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Characterization of temperature-dependent hemin uptake receptors HupA and HvtA in Vibrio vulnificus.MicrobiologyOpen Oct 2019The Gram-negative pathogen Vibrio vulnificus produces several iron-sequestration systems including a hemin uptake system in response to iron limitation as a means to...
The Gram-negative pathogen Vibrio vulnificus produces several iron-sequestration systems including a hemin uptake system in response to iron limitation as a means to acquire this essential element. Strains of this organism are capable of causing serious septicemia in humans and eels, where hemin is abundant and an advantageous source of iron. Vibrio vulnificus hemin uptake systems consist of HupA, a well studied outer membrane protein, and a recently identified HvtA protein receptor. In this study, we confirmed that the expression of the hvtA gene is iron-regulated in a fur-dependent manner. When analyzed for virulence in a hemin-overloaded murine model system, the hupA gene was more important for establishing infection than the hvtA gene. Transcriptional profiling of these genes using strains of two different biotypes, biotype 1 (human pathogen) and biotype 2 (eel pathogen), showed that the expression of the two receptors was also regulated in response to temperature. The expression of hupA was highly induced in elevated temperatures in the human pathogenic strain when tested in iron-depleted conditions. Conversely, hvtA expression was induced significantly in the eel pathogenic strain at a lower temperature, a condition where the hupA locus was relatively repressed. Our results indicate that although both hupA and hvtA are involved for optimal hemin uptake in V. vulnificus, their expression is dually regulated by the environmental cues of iron concentration and temperature. Together, these data suggest that the virulence genes hupA and hvtA are tightly regulated and strictly induced during iron limitation combined with the physiological temperature of the host organism.
Topics: Animals; Bacterial Outer Membrane Proteins; Cold Temperature; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Hemin; Iron; Membrane Transport Proteins; Mice; Protein Transport; Vibrio Infections; Vibrio vulnificus; Virulence Factors
PubMed: 31290613
DOI: 10.1002/mbo3.905 -
Acta Biochimica Polonica 2017Vibrio vulnificus is a virulent human pathogen causing gastroenteritis and possibly life threatening septicemia in patients. Most V. vulnificus are catalase positive and...
Vibrio vulnificus is a virulent human pathogen causing gastroenteritis and possibly life threatening septicemia in patients. Most V. vulnificus are catalase positive and can deactivate peroxides, thus allowing them to survive within the host. In the study presented here, a catalase from V. vulnificus (CAT-Vv) was purified to homogeneity after expression in Escherichia coli. The kinetics and function of CAT-Vv were examined. CAT-Vv catalyzed the reduction of HO at an optimal pH of 7.5 and temperature of 35°C. The V and K values were 65.8±1.2 U/mg and 10.5±0.7 mM for HO, respectively. Mutational analysis suggests that amino acids involved in heme binding play a key role in the catalysis. Quantitative reverse transcription-PCR revealed that in V. vulnificus, transcription of CAT-Vv was upregulated by low salinity, heat, and oxidative stresses. This research gives new clues to help inhibit the growth of, and infection by V. vulnificus.
Topics: Catalase; Chromatography, Affinity; Cloning, Molecular; Computer Simulation; Enzyme Stability; Gene Expression Regulation, Bacterial; Heme; Hydrogen-Ion Concentration; Mutagenesis, Site-Directed; Phylogeny; Protein Engineering; Recombinant Proteins; Temperature; Vibrio vulnificus
PubMed: 28892106
DOI: 10.18388/abp.2017_1530 -
Infection and Immunity Aug 2011As an etiological agent of bacterial sepsis and wound infections, Vibrio vulnificus is unique among the Vibrionaceae. The most intensely studied of its virulence factors...
As an etiological agent of bacterial sepsis and wound infections, Vibrio vulnificus is unique among the Vibrionaceae. The most intensely studied of its virulence factors is the capsular polysaccharide (CPS). Over 100 CPS types have been identified, yet little is known about the genetic mechanisms that drive such diversity. Chitin, the second-most-abundant polysaccharide in nature, is known to induce competence in Vibrio species. Here, we show that the frequency of chitin-induced transformation in V. vulnificus varies by strain and that (GlcNAc)(2) is the shortest chitin-derived polymer capable of inducing competence. Transformation frequencies (TFs) increased 8-fold when mixed-culture biofilms were exposed to a strain-specific lytic phage, suggesting that the lysis of dead cells during lytic infection increased the amount of extracellular DNA within the biofilm that was available for transfer. Furthermore, we show that V. vulnificus can undergo chitin-dependent carbotype conversion following the uptake and recombination of complete cps loci from exogenous genomic DNA (gDNA). The acquisition of a partial locus was also demonstrated when internal regions of homology between the endogenous and exogenous loci existed. This suggested that the same mechanism governing the transfer of complete cps loci also contributed to their evolution by generating novel combinations of CPS biosynthesis genes. Since no evidence that cps loci were preferentially acquired during natural transformation (random transposon-tagged DNA was readily taken up in chitin transformation assays) exists, the phenomenon of chitin-induced transformation likely plays an important but general role in the evolution of this genetically promiscuous genus.
Topics: Bacterial Capsules; Chitin; Humans; Serotyping; Transformation, Bacterial; Vibrio vulnificus
PubMed: 21670169
DOI: 10.1128/IAI.00158-11 -
Microbiology (Reading, England) Dec 2020Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several...
Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, . Different mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen , even though rifampicin-resistant strains of have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of mutations. In this work, effects on growth, stress response and virulence of were investigated using a set of nine spontaneous rifampicin-resistant derivatives of CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.
Topics: Anti-Bacterial Agents; Bacterial Proteins; DNA-Directed RNA Polymerases; Drug Resistance, Bacterial; Genetic Fitness; Locomotion; Microbial Sensitivity Tests; Microbial Viability; Mutation; Rifampin; Stress, Physiological; Vibrio vulnificus
PubMed: 33186092
DOI: 10.1099/mic.0.000991 -
Biocontrol Science Mar 2011Bacteria of the genus Vibrio are normal habitants of the aquatic environment but the some species are believed to be human pathogens. Pathogenic vibrios produce various... (Review)
Review
Bacteria of the genus Vibrio are normal habitants of the aquatic environment but the some species are believed to be human pathogens. Pathogenic vibrios produce various pathogenic factors, and the proteases are also recognized to play pathogenic roles in the infection: the direct roles by digesting many kinds of host proteins or indirect roles by processing other pathogenic protein factors. Especially VVP from Vibrio vulnificus is thought to be a major pathogenic factor of the vibrio. Although HA/P, the V. cholerae hemagglutinin/protease, is not a direct toxic factor of cholera vibrio, its significance is an undeniable fact. Production of HA/P is regulated together with major pathogenic factors such as CT (cholera toxin) or TCP (toxin co-regulated pilus) by a quorum-sensing system. HA/P is necessary for full expression of pathogenicity of the vibrio by supporting growth and translocation in the digestive tract. Processing of protein toxins such as CT or El Tor hemolysin is also an important pathogenic role.
Topics: Bacterial Proteins; Cholera Toxin; Fimbriae, Bacterial; Hemolysin Proteins; Metalloendopeptidases; Peptide Hydrolases; Quorum Sensing; Vibrio cholerae; Vibrio vulnificus; Virulence Factors
PubMed: 21467624
DOI: 10.4265/bio.16.1 -
Molecular and Cellular Probes Aug 2011A novel loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay was developed and evaluated...
A novel loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay was developed and evaluated for the detection of Vibrio vulnificus. Biotinylated LAMP amplicons were produced by a set of six designed primers that recognized the V. vulnificus RNA polymerase subunit sigma factor S (rpoS) gene followed by hybridization with an FITC-labeled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 14 isolates of V. vulnificus but did not detect 25 non-vulnificus Vibrio isolates and 37 non-Vibrio isolates. The sensitivity of LAMP-LFD for V. vulnificus detection in pure culture was 1.5 × 10(3) CFU ml(-1) or equivalent to 2.8 CFU per reaction. In the case of spiked oyster samples without enrichment, the detection limit for V. vulnificus was 1.2 × 10(4) CFU g(-1) or equivalent to 11 CFU per reaction. The results show that this method appears to be accurate, precise and valuable tool for identification of V. vulnificus and can be used efficiently for detection of V. vulnificus in contaminated food sample.
Topics: Bacterial Proteins; DNA Primers; Fluorescein-5-isothiocyanate; Molecular Probes; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Sigma Factor; Temperature; Vibrio vulnificus
PubMed: 21513793
DOI: 10.1016/j.mcp.2011.04.001 -
Applied and Environmental Microbiology Jun 2003While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen...
While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of V. vulnificus in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of Vibrio spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured. V. vulnificus isolates, along with estuarine bacteria, Vibrio spp., Escherichia coli organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including V. vulnificus. Isolates were confirmed as V. vulnificus by using hemolysin gene PCR or colony hybridization. V. vulnificus was isolated only when water temperatures were between 15 and 27 degrees C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of V. vulnificus in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both V. vulnificus (47%) and Vibrio spp. (48%).
Topics: Colony Count, Microbial; Culture Media; Ecosystem; Fresh Water; Hemolysin Proteins; North Carolina; Polymerase Chain Reaction; Regression Analysis; Seasons; Vibrio vulnificus
PubMed: 12788759
DOI: 10.1128/AEM.69.6.3526-3531.2003 -
The Journal of Biological Chemistry Oct 2017The marine bacterium causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in is of...
The marine bacterium causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in is of high priority. We performed a transcriptome analysis on after infection of human intestinal HT29-methotrexate cells and found induction of , encoding a putative phospholipase, PlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that PlpA is a phospholipase A secreted in a type II secretion system-dependent manner. Compared with the wild type, the mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that PlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of Molecular biological analyses revealed that expression is controlled via the promoter, P , and that HlyU and CRP directly bind to P upstream sequences. Taken together, this study demonstrated that PlpA is a type II secretion system-dependent secretory phospholipase A regulated by HlyU and CRP and is essential for the pathogenicity of .
Topics: Bacterial Proteins; Bacterial Secretion Systems; Cell Line; Humans; Intestinal Mucosa; Phospholipases A2; Transcription Factors; Vibrio Infections; Vibrio vulnificus
PubMed: 28855258
DOI: 10.1074/jbc.M117.791657 -
Infection and Immunity May 2014Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and...
Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.
Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Bacterial Toxins; CHO Cells; Cloning, Molecular; Coculture Techniques; Cricetinae; Cricetulus; Female; Gene Expression Regulation, Bacterial; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Vibrio Infections; Vibrio vulnificus; Virulence
PubMed: 24614656
DOI: 10.1128/IAI.00017-14 -
International Journal of Food... Dec 2020Routine handling of oysters is a common industry practice for off-bottom oyster aquaculture, which aims to produce a high-quality oyster. These practices expose oysters...
Effects of tumbling, refrigeration and subsequent resubmersion on the abundance of Vibrio vulnificus and Vibrio parahaemolyticus in cultured oysters (Crassostrea virginica).
Routine handling of oysters is a common industry practice for off-bottom oyster aquaculture, which aims to produce a high-quality oyster. These practices expose oysters to elevated temperatures and interrupt filter feeding, which can increase Vibrio vulnificus and V. parahaemolyticus levels within the oyster. The resubmersion of oysters after exposure to conditions where the time-temperature controls are exceeded is as an effective mitigation strategy to allow elevated levels of Vibrio spp. to "recover", or return to ambient levels, prior to harvest. Previous work examined the effect of desiccation on recovery times; the objective of this study was to evaluate the effect of additional handling treatments [tumbled and refrigerated (TR), tumbled and not refrigerated (TNR), not tumbled and refrigerated (NTR), and not tumbled and not refrigerated (NTNR)] on the time needed for V. vulnificus, total V. parahaemolyticus, and pathogenic V. parahaemolyticus (tdh+/trh+) to recover in oysters. A set of non-treated (control) oysters remained submerged throughout the study to determine the ambient Vibrio spp. (inclusive of genotypes) levels within oysters. Vibrio spp. levels were measured immediately before (pre) and after (post) the treatments, and 1, 2, 4, 7, 10, and 14 days after resubmersion using a three-tube MPN real-time PCR method. The non-refrigerated oysters (TNR, NTNR) had Vibrio spp. levels 1.54 to 2.10 log MPN/g higher than the pre-treatment levels, while the Vibrio spp. levels in refrigerated oysters were not significantly higher than pre-treatment levels. After resubmersion, Vibrio spp. levels increased by 0.84 to 1.78 log MPN/g in the refrigerated oysters (TR, NTR). Vibrio spp. levels in oysters returned to ambient after 1-7 days of resubmersion, depending on the handling treatment and the Vibrio spp. These results provide data on handling treatments not previously reported and further support the seven-day resubmersion requirement for farmers in Alabama using the adjustable longline system.
Topics: Alabama; Animals; Aquaculture; Crassostrea; Food Contamination; Food Handling; Real-Time Polymerase Chain Reaction; Refrigeration; Seafood; Temperature; Time Factors; Vibrio parahaemolyticus; Vibrio vulnificus
PubMed: 33032034
DOI: 10.1016/j.ijfoodmicro.2020.108858