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Sumio Shinoda - One of the best experts on this subject based on the ideXlab platform.
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A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin
Microbiology and immunology, 2014Co-Authors: Tamaki Mizuno, Sumio Shinoda, Yoko Maehara, Ayako Nanko, Shin-ichi MiyoshiAbstract:Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N-terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin-like substrate specificity was purified from the bacterial culture supernatant. The N-terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg(151)-Ser(152) bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.
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Modulation of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease.
The FEBS journal, 2009Co-Authors: Tamaki Mizuno, Sumio Shinoda, Syed Zafar Sultan, Yoshimi Kaneko, Tomonaga Yoshimura, Yoko Maehara, Hiroshi Nakao, Tomofusa Tsuchiya, Shin-ichi MiyoshiAbstract:Vibrio mimicus is a causative agent of human gastroenteritis and food poisoning, and this species produces an enterotoxic hemolysin (V. mimicus hemolysin) as a virulence determinant. Vibrio mimicus hemolysin is secreted as an 80 kDa precursor, which is later converted to the 66 kDa mature toxin through removal of an N-terminal propeptide via cleavage of the Arg151–Ser152 bond. In this article, we investigate the role of the endogenous metalloprotease (V. mimicus protease) in the maturation of V. mimicus hemolysin. In vitro experiments using purified proteins showed that, although it activated the precursor at the early stage via cleavage of the Asn157–Val158 bond, V. mimicus protease finally converted the activated and physiologically maturated toxin to a 51 kDa protein through removal of the C-terminal polypeptide. This 51 kDa derivative was unable to lyse erythrocytes because of its inability to bind to the erythrocyte membrane. Vibrio mimicus protease-negative strains were found to produce high levels of V. mimicus hemolysin at the logarithmic phase of bacterial growth and maintained high hemolytic activity even at the stationary phase. These findings indicate that, although it is not directly related to toxin maturation in vivo, V. mimicus protease can modulate the activity of V. mimicus hemolysin and/or its precursor through limited proteolysis.
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Haemolysin produced by Vibrio mimicus activates two Cl- secretory pathways in cultured intestinal-like Caco-2 cells.
Cellular microbiology, 2006Co-Authors: Akira Takahashi, Shin-ichi Miyoshi, Sumio Shinoda, Noriko Takata, Masayuki Nakano, Akiko Hamamoto, Kazuaki Mawatari, Nagakatsu Harada, Yutaka NakayaAbstract:Summary Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH-induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3′,5′-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl– channel inhibitors 4,4′-disothiocyanatostibene-2,2′-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl– by activating both Ca2+-dependent and cAMP-dependent Cl– secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl– secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl– secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.
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presence of luxs ai 2 based quorum sensing system in Vibrio mimicus luxo controls protease activity
Microbiology and Immunology, 2006Co-Authors: Zafar Sultan, Shin-ichi Miyoshi, Sumio ShinodaAbstract:Presence of the quorum-sensing regulation system in Vibrio mimicus was investigated. The culture supernatants of V. mimicus strains were found to possess AI-2 autoinducer like activity, and the strains were found to harbor the genes which are homologous to luxS, luxO, and luxR of V. harveyi. These genes of V. harveyi have been shown to be important components of V. harveyi-like quorum-sensing system. The luxO gene homologue known to encode LuxO, the central component of the regulation system, was disrupted, and effects on protease and hemolysin activity were studied. Disruption of luxO gene resulted in the increased protease activity, but the hemolysin activity did not vary considerably.
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Proteolytic activation of Vibrio mimicus (Vm) major outer membrane protein haemagglutinin (HA) with Vm-HA/protease: Implication for understanding bacterial adherence.
Microbiology and immunology, 2006Co-Authors: Munirul Alam, Shin-ichi Miyoshi, Kabir Uddin Ahmed, Nur A. Hasan, Ken Ichi Tomochika, Sumio ShinodaAbstract:Vibrio mimicus (Vm) haemagglutinins (HAs), such as an extracellular HA/protease (Vm-HA/protease) and a major outer membrane protein-HA (Vm-OMPHA), have been recognized as the putative adherence factors for the bacterium. However, the mechanism by which HAs coordinate the adherence function of the bacterium remains as yet unknown. We report herein the positive interaction between Vm-HA/protease and Vm-OMPHA resulting in significant enhancement of the haemagglutinating ability. In this interaction, no cleaved polypeptide was detected; however, limited proteolysis of Vm-OMPHA was confirmed by SDS-PAGE. The proteolytic activation of the native cell-associated Vm-OMPHA by limited proteolysis was also demonstrated in several V. mimicus strains. Proteolytic activation of OMPHA was also achieved with various proteases from bacterial and eukaryotic sources. These findings may indicate a novel coordination of V. mimicus HAs in the adherence of the bacterium.
Shin-ichi Miyoshi - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Vibrio mimicus metalloprotease (VMP) production by the quorum-sensing master regulatory protein, LuxR
Journal of basic microbiology, 2016Co-Authors: El Shaymaa Abdel-sattar, Shin-ichi Miyoshi, Abdelaziz ElgamlAbstract:Vibrio mimicus is an estuarine bacterium, while it can cause severe diarrhea, wound infection, and otitis media in humans. This pathogen secretes a relatively important toxin named V. mimicus metalloprotease (VMP). In this study, we clarified regulation of the VMP production according to the quorum-sensing master regulatory protein named LuxR. First, the full length of luxR gene, encoding LuxR, was detected in V. mimicus strain E-37, an environmental isolate. Next, the putative consensus binding sequence of LuxR protein could be detected in the upstream (promoter) region of VMP encoding gene, vmp. Finally, the effect of disruption of luxR gene on the expression of vmp and production of VMP was evaluated. Namely, the expression of vmp was significantly diminished by luxR disruption and the production of VMP was severely altered. Taken together, here we report that VMP production is under the positive regulation of the quorum-sensing master regulatory protein, LuxR.
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A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin
Microbiology and immunology, 2014Co-Authors: Tamaki Mizuno, Sumio Shinoda, Yoko Maehara, Ayako Nanko, Shin-ichi MiyoshiAbstract:Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N-terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin-like substrate specificity was purified from the bacterial culture supernatant. The N-terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg(151)-Ser(152) bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.
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Modulation of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease.
The FEBS journal, 2009Co-Authors: Tamaki Mizuno, Sumio Shinoda, Syed Zafar Sultan, Yoshimi Kaneko, Tomonaga Yoshimura, Yoko Maehara, Hiroshi Nakao, Tomofusa Tsuchiya, Shin-ichi MiyoshiAbstract:Vibrio mimicus is a causative agent of human gastroenteritis and food poisoning, and this species produces an enterotoxic hemolysin (V. mimicus hemolysin) as a virulence determinant. Vibrio mimicus hemolysin is secreted as an 80 kDa precursor, which is later converted to the 66 kDa mature toxin through removal of an N-terminal propeptide via cleavage of the Arg151–Ser152 bond. In this article, we investigate the role of the endogenous metalloprotease (V. mimicus protease) in the maturation of V. mimicus hemolysin. In vitro experiments using purified proteins showed that, although it activated the precursor at the early stage via cleavage of the Asn157–Val158 bond, V. mimicus protease finally converted the activated and physiologically maturated toxin to a 51 kDa protein through removal of the C-terminal polypeptide. This 51 kDa derivative was unable to lyse erythrocytes because of its inability to bind to the erythrocyte membrane. Vibrio mimicus protease-negative strains were found to produce high levels of V. mimicus hemolysin at the logarithmic phase of bacterial growth and maintained high hemolytic activity even at the stationary phase. These findings indicate that, although it is not directly related to toxin maturation in vivo, V. mimicus protease can modulate the activity of V. mimicus hemolysin and/or its precursor through limited proteolysis.
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Role of the Enterotoxic Hemolysin in Pathogenicity of Vibrio mimicus
Journal of Health Science, 2008Co-Authors: Akiko Kobayashi, Tomonaga Yoshimura, Yoko Maehara, Tomofusa Tsuchiya, Noriko Takata, Shin-ichi MiyoshiAbstract:Vibrio mimicus (V. mimicus) is a causative agent of human gastroenteritis and food poisoning. Although several toxic or virulence factors have been isolated from the bacterium, an enterotoxic hemolysin is a sole toxin produced by all clinical isolates. In the present study, we found that the antibody against the hemolysin significantly inhibited the fluid-accumulating action of the living cells inoculated into a rabbit ileal loop, and that the hemolysin gene (vmhA) was probably expressed by the bacterium in the ileal loop. Additionally, in spit of the comparable motility and similar proteome profiles, a vmhA mutant revealed the reduced fluid-accumulating activity. Theses findings suggest that the hemolysin contributes to full virulence of V. mimicus.
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Growth Phase Dependant Activation of the Precursor of Vibrio mimicus Hemolysin (Pro-VMH)
Journal of Health Science, 2007Co-Authors: Zafar Sultan, Tamaki Mizuno, Noriko Takata, Aki Sakurai, Keinosuke Okamoto, Shin-ichi MiyoshiAbstract:Vibrio mimicus (V. mimicus), a causative agent of gastroenteritis and food poisoning, secretes a 63-kDa enterotoxic hemolysin as the most potent virulence factor. ThevmhA gene encoding an 83-kDa precursor of the hemolysin was expressed from the early to late log phase of the bacterial growth, and the 79-kDa inactive protoxin was detected from the culture supernatant in the same growth phase. The N-terminal amino acid sequence of the protoxin was determined to be NH2-Asn-Ile-Ser-Asp-Pro-Val indicating cleavage of the Ala 25 -Asn 26 bond by a signal peptidase. In contrast, the hemolytic activity and the mature hemolysin in the culture supernatant were detected only at th el ate log phase. The maturation of the hemolysin, therefore, is suggested to be achieved by two-step processing, cleavage of the signal peptide followed by the growth phase-dependent removal of the 16-kDa propeptide.
In-soo Kong - One of the best experts on this subject based on the ideXlab platform.
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Expression of a fusion protein containing human epidermal growth factor and the collagen-binding domain of Vibrio mimicus metalloprotease
Biotechnology Letters, 2009Co-Authors: Dong-gyun Kim, Mun-kyeong Min, Soon Cheol Ahn, Joong Kyun Kim, In-soo KongAbstract:Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.
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Expression of a fusion protein containing human epidermal growth factor and the collagen-binding domain of Vibrio mimicus metalloprotease.
Biotechnology letters, 2008Co-Authors: Dong-gyun Kim, Mun-kyeong Min, Soon Cheol Ahn, Joong Kyun Kim, In-soo KongAbstract:Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.
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The FAXWXXT motif in the carboxyl terminus of Vibrio mimicus metalloprotease is involved in binding to collagen.
FEBS letters, 2005Co-Authors: Jong-hee Lee, Sun-hee Ahn, Eun-mi Lee, Seung-ha Jeong, Young-ok Kim, Sang-jun Lee, In-soo KongAbstract:We have shown previously that the C-terminal region of the extracellular metalloprotease of Vibrio mimicus (VMC) is essential for collagenase activity. Here, we demonstrate that deletion of 100 amino acids, but not 67 amino acids, from the C-terminus of the intact VMC protein (VMC61) abolished the collagenase activity. The intervening 33-amino acid region contains a repeated FAXWXXT motif that is essential for insoluble type I collagen binding; the isolated 33-amino acid peptide bound to insoluble type I collagen, while a peptide containing only the first FAXWXXT motif did not. Compared to the VMC61, the 33-amino acid peptide corresponding to the C-terminus exhibited a similar binding affinity and a lower binding capacity.
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Characterization of the enzyme activity of an extracellular metalloprotease (VMC) from Vibrio mimicus and its C‐terminal deletions
FEMS microbiology letters, 2003Co-Authors: Jong-hee Lee, Sun-hee Ahn, Eun-mi Lee, Young-ok Kim, Sang-jun Lee, In-soo KongAbstract:To investigate the enzymatic properties of Vibrio mimicus metalloprotease, the mature metalloprotease gene (vmc) was overexpressed in Escherichia coli and the recombinant protein (rVMC61) was purified by metal affinity chromatography. rVMC61 showed maximum activity at about 37°C, pH 8. The purified rVMC61 was very specific toward collagen substrates, such as gelatin, type I, II, and III collagens and synthetic peptides (Cbz-GPLGP and Cbz-GPGGPA). But it did not show degrading activity toward other biological proteins including lysozyme, lactoferrin and bovine serum albumin. rVMC61 also showed cytotoxicity against CHSE-214 fish cells. To examine the role of the C-terminal region of rVMC61, the 3′ end of the metalloprotease gene (vmc) was digested serially with exonuclease III. The truncated vmc derivatives encoding 57–42 kDa of the protease were isolated and overexpressed in E. coli. The collagenase activities of truncated proteins were investigated using gelatin as substrate. Deletion of 100 amino acids from the C-terminus resulted in loss of gelatin degrading activity. However, deletion of 67 amino acids from the C-terminus did not affect its gelatin degrading activity.
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characterization of the enzyme activity of an extracellular metalloprotease vmc from Vibrio mimicus and its c terminal deletions
Fems Microbiology Letters, 2003Co-Authors: Jong-hee Lee, Sun-hee Ahn, Eun-mi Lee, Young-ok Kim, Sang-jun Lee, In-soo KongAbstract:To investigate the enzymatic properties of Vibrio mimicus metalloprotease, the mature metalloprotease gene (vmc) was overexpressed in Escherichia coli and the recombinant protein (rVMC61) was purified by metal affinity chromatography. rVMC61 showed maximum activity at about 37°C, pH 8. The purified rVMC61 was very specific toward collagen substrates, such as gelatin, type I, II, and III collagens and synthetic peptides (Cbz-GPLGP and Cbz-GPGGPA). But it did not show degrading activity toward other biological proteins including lysozyme, lactoferrin and bovine serum albumin. rVMC61 also showed cytotoxicity against CHSE-214 fish cells. To examine the role of the C-terminal region of rVMC61, the 3′ end of the metalloprotease gene (vmc) was digested serially with exonuclease III. The truncated vmc derivatives encoding 57–42 kDa of the protease were isolated and overexpressed in E. coli. The collagenase activities of truncated proteins were investigated using gelatin as substrate. Deletion of 100 amino acids from the C-terminus resulted in loss of gelatin degrading activity. However, deletion of 67 amino acids from the C-terminus did not affect its gelatin degrading activity.
Yi Geng - One of the best experts on this subject based on the ideXlab platform.
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Complete genome analysis of Vibrio mimicus strain SCCF01, a highly virulent isolate from the freshwater catfish.
Virulence, 2019Co-Authors: Erlong Wang, Yi Geng, Kaiyu Wang, Defang Chen, Xiaoli Huang, Ping Ouyang, Zhicai Zuo, Chao Huang, Jing FangAbstract: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 → environmental strain → 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.
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Multiplex genome editing by natural transformation in Vibrio mimicus with potential application in attenuated vaccine development.
Fish & shellfish immunology, 2019Co-Authors: Erlong Wang, Yi Geng, Kaiyu Wang, Defang Chen, Xiaoli Huang, Ping Ouyang, Zhicai Zuo, Li TangAbstract: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 (△Hem, △TS1, △TS2, △TS1△TS2, and △II), 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.
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Complete genome sequence of Vibrio mimicus strain SCCF01 with potential application in fish vaccine development
Virulence, 2016Co-Authors: Yi Geng, Kaiyu Wang, Defang Chen, Xiaoli Huang, Guangneng PengAbstract:Vibrio mimicus strain SCCF01 was isolated from the yellow catfish (Pelteobagrus fulvidraco) during a mass mortality event at an aquaculture facility. We sequenced the genome of strain SCCF01, then ...
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Outbreaks of Vibriosis associated with Vibrio mimicus in freshwater catfish in China
Aquaculture, 2014Co-Authors: Yi Geng, Xiaoli Huang, D. Liu, S. Han, Yaojia Zhou, K.y. Wang, D.f. Chen, Xi Peng, Weiming LaiAbstract:Abstract An epidemic with a high mortality rate (80%–100%) recently occurred in yellow catfish, southern catfish, and Zhengchuan catfish in China. The clinical and pathological features were similar to those previously described for fish infected with Vibriosis. Six bacterial strains were isolated from the different affected catfish species between 2011 and 2013. Based on the phenotypic characteristics, 16S rRNA and gyrB gene sequence analysis, and multiplex PCR detection, all isolated strains were identified as Vibrio mimicus. The hemolysin gene was detected in all of the V. mimicus isolates, whereas none possessed the ctxA, zot, ace, or tcpA gene. The virulence tests were conducted by intraperitoneally injecting the V. mimicus isolates into healthy fish, and the results showed that all of the isolates were lethal to yellow catfish, southern catfish, and Zhengchuan catfish. The challenged fish showed similar clinical symptoms as those that were naturally infected. The results of this investigation indicate that V. mimicus was the causative agent of the natural epizootic. This is the first report of V. mimicus infection associated with mass mortality in freshwater catfish.
Xiaoli Huang - One of the best experts on this subject based on the ideXlab platform.
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Complete genome analysis of Vibrio mimicus strain SCCF01, a highly virulent isolate from the freshwater catfish.
Virulence, 2019Co-Authors: Erlong Wang, Yi Geng, Kaiyu Wang, Defang Chen, Xiaoli Huang, Ping Ouyang, Zhicai Zuo, Chao Huang, Jing FangAbstract: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 → environmental strain → 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.
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Multiplex genome editing by natural transformation in Vibrio mimicus with potential application in attenuated vaccine development.
Fish & shellfish immunology, 2019Co-Authors: Erlong Wang, Yi Geng, Kaiyu Wang, Defang Chen, Xiaoli Huang, Ping Ouyang, Zhicai Zuo, Li TangAbstract: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 (△Hem, △TS1, △TS2, △TS1△TS2, and △II), 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.
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Complete genome sequence of Vibrio mimicus strain SCCF01 with potential application in fish vaccine development
Virulence, 2016Co-Authors: Yi Geng, Kaiyu Wang, Defang Chen, Xiaoli Huang, Guangneng PengAbstract:Vibrio mimicus strain SCCF01 was isolated from the yellow catfish (Pelteobagrus fulvidraco) during a mass mortality event at an aquaculture facility. We sequenced the genome of strain SCCF01, then ...
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Outbreaks of Vibriosis associated with Vibrio mimicus in freshwater catfish in China
Aquaculture, 2014Co-Authors: Yi Geng, Xiaoli Huang, D. Liu, S. Han, Yaojia Zhou, K.y. Wang, D.f. Chen, Xi Peng, Weiming LaiAbstract:Abstract An epidemic with a high mortality rate (80%–100%) recently occurred in yellow catfish, southern catfish, and Zhengchuan catfish in China. The clinical and pathological features were similar to those previously described for fish infected with Vibriosis. Six bacterial strains were isolated from the different affected catfish species between 2011 and 2013. Based on the phenotypic characteristics, 16S rRNA and gyrB gene sequence analysis, and multiplex PCR detection, all isolated strains were identified as Vibrio mimicus. The hemolysin gene was detected in all of the V. mimicus isolates, whereas none possessed the ctxA, zot, ace, or tcpA gene. The virulence tests were conducted by intraperitoneally injecting the V. mimicus isolates into healthy fish, and the results showed that all of the isolates were lethal to yellow catfish, southern catfish, and Zhengchuan catfish. The challenged fish showed similar clinical symptoms as those that were naturally infected. The results of this investigation indicate that V. mimicus was the causative agent of the natural epizootic. This is the first report of V. mimicus infection associated with mass mortality in freshwater catfish.
