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1.
Aims: To characterize the luxO gene in fish pathogen Vibrio alginolyticus MVP01 and investigate its roles in regulation of extracellular products (ECP) and siderophore production. Methods and Results: The luxO gene was cloned from V. alginolyticus MVP01. Genetic analysis revealed that it encoded a protein with high similarity to other LuxO homologues. The luxO in-frame deletion mutant and rpoN null mutant were constructed with suicide plasmids. We demonstrated that sole deletion in LuxO increased the secretion of extracellular protease and haemolytic products, but decreased siderophore production for V. alginolyticus MVP01. Mutants with null rpoN displayed significantly enhanced protease level and siderophore production while notable reduction in haemolytic activities of ECP. Conclusions: Vibrio alginolyticus harbours functional luxO gene that regulates the secretion of extracellular protease and haemolytic materials as well as siderophore production in either σ54 dependent or independent manners. Significance and Impact of the Study: The current study demonstrated that V. alginolyticus MVP01 produces extracellular protease and haemolytic activity material as well as siderophore, which may be characteristics of the virulence of the strain. Revelations that secretion of these products is under the regulation of LuxO and σ54 as well as the potential quorum sensing systems in V. alginolyticus MVP01 will expedite the understanding of vibriosis pathogenesis. 相似文献
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Chen C Wang QB Liu ZH Zhao JJ Jiang X Sun HY Ren CH Hu CQ 《Antonie van Leeuwenhoek》2012,101(2):281-288
toxR, a conserved virulence-associated gene in vibrios, is identified in Vibrio alginolyticus ZJ51-O, a pathogenic strain isolated from diseased fish. To reveal the role of ToxR in the pathogenicity of V. alginolyticus, a deletion mutant was constructed by allelic exchange. The mutant showed the same level of growth in trypticase soy broth
(TSB) and iron-limiting condition, as the wild type strain. However, deletion of toxR severely reduced resistance against bile salts and the capability of biofilm formation. Outer-membrane protein (OMP) analysis
showed that a 37-kD protein was absent and a 43-kD protein was decreased in the mutant. By MS/MS, the two proteins are identified
as the homologues of OmpT and OmpN, respectively. These data suggest that ToxR might have enhanced the bile resistance and
biofilm formation through modulating the production of OMP without affecting the ability of iron acquisition and the virulence
to the fish via injection. These results indicate that ToxR may assist V. alginolyticus to colonize on the surface of the fish intestine which is crucial for the initiation of the infection, though it may not
be involved in the proliferation of the bacteria in the host tissue. 相似文献
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The bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins and plays pleiotropic roles
in growth, motility, and the secretion of some virulent factors. In this study, the authors identified the Tat gene cluster
in fish pathogen Vibrio alginolyticus and explored its roles in pathogenesis toward fish. Vibrio alginolyticus Tat mutants showed growth deficiency in TMAO medium, while the complement strain restored the ability to grow in the medium,
demonstrating the conservative function of the Tat system in translocation of redox enzymes or cofactors in this bacterium.
In V. alginolyticus, deletion of the tatABC genes led to a drastic decrease in biofilm biogenesis. Interestingly, the secretion of extracellular protease Asp, an established
exotoxin of the bacterium, was significantly decreased in the TatC mutant, suggesting that TatC might play a part in the production
of virulence factors in the bacterium. Furthermore, the Tat mutants displayed attenuated virulence toward the fish model and
EPC cells. These findings suggest that the Tat secretion related to the extracellular protease activity as well as virulence
in V. alginolyticus provided new insights into the pathogenesis of vibriosis in fish. 相似文献
4.
A previous study has shown that Vibrio alginolyticus ZJ-51 undergoes colony phase variation between opaque/rugose (Op) and translucent/smooth (Tr). The AI-2 quorum-sensing master regulator ValR, a homolog to V. harveyi LuxR, was suggested to be involved in the transition. To investigate the role of ValR in the variation and in biofilm formation, an in-frame deletion of valR in both Op and Tr backgrounds was carried out. The mutants in both backgrounds showed an intermediate colony morphotype, where the colonies were less opaque/rugose but not fully translucent/smooth either. They also showed an intermediate level of motility. However, biofilm formation was severely decreased in both mutants and polar flagella were depleted also. Quantitative PCR showed that most of the genes related to flagellar and polysaccharide biosynthesis were upregulated in the mutant of Op background (ΔvalR/Op) but downregulated in the mutant of Tr background (ΔvalR/Tr) compared with their parental wild-type strains. This suggests that ValR may control biofilm formation by regulating flagellar biosynthesis and affect the expression of the genes involved in colony phase variation in V. alginolyticus. 相似文献
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Mejdi Snoussi Emira Noumi Donatella Usai Leonardo Antonio Sechi Stefania Zanetti Amina Bakhrouf 《World journal of microbiology & biotechnology》2008,24(10):2133-2141
This study characterizes 28 Vibrio alginolyticus strains isolated from seawater from the Seacoast of Monastir (Khenis; Tunisia). V. alginolyticus were isolated using the TCBS modified agar plates and the biochemical activities were tested using RapID NF plus Strips.
Proteases activities, hemolysis, antibiotics susceptibility, and adhesion to fish mucus and epithelial cell lines (Hep-2 and
Caco-2) were also investigated. Eight Vibrio
cholerae virulence genes (toxR, toxS, toxRS, toxT, ctxA, vpi, ace, zot) were investigated by PCR in genomes of V. alginolyticus strains. Most of the studied strains were β-haemolytic and produce many proteolytic enzymes. All isolates described here
were resistant to several antibiotics tested. Six strains were able to adhere strongly to both Hep-2 and Caco-2 cell lines.
The PCR investigation of V. cholerae genes showed a large distribution among the genomes of all V. alginolyticus strains. The toxR operon was found in 9 V. alginolyticus strains out of 28 studied. Only one strain was positive for the toxS and toxRS respectively. Five strains showed a positive amplification for the virulence pathogenic island (vpi), seven for the toxT, 3 for the ctxA and 9 for the Zonula occludens toxin (zot). The bay of Khenis harbors different genotypes of V. alginolyticus strains who inheritated several virulence genes from autochthones bacteria such as V. cholerae. These strains were able to produce several virulence enzymes and exhibit a high power to adhere to human epithelial cells
and fish mucus. 相似文献
7.
Md. Furkanur Rahaman Mizan Iqbal Kabir Jahid Minhui Kim Ki-Hoon Lee Tae Jo Kim 《Biofouling》2016,32(4):497-509
Vibrio parahaemolyticus is one of the leading foodborne pathogens causing seafood contamination. Here, 22 V. parahaemolyticus strains were analyzed for biofilm formation to determine whether there is a correlation between biofilm formation and quorum sensing (QS), swimming motility, or hydrophobicity. The results indicate that the biofilm formation ability of V. parahaemolyticus is positively correlated with cell surface hydrophobicity, autoinducer (AI-2) production, and protease activity. Field emission scanning electron microscopy (FESEM) showed that strong-biofilm-forming strains established thick 3-D structures, whereas poor-biofilm-forming strains produced thin inconsistent biofilms. In addition, the distribution of the genes encoding pandemic clone factors, type VI secretion systems (T6SS), biofilm functions, and the type I pilus in the V. parahaemolyticus seafood isolates were examined. Biofilm-associated genes were present in almost all the strains, irrespective of other phenotypes. These results indicate that biofilm formation on/in seafood may constitute a major factor in the dissemination of V. parahaemolyticus and the ensuing diseases. 相似文献
8.
Masatomo Morita Shouji Yamamoto Hirotaka Hiyoshi Toshio Kodama Masatoshi Okura Eiji Arakawa Munirul Alam Makoto Ohnishi Hidemasa Izumiya Haruo Watanabe 《Microbiology and immunology》2013,57(5):334-339
Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS‐related genes were distributed among the various serogroups and pulsed‐field gel electrophoresis of NotI‐digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS‐related genes had similar patterns. Additionally, naturally competent T3SS‐negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS‐related genes occurs among V. cholerae in natural ecosystems. 相似文献
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Aims: The main aims of this study were to clone and express flagellin flaA gene from Vibrio alginolyticus strain HY9901, also to prepare mouse anti‐FlaA polyclonal antibody for future pathogen or vaccine study. Methods and Results: The full‐length flaA gene was amplified by PCR with designed primers. The open reading frame of flaA gene contains 1131 bp, and its putative protein consists of 376 amino acid residues. Alignment analysis indicated that the FlaA protein was highly conserved. SDS–PAGE indicated that the FlaA protein was successfully expressed in Escherichia coli BL21 (DE3). Then, the recombinant FlaA protein was purified by affinity chromatography, and the mouse anti‐FlaA serum was produced. The expression of flaA gene was verified by various immunological methods, including western blotting, enzyme‐linked immunosorbent assay (ELISA) and immunogold electron microscopy (IEM). Conclusions: Flagellin flaA gene was cloned and identified from V. alginolyticus HY9901, the recombinant FlaA protein was expressed and purified, and high‐titre FlaA protein‐specific antibody was produced. Western blot analysis revealed that the prepared antiserum not only specifically react to FlaA fusion protein, but also to natural FlaA protein of V. alginolyticus. The expressed FlaA protein was demonstrated, for the first time, as the component of flagella from V. alginolyticus by IEM. Significance and Impact of the Study: This study may offer important insights into the pathogenesis of V. alginolyticus, provide a base for further studies on the diagnosis and evaluation that whether the FlaA protein could be used as an effective vaccine candidate against infection by V. alginolyticus and other Vibrio species. Additionally, the purified FlaA protein and polyclonal antibody can be used for further functional and structural studies. 相似文献
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Hirano T Aoki M Kadokura K Kumaki Y Hakamata W Oku T Nishio T 《Letters in applied microbiology》2011,53(2):161-166
Aims: To investigate the attractant effect of 4‐O‐(N‐acetyl‐β‐d ‐glucosaminyl)‐d ‐glucosamine (GlcNAc‐GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc‐GlcN from N,N′‐diacetylchitobiose (GlcNAc)2. Methods and Results: The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane‐migration method. The results demonstrated that GlcNAc‐GlcN functions as an effective chemoattractant in the CE family 4 COD‐producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc‐GlcN appears to stimulate polar flagellum rotation. Conclusions: GlcNAc‐GlcN is a specific chemoattractant for the CE family 4 COD‐producing vibrios, V. parahaemolyticus and V. alginolyticus. Significance and Impact of the Study: It was clarified for the first time that GlcNAc‐GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc‐GlcN from (GlcNAc)2. 相似文献
11.
Characterization of V. cholerae T3SS‐dependent cytotoxicity in cultured intestinal epithelial cells 下载免费PDF全文
Kelly A. Miller Mudit Chaand Stacy Gregoire Takeshi Yoshida Lisa A. Beck Andrei I. Ivanov Michelle Dziejman 《Cellular microbiology》2016,18(12):1857-1870
AM‐19226 is a pathogenic, non‐O1/non‐O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co‐culturing the Caco2‐BBE human intestinal epithelial cell line with AM‐19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2‐BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell‐cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM‐19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner. 相似文献
12.
S.H. Cai Y.S. Lu Z.‐H. Wu J.C. Jian B. Wang Y.C. Huang 《Letters in applied microbiology》2010,50(5):480-485
Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 102 CFU ml?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field. 相似文献
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The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an
altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable
minicells state capable to pass membrane pore size 0.45 μm. Outer membrane proteins patterns of stressed bacteria were also
altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level
of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles. 相似文献
15.
Involvement of PorK,a component of the type IX secretion system,in Prevotella melaninogenica pathogenicity 下载免费PDF全文
Yoshio Kondo Keiko Sato Keiji Nagano Miyuki Nishiguchi Tomonori Hoshino Taku Fujiwara Koji Nakayama 《Microbiology and immunology》2018,62(9):554-566
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Many pathogens undergo phase variation between rugose and smooth colony morphology or between opaque and translucent colony morphology, which is mainly due to the variation in the surface polysaccharides. In this study, Vibrio alginolyticus ZJ-51 displayed phase variation between opaque, rugose colonies (Op) and translucent, smooth colonies (Tr). Unlike the vibrios reported previously, Tr cells of ZJ-51 enhanced biofilm formation and motility, but they did not differ from Op cells in the quantity of surface polysaccharides produced. Real time PCR was used to analyze the expression of the genes involved in polysaccharide biosynthesis, flagellar synthesis, and the AI-2 quorum-sensing system. The results revealed that the K-antigen capsule gene cluster (which consists of homologs to the cpsA-K in Vibrio parahaemolyticus) and O-antigen polysaccharide gene cluster (which contains homologs to the wza-wzb-wzc) were significantly more transcribed in Tr cells. The AI-2 quorum-sensing genes showed enhanced expression in the Tr variant which also exhibited greater expression of genes associated with polar flagellar biosynthesis. These results suggest that colony phase variation might affect the virulence and survival ability in the stressful environment inhabited by V. alginolyticus. 相似文献
18.
Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPSC putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPSC in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPSC led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (epsT) in EPSC containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of epsT resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPSC that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios. 相似文献
19.
I. A. Beleneva E. F. Maslennikova T. Yu. Magarlamov 《Russian Journal of Marine Biology》2004,30(2):96-100
One hundred strains of halophilic vibrios were isolated from 16 species of marine invertebrates of Peter the Great Bay. Based on their morphological and biochemical characteristics, the bacteria were identified as Vibrio parahaemolyticus and Vibrio alginolyticus. Bacterial isolates possessed virulence enzymes (DNAase, lecithinase, catalase) and were characterized by a high enterotoxigenicity. It was determined that 76% of the V. parahaemolyticus strains and 43% of the V. alginolyticus strains were Kanagawa-positive. The isolates showed a high adhesive capability, the average adhesion index was 18.06 cells per erythrocyte for V. parahaemolyticus and 12.55 for V. alginolyticus. The results of this study suggest a high pathogenic potential of the isolated halophilic vibrios, which are an epidemic hazard to marine invertebrates and to humans. 相似文献
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【目的】探究生物膜形成中间状态下副溶血弧菌的差异基因表达情况,为今后研究生物膜形成调控机制提供基因信息。【方法】以非生物膜形成条件下为参照,采用Illumina HiSeq测序平台进行转录组测序(RNA sequencing,RNA-seq)研究,分析生物膜形成中间状态下副溶血弧菌的基因表达情况,并采用实时定量PCR (quantitative real-time PCR,qPCR)进行验证。【结果】本研究共获得979个差异显著性表达基因(differentially expressed gene,DEG),其中下调基因379个,上调基因600个。基因本体(gene ontology,GO)分类分析结果显示,共有363个DEGs注释到分子功能、细胞组分和生物学过程3个一级分类和30个二级分类;京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)代谢途径分析结果显示,共有706个DEGs归到37个代谢通路中(Q value<0.05),差异表达基因主要集中在细胞代谢和转运通路上;蛋白相邻类的聚簇(clusters of orthologous groups,COG)分类结果显示,有888个DEGs可归为20个类别,涉及DEGs最多的为氨基酸转运与代谢、一般功能预测基因、能量产生与转换以及未知功能基因。qPCR验证挑选的DEGs变化趋势均与RNA-seq的结果一致。此外,从转录组数据中共筛选出10个c-di-GMP代谢相关基因、1个侧生鞭毛蛋白基因(lafA)、13个极生鞭毛合成相关基因、6个荚膜多糖合成相关基因、6个胞外多糖合成相关基因、5个IV型菌毛合成相关基因、膜融合蛋白(membrane fusion protein,Mfp)基因(cpsQ-mfpABC)、14个III型分泌系统1(T3SS1)相关基因、14个Vp-PAI基因(1个tdh2和13个T3SS2基因)、3个VI型分泌系统1(T6SS1)相关基因、6个T6SS2基因、45个推定调控子基因和15个推定的外膜蛋白基因。【结论】生物膜形成引起副溶血弧菌基因表达谱发生明显变化,差异表达基因中包含生物膜形成相关基因、关键毒力基因和许多推定调控子基因等,这为后续研究生物膜形成调控机制提供重要信息。 相似文献