Vibrio Vulnificus Strain Research Articles

Structural basis of the activation of MARTX cysteine protease domain from Vibrio vulnificus.

The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP6)-mediated activation of the CPD, we determined the crystal structures of unprocessed and β-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP6 at 1.3 and 2.2Å resolution, respectively. The CPD displays a conserved domain with a central seven-stranded β-sheet flanked by three α-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP6-loaded form of the Cys3727Ala mutant. InsP6 interacts with the conserved basic cleft and the β-flap inducing the active conformation of catalytic residues. The β-flap of the post-CPD is flexible in the InsP6-unbound state. The structure of the CPD Δβ-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the β-flap. This study confirms the InsP6-mediated activation of the MARTX CPDs in which InsP6-binding induces conformational changes of the catalytic residues and the β-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.

Complete genome sequence and comparative analysis of a Vibrio vulnificus strain isolated from a clinical patient.

Vibrio vulnificus is an opportunistic, global pathogen that naturally inhabits sea water and is responsible for most vibriosis-related deaths. We investigated the genetic characteristics of V. vulnificus isolated from the clinical blood culture specimen of a patient with hepatitis B virus cirrhosis in 2018 (named as V. vulnificus VV2018) by whole genome sequencing (WGS). VV2018 belonged to a novel sequencing type 620 (ST620) and comprised two circular chromosomes, containing 4,389 potential coding sequences (CDSs) and 152 RNA genes. The phylogenetic tree of single nucleotide polymorphisms (SNPs) using 26 representative genomes revealed that VV2108 grouped with two other V. vulnificus strains isolated from humans. The pan-genome of V. vulnificus was constructed using 26 representative genomes to elucidate their genetic diversity, evolutionary characteristics, and virulence and antibiotic resistance profiles. The pan-genome analysis revealed that VV2018 shared a total of 3,016 core genes (≥99% presence), including 115 core virulence factors (VFs) and 5 core antibiotic resistance-related genes, and 309 soft core genes (≥95 and <99% presence) with 25 other V. vulnificus strains. The varG gene might account for the cefazolin resistance, and comparative analysis of the genetic context of varG revealed that two genes upstream and downstream of varG were conserved. The glycosylation (pgl) like genes were found in VV2018 compared with Pgl-related proteins in Neisseria that might affect the adherence of the strain in hosts. The comparative analysis of VV2018 would contribute to a better understanding of the virulence and antibiotic resistance profiles of V. vulnificus. Meanwhile much work remains to be done to better understand the function of pgl-like genes in V. vulnificus.

New drug target identification in Vibrio vulnificus by subtractive genome analysis and their inhibitors through molecular docking and molecular dynamics simulations

Vibrio vulnificus is a rod shape, Gram-negative bacterium that causes sepsis (with a greater than 50% mortality rate), necrotizing fasciitis, gastroenteritis, skin, and soft tissue infection, wound infection, peritonitis, meningitis, pneumonia, keratitis, and arthritis. Based on pathogenicity V. vulnificus is categorized into three biotypes. Type 1 and type 3 cause diseases in humans while biotype 2 causes diseases in eel and fish. Due to indiscriminate use of antibiotics V. vulnificus has developed resistance to many antibiotics so curing is dramatically a challenge. V. vulnificus is resistant to cefazolin, streptomycin, tetracycline, aztreonam, tobramycin, cefepime, and gentamycin. Subtractive genome analysis is the most effective method for drug target identification. The method is based on the subtraction of homologous proteins from both pathogen and host. By this process set of proteins present only in the pathogen and perform essential functions in the pathogen can be identified. The entire proteome of Vibrio vulnificus strain ATCC 27562 was reduced step by step to a single protein predicted as the drug target. AlphaFold2 is one of the applications of deep learning algorithms in biomedicine and is correctly considered the game changer in the field of structural biology. Accuracy and speed are the major strength of AlphaFold2. In the PDB database, the crystal structure of the predicted drug target was not present, therefore the Colab notebook was used to predict the 3D structure by the AlphaFold2, and subsequently, the predicted model was validated. Potent inhibitors against the new target were predicted by virtual screening and molecular docking study. The most stable compound ZINC01318774 tightly attaches to the binding pocket of bisphosphoglycerate-independent phosphoglycerate mutase. The time-dependent molecular dynamics simulation revealed compound ZINC01318774 was superior as compared to the standard drug tetracycline in terms of stability. The availability of V. vulnificus strain ATCC 27562 has allowed in silico identification of drug target which will provide a base for the discovery of specific therapeutic targets against Vibrio vulnificus.

Genome sequence and pathogenicity of Vibrio vulnificus strain MCCC 1A08743 isolated from contaminated prawns.

ABSTRACTVibrio vulnificus is an opportunistic pathogen that naturally inhabits sea water globally and is responsible for most vibriosis-related deaths. The consumption of V. vulnificus contaminated seafood and exposure of wounds to Vibrio can result in systemic infection, with increased risks of amputation and extremely high rates of mortality. However, the pathogenicity and virulence factors of V. vulnificus are not fully understood. The genomic characterization of V. vulnificus will be helpful to extend our understanding on V. vulnificus at a genomic level. In this manuscript, the genome of V. vulnificus strain MCCC 1A08743 isolated from contaminated prawns from Zhanjiang, China, was sequenced using Illumina HiSeq X Ten system and annotated through multiple databases. The strain MCCC 1A08743 genome included 4371 protein-coding genes and 117 RNA genes. Average nucleotide identity analysis and core genome phylogenetic analysis revealed that MCCC 1A08743 was most closely related to strains from clinical samples from the United States. Pathogenicity annotation of the MCCC 1A08743 genome, using Virulence Factor Database and Pathogen-Host Interactions database, predicted the pathogenicity of the strain, and this was confirmed using mice infection experiments, which indicated that V. vulnificus strain MCCC 1A08743 could infect C57BL/6J mice and cause liver lesions. This article has an associated First Person interview with the first author of the paper.

First person – Jie Li

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Jie Li is first author on ‘ Genome sequence and pathogenicity of Vibrio vulnificus strain MCCC 1A08743 isolated from contaminated prawns’, published in BiO. Jie is an assistant professor (lecturer in China university system) in the lab of Fu Yang at the Department of Medical Genetics, Naval Medical University, Shanghai, China, investigating bacterial infection and liver injury.

Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR-Cas system in Vibrio vulnificus.

The association of reverse transcriptases (RTs) with CRISPR–Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5′and 3′ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.

Comparison of DNA Methylation in Vibrio vulnificus Cells Grown in Human Serum with Those Grown in Seawater.

The chromosomal methylation statuses of the highly virulent Vibrio vulnificus strain CMCP6 grown in human serum and in seawater are compared here. Growth in seawater resulted in ∼4 times as much methylation as that in human serum, primarily N4-methylcytosines.

Correction for Tiba-Casas et al., "Draft Genome Sequence of a Pathogenic Vibrio vulnificus Strain Isolated in Brazil".

Volume 6, no. 7, e01274-17, 2018, [https://doi.org/10.1128/genomeA.01274-17][1]. Page 1: The following statement should be added to Acknowledgments. “Publication of this study was supported by grant no. 2017/22402-4, Sao Paulo Research Foundation (FAPESP).” [1]: /lookup/doi/10.1128/genomeA.

NMR study of the O-specific polysaccharide and the core oligosaccharide from the lipopolysaccharide produced by Plesiomonas shigelloides O24:H8 (strain CNCTC 92/89).

The structures of the O-specific polysacccharide and core oligosaccharide of the lipopolysaccharide from Plesiomonas shigelloides O24:H8, strain CNCTC 92/89, have been investigated by NMR spectroscopy and ESI mass spectrometry. The O-specific polysaccharide was found to be composed of a tetrasaccharide repeating unit consisting of [→3)-α-FucpNAc-(1→3)-α-GalpNAcA-(1→3)-α-QuipNAc-(1→] and of α-RhapNAc (1→4) linked to the GalpNAcA residue. An identical structure has been reported for the capsular polysaccharide of the clinical isolate of Vibrio vulnificus strain BO62316 [1]. The core oligosaccharide was composed of a decasaccharide which structure is identical with these in P. shigelloides serotype O54 [2] and serotype O37 [3].

Comparative genomic analysis of clinical and environmental Vibrio vulnificus isolates revealed biotype 3 evolutionary relationships.

In 1996 a common-source outbreak of severe soft tissue and bloodstream infections erupted among Israeli fish farmers and fish consumers due to changes in fish marketing policies. The causative pathogen was a new strain of Vibrio vulnificus, named biotype 3, which displayed a unique biochemical and genotypic profile. Initial observations suggested that the pathogen erupted as a result of genetic recombination between two distinct populations. We applied a whole genome shotgun sequencing approach using several V. vulnificus strains from Israel in order to study the pan genome of V. vulnificus and determine the phylogenetic relationship of biotype 3 with existing populations. The core genome of V. vulnificus based on 16 draft and complete genomes consisted of 3068 genes, representing between 59 and 78% of the whole genome of 16 strains. The accessory genome varied in size from 781 to 2044 kbp. Phylogenetic analysis based on whole, core, and accessory genomes displayed similar clustering patterns with two main clusters, clinical (C) and environmental (E), all biotype 3 strains formed a distinct group within the E cluster. Annotation of accessory genomic regions found in biotype 3 strains and absent from the core genome yielded 1732 genes, of which the vast majority encoded hypothetical proteins, phage-related proteins, and mobile element proteins. A total of 1916 proteins (including 713 hypothetical proteins) were present in all human pathogenic strains (both biotype 3 and non-biotype 3) and absent from the environmental strains. Clustering analysis of the non-hypothetical proteins revealed 148 protein clusters shared by all human pathogenic strains; these included transcriptional regulators, arylsulfatases, methyl-accepting chemotaxis proteins, acetyltransferases, GGDEF family proteins, transposases, type IV secretory system (T4SS) proteins, and integrases. Our study showed that V. vulnificus biotype 3 evolved from environmental populations and formed a genetically distinct group within the E-cluster. The unique epidemiological circumstances facilitated disease outbreak and brought this genotype to the attention of the scientific community.

Regulation system of serine protease production in Vibrio vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel

Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in cultured eels or shrimps, as well as in immunocompromised humans. An extracellular metalloprotease has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA. In the present study, we first clarified the regulatory characteristics of the VvsA production in V. vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel. V. vulnificus coordinates expression of virulence genes in response to the bacterial cell density, which is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). When cultivated at 26°C, the vvsA expression was closely related with expression of the luxS gene encoding the synthase of the AI-2 precursor LuxS. Both VvsA and AI-2 were sufficiently secreted at early stationary phase of the bacterial growth. In contrast, when cultivated at 37°C, far less amounts of the AI-2 and VvsA were produced even at the stationary phase. Disruption of the luxS gene was found to decrease significantly the vvsA expression and VvsA production. Disruption of luxO encoding the central response regulator of the QS circuit caused an increase in the vvsA expression and VvsA production at the logarithmic growth phase. These findings indicate that VvsA production is positively regulated by the V. vulnificus LuxS-dependent QS system, which operated more effectively at 26°C than at 37°C.

Effects of temperature, growth phase and luxO-disruption on regulation systems of toxin production in Vibrio vulnificus strain L-180, a human clinical isolate

Vibrio vulnificus is a halophilic estuarine bacterium while it causes fatal septicemia or necrotizing wound infections in humans. This pathogen secretes the metalloprotease (V. vulnificus protease: VVP) and the cytolysin (V. vulnificus hemolysin: VVH) as protein toxins; however, their production was coordinated in response to the bacterial cell density. This regulation is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). In the present study, we investigated effects of disruption of luxO encoding a central response regulator of the QS circuit, as well as effects of temperature and growth phase, on the toxin production by V. vulnificus. Disruption of luxO was found to increase VVP production and expression of its gene vvpE. The expression of smcR, crp and rpoS, of which products positively regulate vvpE expression, and luxS encoding the AI-2 synthetase were also significantly increased. On the other hand, the luxO disruption resulted in reduction of VVH production and expression of its gene vvhA. Expression of other two genes affecting the QS circuit, luxT and rpoN, were also significantly decreased. The regulation systems of VVP production were found to exert their action during the stationary phase of the bacterial growth and to be operated strongly at 26 °C. By contrast, those of VVH production apparently started at the log phase and were operated more effectively at 37 °C.

Genetic and quantitative assessment of Vibrio vulnificus populations in oyster (Crassostrea virginica) tissues

Vibrio vulnificus is a leading cause of shellfish-associated food-borne illness. US regulations stipulate shellfish processing procedures to limit V. vulnificus densities; however, the effect of these procedures on V. vulnificus strain distribution and/or genetic diversity is unknown. Vibrio vulnificus concentrations and strain diversity were analysed in various oyster tissues stored overnight at 26°C that were subsequently divided into two treatment groups: one received post-harvest processing (PHP) via individual quick freeze and one was stored on ice. Vibrio vulnificus densities were 10-fold lower in all PHP-treated tissues compared with untreated tissues. Genetic diversity of V. vulnificus was assessed by BOX-PCR genotyping and was high in all oyster tissues, but was significantly lower in untreated compared with PHP-treated oysters. BOX-PCR discriminated strains into BOX-C (clinical-associated) and BOX-E (environmental-associated) types based on a 1.1 kb DNA band, which correlated well (83% agreement) with 16S rRNA (A/B) typing. A significantly higher proportion of BOX-C isolates were recovered from PHP oysters compared with untreated oysters (24% of all isolates versus 12%) suggesting that BOX-C strains may be more resistant to treatment. These results reveal highly diverse populations of V. vulnificus in oysters with different responses to PHP, emphasizing the need to better understand the organism's ecology and population genetics to optimize food safety practices.

Structure of a polysaccharide from the lipopolysaccharides of Vibrio vulnificus strains CECT 5198 and S3-I2-36, which is remarkably similar to the O-polysaccharide of Pseudoalteromonas rubra ATCC 29570

High-molecular-mass polysaccharides were released by mild acid degradation of the lipopolysaccharides of two wild-type Vibrio vulnificus strain, a flagellated motile strain CECT 5198 and a non-flagellated non-motile strain S3-I2-36. Studies by sugar analysis and partial acid hydrolysis along with 1H and 13C NMR spectroscopies showed that the polysaccharides from both strains have the same trisaccharide repeating unit of the following structure: →4)-β- d-Glc pNAc3NAcylAN-(1→4)-α- l-Gal pNAmA-(1→3)-α- d-Qui pNAc-(1→ where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose, GalNAmA for 2-acetimidoylamino-2-deoxygalacturonic acid, GlcNAc3NAcylAN for 2-acetamido-3-acylamino-2,3-dideoxyglucuronamide and acyl for 4- d-malyl (∼30%) or 2- O-acetyl-4- d-malyl (∼70%). The structure of the polysaccharide studied resembles much that of a marine bacterium Pseudoalteromonas rubra ATCC 29570 reinvestigated in this work. The latter differs in (i) the absolute configuration of malic acid ( l vs d), (ii) 3-O-acetylation of GalNAmA and (iii) replacement of QuiNAc with its 4-keto biosynthetic precursor.

Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus

The viable but nonculturable (VBNC) suppression mutant formed platable cells at low temperature stress after inoculation in artificial seawater (ASW). Suppression subtractive hybridization was used to identify differentially expressed genes among cDNAs of the VBNC suppression mutant and the wild-type Vibrio vulnificus strain. Glutathione S-transferase was identified as a responsive gene of the VBNC suppression mutant in our assay, and was highly expressed from the VBNC suppression mutant at low temperature stress. Culturability tests revealed that the wild-type cells were sensitive to oxidative stress in the hydrogen peroxide (H(2)O(2)) and to 1-chloro-2,4-dinitrobenzene (CDNB) compared with the VBNC suppression mutant cells. Adding glutathione showed that many wild-type V. vulnificus cells maintained culturability in cold ASW. These results suggest that non-nutritional growth inhibitors, such as peroxide that accumulates at low temperatures, influence VBNC in V. vulnificus cells.

The Vibrio seventh pandemic island-II is a 26{middle dot}9 kb genomic island present in Vibrio cholerae El Tor and O139 serogroup isolates that shows homology to a 43{middle dot}4 kb genomic island in V. vulnificus

Vibrio cholerae is the aetiological agent of the deadly diarrhoeal disease cholera. In this study the 7.5 kb Vibrio seventh pandemic island-II (VSP-II) that is unique to V. cholerae El Tor and O139 serogroups was analysed and it was found to be part of a novel 26.9 kb genomic island (GEI) encompassing VC0490-VC0516. The low-GC-content VSP-II encompassed 24 predicted ORFs, including DNA repair and methyl-accepting chemotaxis proteins, a group of hypothetical proteins and a bacteriophage-like integrase adjacent to a tRNA gene. Interestingly, V. cholerae ORFs VC0493-VC0498, VC0504-VC0510 and VC0516, which encodes an integrase, were homologous to Vibrio vulnificus strain YJ016 ORFs VV0510-VV0516, VV0518-VV0525 and VV0560, which also encodes an integrase, respectively. Some ORFs showed amino acid identities greater than 90 % between the two species in these regions. In V. vulnificus strain YJ016, a 43.4 kb low-GC-content (43 %) GEI encompassing ORFs VV0509-VV0560 was identified and named V. vulnificus island-I (VVI-I). The 52 ORFs of VVI-I included a phosphotransferase system gene cluster, genes required for sugar metabolism and transposase genes. There was synteny and homology between the 5' region of V. cholerae VSP-II and the 5' region of V. vulnificus VVI-I; however, VVI-I contained an additional 31.5 kb of DNA between VV0526 and VV0560 in strain YJ016. A second V. vulnificus strain, CMCP6, did not contain the 43.4 kb VVI-I; in this strain two ORFs were found between the 5' and 3' flanking genes VV10636 and VV10632, showing 100 % identity to VV0508 and VV0561, respectively, which flank VVI-I.

Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds

Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron–tetra(4-sulfonatophenyl)porphyrin (Fe–TPPS), as well as from a natural heme, protoheme. This assimilation of iron bound to TPPS was demonstrated to be a common property of V. vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources. Strain L-180 could also utilize Fe–TCPP, but not Fe–TMPyP, as a sole iron source. TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe–TPPS. When inoculated into human serum supplemented with Fe–TCPP, L-180 could grow only in the presence of a protease from the same bacterium. In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge. Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V. vulnificus.

Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds.

Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron-tetra(4-sulfonatophenyl)porphyrin (Fe-TPPS), as well as from a natural heme, protoheme. This assimilation of iron bound to TPPS was demonstrated to be a common property of V. vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources. Strain L-180 could also utilize Fe-TCPP, but not Fe-TMPyP, as a sole iron source. TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe-TPPS. When inoculated into human serum supplemented with Fe-TCPP, L-180 could grow only in the presence of a protease from the same bacterium. In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge. Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V. vulnificus.

Disease resistance and humoral immunomodulatory effects of vitamin C on grouper,Epinephelus awoara

The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper,Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/kg were investigated. After 20 weeks, the growth rates of the groups with high level of vitamin C apparently increased. The untreated fish had symptoms of vitamin C deficiency. The endogenous liver tissue vitamin C levels were found to reflect well the dietary treatments. After intraperitoneal injection or bath challenge with a virulent strain ofVibrio vulnificus, fish fed with high level vitamin C showed significantly higher survival rate compared with the normal control group. Vaccination with formalin inactivatedV. vulnificus significantly enhanced the specific antibody production in fish treated with vitamin C, and completely protected from strong bacterial challenge the groups fed on fish with vitamin C 1500 and 2000 mg/kg diet.

In vitro synergism between cefotaxime and minocycline against Vibrio vulnificus

We conducted time-kill studies to evaluate the inhibitory activities of either cefotaxime or minocycline alone and the two drugs in combination against a clinical strain of Vibrio vulnificus. The MICs of cefotaxime and minocycline were 0.03 and 0.06 microg/ml, respectively. When approximately 5 x 10(5) CFU of V. vulnificus per ml was incubated with cefotaxime at 0.03 or 0.05 microg/ml, the bacterial growth was inhibited during the initial 2 and 8 h, respectively. Thereafter, V. vulnificus regrew and the level of growth reached that of the control. Within the dose range of less than five times the MIC, the duration of the inhibitory effect of cefotaxime was proportional to its concentration. When minocycline at 0.015, 0.03, 0.045, and 0.06 microg/ml was used to evaluate the inhibitory effect, a similar trend was observed. Either antibiotic at a concentration of five times the MIC or greater prevented the regrowth of V. vulnificus for at least 48 h. When cefotaxime at 0.05 microg/ml and minocycline at 0.045 microg/ml were combined in the same culture, the inhibitory effect against V. vulnificus persisted for more than 48 h, with no regrowth noted. The use of a combination of these two antibiotics resulted in the reduction of growth by 6 orders of magnitude compared to the use of either of the two antibiotics alone, and the number of surviving organisms in the presence of the antibiotics combined was approximately 3 orders of magnitude less than that in the starting inoculum. We conclude that cefotaxime and minocycline acted synergistically in inhibiting V. vulnificus in vitro.