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Infection and Immunity Oct 2003Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to...
Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.
Topics: Animals; Antigens, Bacterial; Bacterial Proteins; Base Sequence; DNA, Bacterial; Female; Gene Expression; Genes, Bacterial; HeLa Cells; Humans; Male; Mice; Middle Aged; Molecular Sequence Data; Mutation; Open Reading Frames; Sepsis; Vibrio Infections; Vibrio vulnificus; Virulence
PubMed: 14500463
DOI: 10.1128/IAI.71.10.5461-5471.2003 -
Biochimica Et Biophysica Acta. General... Jan 2021The products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are essential for bacterial survival. This paper focuses on the structural and... (Comparative Study)
Comparative Study
BACKGROUND
The products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are essential for bacterial survival. This paper focuses on the structural and mechanistic characterization of 4-hydroxy-tetrahydrodipicolinate reductase (DapB), which is one of the enzymes from the lysine biosynthesis pathway. DapB catalyzes the conversion of (2S, 4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate (HTPA) to 2,3,4,5-tetrahydrodipicolinate in an NADH/NADPH dependent reaction. Genes coding for DapBs were identified as essential for many pathogenic bacteria, and therefore DapB is an interesting new target for the development of antibiotics.
METHODS
We have combined experimental and computational approaches to provide novel insights into mechanism of the DapB catalyzed reaction.
RESULTS
Structures of DapBs originating from Mycobacterium tuberculosis and Vibrio vulnificus in complexes with NAD, NADP, as well as with inhibitors, were determined and described. The structures determined by us, as well as currently available structures of DapBs from other bacterial species, were compared and used to elucidate a mechanism of reaction catalyzed by this group of enzymes. Several different computational methods were used to provide a detailed description of a plausible reaction mechanism.
CONCLUSIONS
This is the first report presenting the detailed mechanism of reaction catalyzed by DapB.
GENERAL SIGNIFICANCE
Structural data in combination with information on the reaction mechanism provide a background for development of DapB inhibitors, including transition-state analogues.
Topics: Biosynthetic Pathways; Catalytic Domain; Humans; Lysine; Models, Molecular; Mycobacterium tuberculosis; Oxidoreductases; Protein Conformation; Substrate Specificity; Tuberculosis; Vibrio Infections; Vibrio vulnificus
PubMed: 32980502
DOI: 10.1016/j.bbagen.2020.129750 -
Infection and Immunity Apr 2008Capsular polysaccharide (CPS) is a major virulence factor in Vibrio vulnificus, and encapsulated strains have an opaque, smooth (OpS) colony morphology, while...
Capsular polysaccharide (CPS) is a major virulence factor in Vibrio vulnificus, and encapsulated strains have an opaque, smooth (OpS) colony morphology, while nonencapsulated strains have a translucent, smooth (TrS) colony morphology. Previously, we showed that OpS and TrS parental strains can yield a third colony type, rugose (R), and that the resulting strains, with the OpR and TrR phenotypes, respectively, form copious biofilms. Here we show that while OpR and TrR strains both produce three-dimensional biofilm structures that are indicative of rugose extracellular polysaccharide (rEPS) production, OpR strains also retain expression of CPS and are virulent in an iron-supplemented mouse model, while TrR strains lack CPS and are avirulent. Chlorine resistance assays further distinguished OpR and TrR isolates as exposure to 3 microg/ml NaOCl eradicated both OpS and OpR strains, while both TrS and TrR strains survived, but at rates which were significantly different from one another. Taken together, these results further emphasize the importance of CPS for virulence of V. vulnificus and establish a correlation between CPS expression and chlorine sensitivity in this organism. Using reverse transcriptase PCR, we also identified a nine-gene cluster associated with both CPS and rEPS expression in V. vulnificus, designated the wcr (capsular and rugose polysaccharide) locus, with expression occurring primarily in R variants. The latter results set the stage for characterization of functional determinants which individually or collectively contribute to expression of multiple EPS forms in this pathogen.
Topics: Animals; Chlorine; Iron, Dietary; Male; Mice; Mice, Inbred ICR; Multigene Family; Polysaccharides, Bacterial; Vibrio Infections; Vibrio vulnificus; Virulence
PubMed: 18212074
DOI: 10.1128/IAI.01289-07 -
Applied and Environmental Microbiology Sep 2004The abundance of Vibrio vulnificus in coastal environments has been linked to water temperature, while its relationship to salinity is less clear. We have developed a...
The abundance of Vibrio vulnificus in coastal environments has been linked to water temperature, while its relationship to salinity is less clear. We have developed a culture-independent, most-probable-number quantitative PCR approach to examine V. vulnificus population dynamics in Barnegat Bay, N.J. Based on the combined analysis of our results from Barnegat Bay and from the literature, the present data show that (i) V. vulnificus population dynamics are strongly correlated to water temperature and (ii) although the general trend is for V. vulnificus abundance to be inversely correlated with salinity, this relationship depends on salinity levels. Irrespective of temperature, high abundances of V. vulnificus are observed at 5 to 10 ppt, which thus appears to be the optimal salinity regime for their survival. At 20 to 25 ppt, V. vulnificus abundances show a positive correlation to salinity. Unsuccessful attempts to resuscitate V. vulnificus, combined with our inability to detect cells during the winter despite an assay adapted to detect viable but nonculturable (VBNC) cells, suggest that the decline and eventual disappearance of V. vulnificus from the water column during the winter months is due primarily to a significant reduction in population size and is not only the consequence of cells entering the VBNC state. These findings are in line with the hypothesis that the sediment serves as a refuge for a subpopulation of V. vulnificus over the winter and weather-driven mixing events during the spring initiate a summer bloom in the water column.
Topics: Chlorophyll; Chlorophyll A; DNA Primers; DNA, Bacterial; Geography; Osmolar Concentration; Polymerase Chain Reaction; Seasons; Sodium Chloride; Temperature; Thermodynamics; Vibrio vulnificus; Water Microbiology
PubMed: 15345434
DOI: 10.1128/AEM.70.9.5469-5476.2004 -
Journal of Virology Jul 2012Vibrio vulnificus phages are abundant in coastal marine environments, shellfish, clams, and oysters. SSP002, a V. vulnificus-specific bacteriophage, was isolated from...
Vibrio vulnificus phages are abundant in coastal marine environments, shellfish, clams, and oysters. SSP002, a V. vulnificus-specific bacteriophage, was isolated from oysters from the west coast of South Korea. In this study, the complete genome of SSP002 was sequenced and analyzed for the first time among the V. vulnificus-specific bacteriophages.
Topics: Animals; Bacteriophages; Base Sequence; Genome, Viral; Molecular Sequence Data; Ostreidae; Sequence Analysis, DNA; Vibrio vulnificus
PubMed: 22733877
DOI: 10.1128/JVI.00972-12 -
PLoS Pathogens Sep 2015A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon....
A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3',5'-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose.
Topics: Amino Acid Sequence; Biofilms; Calcium-Binding Proteins; Genes, Bacterial; Humans; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Operon; Polysaccharides, Bacterial; Real-Time Polymerase Chain Reaction; Vibrio vulnificus
PubMed: 26406498
DOI: 10.1371/journal.ppat.1005192 -
MBio Aug 2018Poor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic...
Poor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic human pathogen , as well as the bivalves (oysters) it naturally colonizes, is indigenous to estuaries and human-inhabited coastal regions and must endure constantly changing environmental conditions as freshwater and seawater enter, mix, and exit the water column. Elevated cellular c-di-GMP levels trigger biofilm formation, but relatively little is known regarding the environmental signals that initiate this response. Here, we show that calcium is a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers expression of the extracellular polysaccharide that enhances biofilm formation. A transposon screen for the loss of calcium-induced expression revealed CysD, an enzyme in the sulfate assimilation pathway. Targeted disruption of the pathway indicated that the production of a specific metabolic intermediate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), was required for calcium-induced expression and that PAPS was separately required for development of the physiologically distinct rugose phenotype. Thus, PAPS behaves as a second messenger in Moreover, c-di-GMP and BrpT (the activator of expression) acted in concert to bias expression of the sulfate assimilation pathway toward PAPS and c-di-GMP accumulation, establishing a feed-forward regulatory loop to boost expression. Thus, this signaling network links extracellular calcium and sulfur availability to the intracellular second messengers PAPS and c-di-GMP in the regulation of biofilm formation and rugosity, survival phenotypes underpinning its evolution as a resilient environmental organism. The second messenger c-di-GMP is a key regulator of bacterial physiology. The genome encodes nearly 100 proteins predicted to make, break, and bind c-di-GMP. However, relatively little is known regarding the environmental signals that regulate c-di-GMP levels and biofilm formation in Here, we identify calcium as a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers -mediated biofilm formation. We show that PAPS, a metabolic intermediate of the sulfate assimilation pathway, acts as a second messenger linking environmental calcium and sulfur source availability to the production of another intracellular second messenger (c-di-GMP) to regulate biofilm and rugose colony formation, developmental pathways that are associated with environmental persistence and efficient bivalve colonization by this potent human pathogen.
Topics: Bacterial Proteins; Biofilms; Calcium; Cyclic GMP; Environment; Gene Expression Regulation, Bacterial; Phenotype; Phosphoadenosine Phosphosulfate; Signal Transduction; Vibrio vulnificus
PubMed: 30154262
DOI: 10.1128/mBio.01377-18 -
Prevalence and population structure of Vibrio vulnificus on fishes from the northern Gulf of Mexico.Applied and Environmental Microbiology Nov 2012The prevalence of Vibrio vulnificus on the external surfaces of fish from the northern Gulf of Mexico was determined in this study. A collection of 242 fish comprising...
The prevalence of Vibrio vulnificus on the external surfaces of fish from the northern Gulf of Mexico was determined in this study. A collection of 242 fish comprising 28 species was analyzed during the course of 12 sampling trips over a 16-month period. The prevalence of V. vulnificus was 37% but increased up to 69% in summer. A positive correlation was found between the percentages of V. vulnificus-positive fish and water temperatures, while salinity and V. vulnificus-positive fish prevalence were inversely correlated. A general lineal model (percent V. vulnificus-positive fish = 0.5930 - 0.02818 × salinity + 0.01406 × water temperature) was applied to best fit the data. Analysis of the population structure was carried out using 244 isolates recovered from fish. Ascription to 16S rRNA gene types indicated that 157 isolates were type A (62%), 72 (29%) were type B, and 22 (9%) were type AB. The percentage of type B isolates, considered to have greater virulence potential, was higher than that previously reported in oyster samples from the northern Gulf of Mexico. Amplified fragment length polymorphism (AFLP) was used to resolve the genetic diversity within the species. One hundred twenty-one unique AFLP profiles were found among all analyzed isolates, resulting in a calculated Simpson's index of diversity of 0.991. AFLP profiles were not grouped on the basis of collection date, fish species, temperature, or salinity, but isolates were clustered into two main groups that correlated precisely with 16S rRNA gene type. The population of V. vulnificus associated with fishes from the northern Gulf of Mexico is heterogeneous and includes strains of great virulence potential.
Topics: Amplified Fragment Length Polymorphism Analysis; Animals; Fishes; Genes, rRNA; Genetic Variation; Gulf of Mexico; RNA, Ribosomal, 16S; Salinity; Temperature; Vibrio vulnificus
PubMed: 22923394
DOI: 10.1128/AEM.01646-12 -
Journal of Bacteriology Apr 2018Septicemia-causing produces at least three exoproteases, VvpE, VvpS, and VvpM, all of which participate in interactions with human cells. Expression of VvpE and VvpS is...
Septicemia-causing produces at least three exoproteases, VvpE, VvpS, and VvpM, all of which participate in interactions with human cells. Expression of VvpE and VvpS is induced in the stationary phase by multiple transcription factors, including sigma factor S, SmcR, and the cAMP-cAMP receptor protein (cAMP-CRP) complex. Distinct roles of VvpM, such as induction of apoptosis, lead us to hypothesize VvpM expression is different from that of the other exoproteases. Its transcription, which was found to be independent of sigma S, is induced at the early exponential phase and then becomes negligible upon entry into the stationary phase. SmcR and CRP were studied regarding the control of expression. Transcription of was repressed by SmcR and cAMP-CRP complex individually, which specifically bound to the regions -2 to +20 and +6 to +27, respectively, relative to the transcription initiation site. Derepression of gene expression was 10- to 40-fold greater in an double mutant than in single-gene mutants. Therefore, these results show that the expression of exoproteases is differentially regulated, and in this way, distinct proteases can engage in specific interactions with a host. An opportunistic human pathogen, produces multiple extracellular proteases that are involved in diverse interactions with a host. The total exoproteolytic activity is detected mainly in the supernatants of the high-cell-density cultures. However, some proteolytic activity derived from a metalloprotease, VvpM, was present in the supernatants of the low-cell-density cultures sampled at the early growth period. In this study, we present the regulatory mechanism for VvpM expression via repression by at least two transcription factors. This type of transcriptional regulation is the exact opposite of those for expression of the other exoproteases. Differential regulation of each exoprotease's production then facilitates the pathogen's participation in the distinct interactions with a host.
Topics: Apoptosis; Bacterial Proteins; Cyclic AMP Receptor Protein; Enzyme Repression; Gene Expression Regulation, Bacterial; Humans; Metalloendopeptidases; Proteolysis; Quorum Sensing; Transcription Factors; Vibrio vulnificus
PubMed: 29339417
DOI: 10.1128/JB.00526-17 -
MicrobiologyOpen Jun 2019The OMPs A (OmpA)-of Edwardsiella anguillarum and OmpU of V. vulnificus have been proven to be good antigens. In this study, after construction of a vector, a new...
The OMPs A (OmpA)-of Edwardsiella anguillarum and OmpU of V. vulnificus have been proven to be good antigens. In this study, after construction of a vector, a new recombinant Omp (rOMP) containing both OmpA and OmpU was expressed and purified. Then, the Japanese eels (Anguilla japonica) were intraperitoneally (i.p.) injected with the phosphate-buffered saline (PBS group), formalin-killed-cell (FKC group) or the recombinant Omp (rOMP group). The stimulation index of the whole blood cells in eels from FKC group was significantly higher than the eels from PBS and rOMP groups at 28 dpi; serum titers of anti-E. anguillarum and anti-V. vulnificus antibody of eels from FKC and rOMP group increased significantly at 21 and 28 dpi; in the rOMP group, eels serum titer stayed at a high level on 42 dpi. The activities of lysozyme in skin mucus, liver, kidney, and serum in three groups exhibited considerable changes. The relative percent survival (RPS) rate of eels from rOMP group were 100% and 83% when challenged with V. vulnificus or E. anguillarum. These results indicated that inoculation of rOMP would protect Japanese eels against the infection by E. anguillarum and V. vulnificus.
Topics: Anguilla; Animals; Bacterial Proteins; Bacterial Vaccines; Edwardsiella; Enterobacteriaceae Infections; Fish Diseases; Vibrio Infections; Vibrio vulnificus
PubMed: 30444580
DOI: 10.1002/mbo3.766