Cloning of the cytotoxin-hemolysin gene of Vibrio vulnificus

Wright, AC; Morris, J. Glenn; Maneval, David R.; Richardson, K. C.; Kaper, James B.

doi:10.1128/iai.50.3.922-924.1985

Cited by 94 publications

(35 citation statements)

References 21 publications

(14 reference statements)

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“…All tissue culture cells were grown in Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum and 200 mM L-glutamine at 37°C in 5% CO 2 . Cytotoxicity assays were carried out on Chinese hamster ovary cells (CHO) cells, using the procedures outlined by Guerrant et al (13) that were previously used to measure the cytolysin activity present in supernatants of V. vulnificus cultures (65). Briefly, cultures were grown overnight in BHI broth (with chloramphenicol and IPTG where appropriate) at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…Culture supernatants from V. vulnificus are cytotoxic to cultured epithelial cells, an activity attributed to the cytolysin, a 51-kDa protein that is hemolytic to mammalian erythrocytes. Purification and subsequent cloning and sequencing indicated that this cytotoxic and hemolytic activity was caused by the protein, designated VvhA or cytolysin, which has some homology to the V. cholerae El Tor hemolysin (12,65,68). To determine the effect of the vvpD mutation on cytoxicity, serial twofold dilutions of filtered supernatants from overnight cultures were added to monolayers of CHO cells, incubated for 6 h at 37°C, and monitored for cell rounding and destruction of the monolayer (65).…”

Section: Loss Of Vvpd Does Not Affect Serum Resistance and Encap-mentioning

confidence: 99%

“…Purification and subsequent cloning and sequencing indicated that this cytotoxic and hemolytic activity was caused by the protein, designated VvhA or cytolysin, which has some homology to the V. cholerae El Tor hemolysin (12,65,68). To determine the effect of the vvpD mutation on cytoxicity, serial twofold dilutions of filtered supernatants from overnight cultures were added to monolayers of CHO cells, incubated for 6 h at 37°C, and monitored for cell rounding and destruction of the monolayer (65). As expected from the results obtained in the cytolysin localization assays, the supernatant from C7184D 12 ⍀ was able to cause a cytopathic effect only at the highest concentration, while the supernatant from wild-type C7184 had to be diluted more than 64-fold before no effect on the CHO cells was observed (Table 4).…”

Section: Loss Of Vvpd Does Not Affect Serum Resistance and Encap-mentioning

confidence: 99%

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The Type IV Leader Peptidase/ N -Methyltransferase of Vibrio vulnificus Controls Factors Required for Adherence to HEp-2 Cells and Virulence in Iron-Overloaded Mice

et al. 1998

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Vibrio vulnificus expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts. We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/N-methyltransferase (PilD) mutant of Pseudomonas aeruginosa with a V. vulnificus genomic library. One of the genes (vvpD) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a P. aeruginosa mutant deficient in the expression of PilD. The other gene (vvpC) encodes a homolog of PilC from P. aeruginosa, where it is essential for assembly of type IV pili. Phenotypic characterization of a V. vulnificus vvpD mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on V. vulnificus are of the type IV class. This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway. Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance were not affected in the vvpD mutant, indicating that in addition to capsule, virulence of V. vulnificus requires type IV pili and/or extracellular secretion of several exoenzymes.Vibrio vulnificus biotype 1 is an estuarine bacterium that can cause primary septicemia as well as serious wound infections (34,35). While septicemia occurs primarily in immunocompromised individuals or those that suffer from cirrhosis or hemochromatosis, healthy people can become infected through wounds. Septicemia can develop after ingestion of shellfish carrying the organism, with the greatest risk coming from the consumption of raw oysters (5,21). Mortality in these cases exceeds 50%, increasing to greater than 90% in people who go into shock or become hypotensive shortly after admission to a hospital (22). As many as 50% of all vibrio-related illnesses in the United States are caused by V. vulnificus (7). Recently, a second biotype of V. vulnificus, biotype 2, has been implicated in septicemic infections of cultured eels (63). Animal studies have shown that biotype 2 is also capable of causing septicemia in mammals, including opportunistic infections of humans (3,4).Among the many factors implicated as possible virulence determinants for V. vulnificus are extracellular toxins and enzymes (e.g., cytolysin and elastolytic protease) (25, 33), a polysaccharide capsule (67), resistance to phagocytosis (19, 70), resistance to the bactericidal effects of human sera (19,67,70), and the ability to acquire iron from transferrin (51). V. vulnificus undergoes a phase variation between viru...

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Section: Methodsmentioning

confidence: 99%

Section: Loss Of Vvpd Does Not Affect Serum Resistance and Encap-mentioning

confidence: 99%

Section: Loss Of Vvpd Does Not Affect Serum Resistance and Encap-mentioning

confidence: 99%

See 1 more Smart Citation

The Type IV Leader Peptidase/ N -Methyltransferase of Vibrio vulnificus Controls Factors Required for Adherence to HEp-2 Cells and Virulence in Iron-Overloaded Mice

et al. 1998

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“…which is unique to this species (Wright et al 1985 ;Morris et al 1987). This gene has been sequenced (Yamamoto et al 1990) and used as the basis of an oligonucleotide probe for the identification of V. vulnzjicus (Wright et al 1993).…”

Section: Vibrio Vulnzjicus Contains a Cytotoxin-haemolysin Genementioning

confidence: 99%

Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

Dalsgaard¹,

Dalsgaard²,

Høi³

et al. 1996

Lett Appl Microbiol

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Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B-cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish samples. When comparing the identification of putative V. vulnificus obtained with the API 20E assay and an oligonucleotide probe, 29 API 20E profiles were obtained with only four profiles (representing 20 isolates) reaching the identification threshold of V. vulnificus among a total of 66 isolates hybridizing with the probe. The results indicated that, compared with colony hybridization, the API 20E assay was not adequate for the identification of environmental isolates of V. vulnificus.

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“…The specificity of the nested PCR proce- dure using JY-1, JY-2, JY-3 and JY-4 primers was assessed with DNAs recovered from pure cultures of the strains listed in Table 1. Previous works revealed that the vvhA gene was unique to this organism by DNA hybridization (Wright et al, 1985). However, there are regions where the nucleotide sequences of the vvhA gene and the hly gene encoding hemolysin of V. cholerae showed significant homologies (Yamamoto et al, 1990).…”

Section: Specificity Of the Nested Pcr Amplificationmentioning

confidence: 97%

Two‐stage Nested PCR Effectiveness for Direct Detection of Vibrio vulnificus in Natural Samples

Lee

Bang

Rhee

et al. 1999

Journal of Food Science

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ABSTRCTThe nested primers designed to amplify a 222-base pair portion of the hemolysin gene, vvhA, were specific for all V. vulnificus strains tested. The nested PCR amplification, coupled with direct extraction of template DNA, revealed improved sensitivity sufficient for detection of 1 to 10 CFU V. vulnificus in 1 mL of seafood homogenates, and eliminated the need for enrichment culturing. Thereby, the nested PCR method achieved a broader applicability, making it effective for extensive use in identification of the pathogen in natural samples such as raw seafoods, seawater and sediments.

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Cloning of the cytotoxin-hemolysin gene of Vibrio vulnificus

Cited by 94 publications

References 21 publications

The Type IV Leader Peptidase/ N -Methyltransferase of Vibrio vulnificus Controls Factors Required for Adherence to HEp-2 Cells and Virulence in Iron-Overloaded Mice

The Type IV Leader Peptidase/ N -Methyltransferase of Vibrio vulnificus Controls Factors Required for Adherence to HEp-2 Cells and Virulence in Iron-Overloaded Mice

Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

Two‐stage Nested PCR Effectiveness for Direct Detection of Vibrio vulnificus in Natural Samples

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