, and cholera toxin B subunit (CTB) as a molecular adjuvant; furthermore, the immunological effects of recombinant L.casei in Carassius auratus (C. auratus) were assessed. The results indicated that oral recombinant L.casei Lc-pPG-OmpK and Lc-pPG-OmpK-CTB stimulated higher levels of serum-specific immunoglobulin M (IgM) and increased the activity of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LYS), lectin, C3, and C4 in C. auratus, compared with control groups (Lc-pPG group and PBS group). Furthermore, the expression of interleukin-1ß (IL-1ß), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and transforming growth factor-ß (TGF-ß) in the liver, spleen, head kidney, hind intestine and gills of C. auratus was significantly increased, compared with that in the controls. These results demonstrated that the two recombinant L. casei strains could effectively trigger humoral and cellular immunity in C. auratus. In addition, two recombinant L.casei strains were able to survive and colonize the intestine of C. auratus. Importantly, after being challenged with V. mimicus, C. auratus fed Lc-pPG-OmpK and Lc-pPG-OmpK-CTB exhibited greater survival rates than the controls (52.08% and 58.33%, respectively). The data showed that recombinant L. casei could elicit a protective immunological response in C. auratus. The effect of the Lc-pPG-OmpK-CTB group was better than that of the Lc-pPG-OmpK group, and Lc-pPG-OmpK-CTB was found to be an effective candidate for oral vaccination.
Subject(s)
Lacticaseibacillus casei , Vibrio mimicus , Humans , Animals , Lacticaseibacillus casei/genetics , Goldfish , Vaccination , Adjuvants, Immunologic , Recombinant ProteinsABSTRACT
Vibrio mimicus is a zoonotic pathogen that is widely distributed in aquatic habitats/environments (marine coastal water, estuaries, etc). The development of biocontrol agents for V. mimicus is imperative for the prevention and control of aquatic animal diseases and human food-borne infections. In this study, a broad-spectrum bacteriophage Vmp-1 was isolated from dealt aquatic product in a local market by double-layer agar plate method using V. mimicus CICC21613 as the host bacteria. Results indicated that Vmp-1, which belongs to the family Podoviridae, showed good pH tolerance (pH 3.0-12.0) and thermal stability (30-50 °C). The optimal multiplicity of infection (MOI) of Vmp-1 was 0.001 for a 20-min incubation and 100-min lysis period. Vmp-1 effectively controlled V. mimicus CICC21613 in LBS model (MOI = 0.0001, 0.001, 0.01, 0.1, 1) within 8 h. The full length of the Vmp-1 genome was 43,312 bp, with average GC content of 49.5%, and a total of 44 protein-coding regions. This study provides a novel phage strain that has the highest homology with vB_VpP_HA5 (GenBank: OK585159.1, 95.96%) for the development of biocontrol agents for V. mimicus.
Subject(s)
Bacteriophages , Vibrio mimicus , Vibrio , Animals , Humans , Bacteriophages/genetics , Genomics , Vibrio/genetics , Vibrio mimicus/genetics , Membrane Proteins/metabolismABSTRACT
HtpG, a bacterial homolog of the eukaryotic 90 kDa heat-shock protein (Hsp90), represents the simplest member of the heat shock protein family. While the significance of Hsp90 in fungal and cancer drug resistance has been confirmed, the role of HtpG in bacterial antibiotic resistance remains largely unexplored. This research aims to investigate the impact of the htpG gene on antibiotic resistance in Vibrio mimicus. Through the creation of htpG gene deletion and complementation strains, we have uncovered the essential role of htpG in regulating the structural integrity of the bacterial cell envelope. Our transcriptomics analysis demonstrates that the deletion of htpG increases the sensitivity of V. mimicus to antimicrobial peptides, primarily due to upregulated lipopolysaccharide synthesis, reduced glycerophospholipid content, and weakened efflux pumps activity. Conversely, reduced sensitivity to ß-lactam antibiotics in the ΔhtpG strain results from decreased peptidoglycan synthesis and dysregulated peptidoglycan recycling and regulation. Further exploration of specific pathway components is essential for a comprehensive understanding of htpG-mediated resistance mechanisms, aiding in the development of antimicrobial agents. To our knowledge, this is the first effort to explore the relationship between htpG and drug resistance in bacteria.
ABSTRACT
Vibrio mimicus is a bacterium that causes gastroenteritis in humans. This pathogen produces an enterotoxic hemolysin called V. mimicus hemolysin (VMH), which is secreted extracellularly as an inactive 80-kDa protoxin and converted to a 66-kDa mature toxin through cleavage between Arg151 and Ser152. The 56-kDa serine protease termed V. mimicus trypsin-like protease (VmtA) is known to mediate this maturating process. However, some strains including strain ES-20 does not possess the vmtA gene. In the present study, the vmtA-negative strains were found to have a replaced gene that encodes a 43-kDa (403 aa) precursor of a serine protease designated by VmtX (V. mimicus trypsin-like protease X). To examine whether VmtX is also involved in the maturation of VMH, VmtX was isolated from the culture supernatant of V. mimicus strain NRE-20, a metalloprotease-negative mutant constructed from strain ES-20. Concretely, the culture supernatant was fractionated with 70% saturated ammonium sulfate and subjected to affinity column chromatography using a HiTrap Benzamidine FF column. The analysis of the N-terminal amino acid sequences of the proteins in the obtained VmtX preparation indicated that the 39-kDa protein was active VmtX consisting of 371 aa (Ile33-Ser403). The VmtX preparation was found to activate pro-VMH through generation of the 66-kDa protein. Additionally, treatment of the VmtX preparation with serine protease inhibitors, such as leupeptin and phenylmethylsulfonyl fluoride, significantly suppressed the activities to hydrolyze the specific peptide substrate and to synthesize the 66-kDa toxin. These findings indicate that VmtX is the second protease that mediats the maturation of VMH.
Subject(s)
Hemolysin Proteins , Vibrio , Humans , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Peptide Hydrolases/genetics , Leupeptins , Ammonium Sulfate , Trypsin , Phenylmethylsulfonyl Fluoride , Metalloproteases , Serine Proteinase Inhibitors , Benzamidines , Vibrio/metabolismABSTRACT
Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in almost every aspect of metabolism, of both eukaryotes and prokaryotes. Several global lysine acetylome studies have been carried out in various bacteria, but thus far, there have been no reports of lysine acetylation for the commercially important aquatic animal pathogen Vibrio mimicus. In the present study, we used anti-Ac-K antibody beads to highly sensitive immune-affinity purification and combined high-resolution LC-MS/MS to perform the first global lysine acetylome analysis in V. mimicus, leading to the identification of 1,097 lysine-acetylated sites on 582 proteins, and more than half (58.4%) of the acetylated proteins had only one site. The analysis of acetylated modified peptide motifs revealed six significantly enriched motifs, namely, KacL, KacR, L(-2) KacL, LKacK, L(-7) EKac, and IEKac. In addition, bioinformatic assessments state clearly that acetylated proteins have a hand in many important biological processes in V. mimicus, such as purine metabolism, ribosome, pyruvate metabolism, glycolysis/gluconeogenesis, the TCA cycle, and so on. Moreover, 13 acetylated proteins were related to the virulence of V. mimicus. To sum up, this is a comprehensive analysis whole situation protein lysine acetylome in V. mimicus and provides an important foundation for in-depth study of the biological function of lysine acetylation in V. mimicus.
ABSTRACT
Vibrio mimicus (V. mimicus) is a pathogen causing serious vibriosis in aquatic animals. Hepcidin and ß-Defensin1 are two important antibacterial peptides (AMPs) with broad-spectrum antibacterial activity in fish. In mammals, some evidences demonstrated that interleukin-1ß (IL-1ß) primarily promote AMPs expression via activating classical NF-κB pathway, but it still remains unclear in fish. Here, the temporal and spatial expression patterns of grass carp IL-1ß (gcIL-1ß) gene and two AMPs genes (gchepcidin and gcß-defensin1) in tissues post-V. mimicus infection and anti-V. mimicus activity of these two AMPs in vitro were detected, showing that V. mimicus infection significantly elevated the mRNA levels of these three genes in the immune-related tissues although their expression patterns were not entirely consistent, and both gcHepcidin and gcß-Defensin1 possessed anti-V. mimicus activity in vitro. Subsequently, the recombinant gcIL-1ß (rgcIL-1ß) was expressed prokaryotically in an inclusion body, which could promote proliferation of grass carp head kidney leukocytes (gcHKLs) and enhance respiratory burst activity and phagocytic activity of head kidney macrophages. Stimulation with rgcIL-1ß was able to significantly regulate the mRNA expression of key regulatory genes (il-1RI, traf6, tak1, ikkß, iκBα and p65) involved in the activation of classical NF-κB pathway, and then induce gcTAK1 phosphorylation, promote gcp65 nuclear translocation and enhance endogenous gcIL-1ß expression at both mRNA and protein levels, implying NF-κB pathway was activated. More importantly, exogenous rgcIL-1ß stimulation also significantly up-regulated both gcHepcidin and gcß-Defensin1 mRNA levels against V. mimicus, and the regulatory effect was blocked or inhibited by NF-κB inhibitor PDTC. Taken together, our results demonstrated for the first time that grass carp IL-1ß stimulation could significantly enhance the expression of these two anti-V.mimicus AMPs via activating classical NF-κB pathway.
Subject(s)
Carps , Fish Diseases , Vibrio mimicus , Animals , Antimicrobial Peptides , Carps/genetics , Carps/metabolism , Fish Diseases/microbiology , Fish Proteins/genetics , Fish Proteins/metabolism , Interleukin-1beta/genetics , Mammals/metabolism , NF-kappa B/metabolism , Signal Transduction/geneticsABSTRACT
Vibrio mimicus collagenase (VMC), a Class II Vibrio metalloprotease, contains an HEXXH motif in a zinc-binding catalytic domain, and two FAXWXXT motifs in its C-terminal domain, which is its collagen binding domain (CBD). To understand the functional role of the individual CBD motifs in the activity of VMC, if any, we created and characterized a series of VMC variants: i) VMA, with 51 amino acids deleted from the C-terminal end of full-length VMC; ii) VMT1, a form of VMA mutated in the first CBD motif; iii) VMT2, a form of VMA mutated in the second CBD motif; iv) DM, a form of VMA with both CBD motifs mutated; v) CT, a truncated form of VMA, lacking the entire CBD region; and vi) CBD, a construct containing the collagen binding domain alone. The activity of each variant was assessed by multiple means, in relation to VMA. We report that VMT1 and VMT2 show 1.6-fold and 10-fold reduced activity, respectively. The reduced activity of VMT2 correlates with reduced binding to insoluble collagen as well as an inability to cause structural perturbation of collagen. VMC appears to cause unwinding and structural alteration of the collagen triple helix prior to hydrolysis of the substrate (using both motifs for collagen binding), like Clostridium collagenases. In the absence of a known structure for VMC, our findings suggest that Vibrio collagenase, functions like Clostridium collagenases, although the two show very little sequence similarity. Also, VMC shows reduced activity with respect to Clostridium collagenases, making it an ideal enzyme for therapeutic applications.
Subject(s)
Vibrio mimicus , Vibrio , Collagen/genetics , Collagenases/genetics , Hydrolysis , Vibrio mimicus/geneticsABSTRACT
The outer membrane protein U (OmpU) is a conserved outer membrane protein in a variety of pathogenic Vibrio species and has been considered as a vital protective antigen for vaccine development. Vibrio mimicus (V. mimicus) is the pathogen causing ascites disease in aquatic animals. In this study, the prokaryotically expressed and purified His-tagged OmpU of V. mimicus (His-OmpU) was used as a subunit vaccine. The formalin inactivated V. mimicus, purified His tag (His-tag), and PBS were used as controls. The vaccinated yellow catfish were challenged with V. mimicus at 28 days post-vaccination, and the results showed that the His-OmpU and inactivated V. mimicus groups exhibited much higher survival rates than the His-tag and PBS groups. To fully understand the underlying mechanism, we detected the expression levels of several immune-related genes in the spleen of fish at 28 days post-vaccination and 24 h post-challenge. The results showed that most of the detected immune-related genes were significantly upregulated in His-OmpU and inactivated V. mimicus groups. In addition, we performed the serum bactericidal activity assay, and the results showed that the serum from His-OmpU and inactivated V. mimicus groups exhibited much stronger bactericidal activity against V. mimicus than those of His-tag and PBS groups. Finally, the serum agglutination antibody was detected, and the antibody could be detected in His-OmpU and inactivated V. mimicus groups with the antibody titers increasing along with the time post-vaccination, but not in His-tag or PBS group. Our data reveal that the recombinant OmpU elicits potent protective immune response and is an effective vaccine candidate against V. mimicus in yellow catfish.
Subject(s)
Adhesins, Bacterial/immunology , Bacterial Vaccines/immunology , Fish Diseases/immunology , Immunogenicity, Vaccine , Vibrio Infections/veterinary , Vibrio mimicus/immunology , Animals , Catfishes , Vaccines, Subunit/immunology , Vibrio Infections/immunologyABSTRACT
Vibrio mimicus is a foodborne pathogen, which is widely distributed in the aquatic environment. Moreover, it is often involved in aquatic animal diseases. In recent years, V. mimicus is an emerging pathogen in some species of Siluriformes. The strain SCCF01 was isolated from yellow catfish (Pelteobagrus fulvidraco). In this study, we aimed to perform genomic analysis of V. mimicus strain SCCF01 to identify genetic features and evolutionary relationships. Information on gene function and classification was obtained by functional annotation, and circular graph of strain SCCF01 genome, which was created by Circos v0.64. Information on virulence genes (adhesion, flagellum system, exotoxin, and secretory system, etc.) was obtained by virulence genes annotation. Genome element prediction showed that most of the mobile elements were distributed in chromosome I. Therefore, chromosome I of SCCF01 genome has more plasticity than chromosome II and might be larger in size. Genomic linear relationship between the strain of V. mimicus and strain SCCF01 was analyzed by linear pairwise comparison but was unable to determine the relationship. Gene family analysis predicted that the evolutionary direction of strain SCCF01 was: clinical strain â environmental strain â SCCF01 strain. Phylogenetic analysis showed that the strain SCCF01 was more closely related to environmental strains. According to gene family analysis and phylogenetic analysis, we speculated that strain SCCF01 has probably diverged from environmental strains.
Subject(s)
Catfishes/microbiology , Vibrio mimicus/genetics , Vibrio mimicus/isolation & purification , Virulence Factors/genetics , Animals , Bacterial Adhesion/genetics , Exotoxins/genetics , Flagella/genetics , Fresh Water , Genes, Bacterial/genetics , Genomics , Interspersed Repetitive Sequences/genetics , Phylogeny , Vibrio mimicus/classification , Vibrio mimicus/pathogenicity , Virulence/geneticsABSTRACT
Vibrio mimicus (V. mimicus) is a significant pathogen in freshwater catfish, though knowledge of virulence determinants and effective vaccine is lacking. Multiplex genome editing by natural transformation (MuGENT) is an easy knockout method, which has successfully used in various bacteria except for V. mimicus. Here, we found V. mimicus strain SCCF01 can uptake exogenous DNA and insert it into genome by natural transformation assay. Subsequently, we exploited this property to make five mutants (â³Hem, â³TS1, â³TS2, â³TS1â³TS2, and â³II), and removed the antibiotic resistance marker by Flp-recombination. Finally, all of the mutants were identified by PCR and RT-PCR. The results showed that combination of natural transformation and FLP-recombination can be applied successfully to generate targeted gene disruptions without the antibiotic resistance marker in V. mimicus. In addition, the five mutants showed mutant could be inherited after several subcultures and a 668-fold decrease in the virulence to yellow catfish (Pelteobagrus fulvidraco). This study provides a convenient method for the genetic manipulation of V. mimicus. It will facilitate the identification and characterization of V. mimicus virulence factors and eventually contribute to a better understanding of V. mimicus pathogenicity and development of attenuated vaccine.