High growing ability of Vibrio vulnificus biotype 1 is essential for production of a toxic metalloprotease causing systemic diseases in humans

doi:10.1016/j.micpath.2003.10.001

Microbial Pathogenesis

Volume 36, Issue 3, March 2004, Pages 117-123

https://doi.org/10.1016/j.micpath.2003.10.001 Get rights and content

Abstract

Vibrio vulnificus biotype 1, a causative agent of fatal septicemia or wound infection in humans, is known to produce a toxic metalloprotease as an important virulence determinant. V. vulnificus biotype 2 (serovar E), a primary eel pathogen, was found to elaborate an extracellular metalloprotease that was indistinguishable from that of biotype 1. The potential of V. vulnificus biotype 1 for production of the metalloprotease was compared with biotype 2 and other human non-pathogenic Vibrio species (Vibrio anguillarum and Vibrio proteolyticus). When cultivated at 25 °C in tryptone–yeast extract broth supplemented with 0.9% NaCl, all bacteria multiplied sufficiently and secreted significant amounts of the metalloprotease. However, at 37 °C with 0.9% NaCl, V. anguillarum neither grew nor produced the metalloprotease. In human serum, only V. vulnificus biotype 1 revealed a steady multiplication accompanied with production of the extracellular metalloprotease. This prominent ability of biotype 1 in growth and protease production may contribute to cause serious systemic diseases in humans.

Introduction

Vibrio vulnificus biotype 1 is a human pathogen causing fatal septicemia or wound infection, and the infection is characterized by formation of hemorrhagic and edematous lesions on the limbs [1]. This bacterium produces a 45 kDa thermolysin-like metalloprotease (V. vulnificus protease (VVP)) as an important virulence determinant, which increases vascular permeability and causes serious hemorrhagic damage [2], [3]. V. vulnificus biotype 2 that is also named as serovar E [4] is a primary causative agent of epizooticus in eel farms [5], [6], while it has isolated occasionally from human clinical sources [7]. Our preliminary experiments using partially purified preparations indicated that an extracellular protease produced by biotype 2 might be indistinguishable from VVP. In spite of their being avirulent to humans, other vibrios such as Vibrio anguillarum [8], [9] and Vibrio proteolyticus [10] also produce closely related metalloproteases those exhibit 70–80% identities in the primary structures and have comparable biological activities. Additionally, these proteolytic enzymes are neutralized with the antibody against VVP [11].

In various microorganisms, production of extracellular proteases, as well as other factors, is coordinated by the quorum sensing or cell density-dependent regulation system [12], [13]. Two kinds of quorum-sensing systems have been reported in V. anguillarum. One system is operated by a small substance, N-(3-oxodecanoyl)-l-homoserine lactone [14], while another system is linked to a substance related to Vibrio harveyi autoinducer 2 (AI-2) [15]. In V. vulnificus biotype 1, VVP production is positively regulated by the system dependent on the AI-2-like substance [16], [17], [18]. Namely, expression of the vvp gene is augmented at stationary growth phase because of sufficient bacterial multiplication followed by accumulation of the AI-2-like substance.

The present study showed that V. vulnificus biotype 1 was able to multiply in human plasma. Therefore, although both biotypes have equal capability of producing toxic metalloproteases in vitro, only biotype 1 is capable of growing and therefore secreting the metalloprotease in humans.

Section snippets

Extracellular protease produced by biotype 2

For purification of a metalloprotease (E86 protease), the supernatant of the 24 h culture of V. vulnificus E86, a biotype 2 strain, was fractionated by ammonium sulfate precipitation. Thereafter, the preparation was subjected to gel filtration on a HiLoad 26/60 Sephacryl S-200 HR column and hydrophobic interaction chromatography on a HiLoad 10/2 Phenyl Sepharose column. The final preparation thus obtained revealed homogeneity on SDS–PAGE. Namely, in the presence or absence of a reducing agent,

Protease preparations

V. vulnificus E86 provided kindly by Dr C. Amaro (Universidad de Valencia, Spain) was cultivated in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) containing 1.5% NaCl at 25 °C for 24 h with shaking (120 cycles/min). After cultivation, the culture supernatant was collected by centrifugation at 7000g for 40 min, and ammonium sulfate was added to 60% saturation. The resulting precipitate was collected, dissolved in distilled water, and applied to a HiLoad 26/60 Sephacryl S-200 HR column

Acknowledgements

We are grateful to Dr Carmen Amaro (Departamento Microbiologia y Ecologia, Universidad de Valencia, Spain) for providing bacterial strains. This study was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.

References (39)

S. Miyoshi et al.
Microbial metalloproteases and pathogenesis
Microbes Infect
(2000)
V.A. David et al.
Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus
Gene
(1992)
H. Withers et al.
Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria
Curr Opin Microbiol
(2001)
H.S. Jeong et al.
Differential expression of Vibrio vulnificus elastase gene in a growth phase-dependent manner by two different types of promoters
J Biol Chem
(2001)
S. Miyoshi et al.
Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds
FEMS Microbiol Lett
(2002)
J.M. Janda et al.
Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp
Clin Microbiol Rev
(1988)
S. Miyoshi et al.
Exocellular toxic factors produced by Vibrio vulnificus
J Toxicol Toxin Rev
(1993)
E.G. Biosca et al.
Phenotypic and genotypic characterization of Vibrio vulnificus: proposal for the substitution of the subspecific taxon biotype for serovar
Appl Environ Microbiol
(1997)
D.L. Tison et al.
Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels
Appl Environ Microbiol
(1982)
E.G. Biosca et al.
First record of Vibrio vulnificus biotype 2 from diseased European eels (Anguilla anguilla)
J Fish Disease
(1991)

C. Amaro et al.

Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans

Appl Environ Microbiol

(1996)

A. Norqvist et al.

Identification and characterization of a zinc metalloprotease associated with invasion by the fish pathogen Vibrio anguillarum

Infect Immun

(1990)

D.L. Milton et al.

Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum

J Bacteriol

(1992)

M. Alam et al.

Production of antigenically related exocellular elastolytic proteases mediating haemagglutination by vibrios

Microbiol Immunol

(1995)

M.R. Parsek et al.

Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: a signaling mechanism involved in associations with higher organisms

Proc Natl Acad Sci USA

(2000)

D.L. Milton et al.

Quorum sensing in Vibrio anguillarum: characterization of vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-l-homoserine lactone

J Bacteriol

(1997)

A. Croxatto et al.

VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum

J Bacteriol

(2002)

D. McDougald et al.

SmcR-dependent regulation of adaptive phenotypes in Vibrio vulnificus

J Bacteriol

(2001)

C.P. Shao et al.

Regulation of metalloprotease gene expression in Vibrio vulnificus by a Vibrio harveyi LuxR homologue

J Bacteriol

(2001)

Cited by (36)

Molecular genotyping and phenotyping of Vibrio vulnificus isolated from diseased, brown-marbled grouper (Epinephelus fuscoguttatus) in Thailand with preliminary vaccine efficacy analysis
2021, Aquaculture
Citation Excerpt :
In correlation with the previous study, the high growth characteristic of V. vulnificus biotype 1 (human isolate) has been accompanied by the production of the virulence gene as the extracellular metalloprotease. This prominent ability of their growth and protease production may contribute to causing serious systemic diseases in humans (Watanabe et al., 2004). Interestingly, in the current study, the VV03 harboring morevirulence genes (20 genes) than the VV06 and VV13, so it should provide a faster growth rate.
Vibrio vulnificus is the major pathogenic bacterium causing mass mortality in groupers (Epinephelus spp.) culture with symptoms as skin and fins hemorrhages and ulcerative necrosis. Microbiological and biochemical characterizations were used to identify Vibrio spp., but it is ambiguous to distinguish V. vulnificus and others. Therefore, molecular genotypic analysis was applied to identify and typing Vibrio spp. isolated from the diseased grouper. Firstly, the identification of Vibrio spp. was assessed by 16 s rRNA before generating the phylogenetic tree. It revealed that 40 samples from 63 samples were V. vulnificus biotype 1. After that, all V. vulnificus samples were classified into subgroup by molecular biotyping based on the 22 virulence genes identification. Accordingly, V. vulnificus can be categorized as subgroup 1, 2, and 3 (marked as G1, G2, and G3, respectively) based on the presence of those studied virulence genes. V. vulnificus isolates containing 16 virulence genes of vvpE, gloB, tlh, Mann Hemo, ManIIA, rimO, vllY, OmpU, vvhA, rtx, hlyIII, pilF, Mann TRAP, vcgC, 16S rRNA type B and nanA has been classified as subgroup 1, whereas subgroup 2 has shown the additional 4 genes including YJ-like nab 1, YJ-like nab 2, cps and sodA, as well as subgroup 3 harboring CM-like nab 1, CM-like nab2, and sodA. Surprisingly, different identification method using 10 house-keeping genes in multi-locus sequence typing (MLST) represented the new three different sequence type (ST) profile. Additionally, phenotypic analysis of isolated V. vulnificus was also performed through the disk diffusion method. This result demonstrated that G1 and G2 were susceptible to tetracycline at 44.4% and 53.8%, respectively, while G3 was susceptible to oxytetracycline (46.2%). Interestingly, G2 which lacking CM-like nab1 and CM-like nab2 alleles exhibited the highest virulent pathogenicity, despite showing a slower growth rate than G1 and G3. Meanwhile, G3, lacking cps gene but containing CM-like nab1 and CM-like nab2 alleles, did not cause mortality in experimental challenge fish. In the case of inactivated vaccine efficiency trial, the FKC-Mixed vaccine revealed the best efficacy when compared with other vaccinated groups (P < 0.05). Altogether, our findings suggested that the molecular genotyping analysis is useful for identifying Vibrio spp. and may apply as the appropriate platform for further characterization of other bacterial species.
Regulation system of serine protease production in Vibrio vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel
2013, Aquaculture
Citation Excerpt :
This regulation is termed QS, which is mediated by the small diffusible signal molecule called AI-2 (Federle and Bassler, 2003; Henke and Bassler, 2004). However, it should be noted that the QS system is much more effective at 26 °C than at 37 °C (Kawase et al., 2004; Lee et al., 2007; Watanabe et al., 2004). Our present study revealed that the cultivation temperature and growth phase are very critical determinants for the regulation of VvsA production in V. vulnificus.
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.
Accurate diagnosis and treatment of Vibrio vulnificus infection: A retrospective study of 12 cases
2013, Brazilian Journal of Infectious Diseases
Vibrio vulnificus causes an infectious disease that has extremely poor convalescence and leads to necrotic fasciitis. In this study, we sought to define the characteristic epidemiology of V. vulnificus infection and clarify its diagnosis at the global level.
Over a period of 10 years, we investigated the appearance of symptoms, underlying conditions, treatment, and mortality in 12 patients (eight men, four women; >50 years old; average age, 66 years,) infected with V. vulnificus.
The development of symptoms occurred primarily between June and September, a period during which seawater temperature rises and the prevalence of V. vulnificus increases. All patients had underlying diseases, and seven patients reported a history of consuming fresh fish and uncooked shellfish. The patients developed sepsis and fever with sharp pain in the limbs. Limb abnormalities were observed on visual examination. All patients underwent debridement; however, in the survival group, the involved limb was amputated early in 80% patients. The mortality rate was 58.3%.
Recognition of the characteristic epidemiology and clinical features of this disease is important, and positive debridement should be performed on suspicion. When the illness reaches an advanced stage, however, amputation should be the immediate treatment of choice.
Regulation of the Vibrio vulnificus vvpE expression by cyclic AMP-receptor protein and quorum-sensing regulator SmcR
2010, Microbial Pathogenesis
Citation Excerpt :
In general, however, CRP functions in response to decreasing glucose availability, whereas SmcR functions after the density of growing bacteria reaches a threshold [2,4]. In glucose-insufficient conditions, CRP can affect the metabolic status or RpoS expression of V. vulnificus and, eventually, the growing ability or density-dependent AI-2-QSS activity of V. vulnificus, all of which may directly or indirectly affect RpoS-dependent vvpE expression [10–12]. It was recently demonstrated that CRP negatively affects RpoS expression in V. vulnificus [13].
In Vibrio vulnificus, cAMP-receptor protein (CRP) and the quorum-sensing regulator SmcR are simultaneously and cooperatively required for the metalloprotease vvpE gene expression, rather than sequentially in a regulatory cascade. However, this study shows a new temporal and functional sequence between the two factors in regulating vvpE expression. A crp mutation inhibited vvpE expression with growth impairment from early stage. In contrast, a smcR mutation inhibited vvpE expression only at the late stage with no effect on growth. A crp–smcR double mutation severely inhibited vvpE expression with growth impairment from early stage. The inhibited vvpE expression was restored only at the early stage by a crp single complementation, but not at all by a smcR complementation. These results indicate that CRP functions as an essential activator, whereas SmcR functions in the presence of CRP for full vvpE expression.
Variation of extracellular proteases produced by Vibrio vulnificus clinical isolates: Genetic diversity of the metalloprotease gene (vvp), and serine protease secretion by vvp-negative strains
2008, Microbial Pathogenesis
Citation Excerpt :
It should be emphasized that no amino acid residue in and around the zinc-binding motif [15] was changed. This finding may consist with our recent reports [14,16]. Namely, a metalloprotease from strain E86 was revealed to have comparable proteolytic, permeability-enhancing and hemorrhagic activity to the enzyme from strain L-180.
Vibrio vulnificus is a causative agent of septicemia or wound infection in human and eel; however, the genetic variation between human and eel isolates has been reported. In the present study, the difference in the vvp gene encoding a tissue-damaging metalloprotease was investigated. The gene of strain E86 from a diseased eel (type B vvp) was 95.2% identical with that of strain L-180 from human blood (type A vvp). PCR using oligonucleotide primers designed to differentiate two types of the gene showed that eel avirulent strains (9 isolates) commonly carry type A vvp, whereas eel virulent strains (18 isolates) revealed significant genetic variation. The vvp genes from 12 strains including strain E86 were placed on type B while those from 3 strains were on type A. Other strains were found to be vvp-negative, but PAGE and amino acid sequencing analysis showed that they secreted a serine protease (VVA0302) instead of the metalloprotease. This protease is an orthologue of a toxic protease from Vibrio parahaemolyticus, a human pathogen causing wound infection as well as gastroenteritis. These findings suggest that, in addition to metalloprotease, the extracellular serine protease may contribute to pathogenicity of V. vulnificus.
Identification and whole-genome sequencing analysis of Vibrio vulnificus strains causing pearl gentian grouper disease in China
2022, BMC Microbiology

View all citing articles on Scopus

View full text

High growing ability of Vibrio vulnificus biotype 1 is essential for production of a toxic metalloprotease causing systemic diseases in humans

Abstract

Introduction

Section snippets

Extracellular protease produced by biotype 2

Protease preparations

Acknowledgements

Microbes Infect

Gene

Curr Opin Microbiol

J Biol Chem

FEMS Microbiol Lett

Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp

Clin Microbiol Rev

Exocellular toxic factors produced by Vibrio vulnificus

J Toxicol Toxin Rev

Phenotypic and genotypic characterization of Vibrio vulnificus: proposal for the substitution of the subspecific taxon biotype for serovar

Appl Environ Microbiol

Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels

Appl Environ Microbiol

First record of Vibrio vulnificus biotype 2 from diseased European eels (Anguilla anguilla)

J Fish Disease

Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans

Appl Environ Microbiol

Identification and characterization of a zinc metalloprotease associated with invasion by the fish pathogen Vibrio anguillarum

Infect Immun

Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum

J Bacteriol

Production of antigenically related exocellular elastolytic proteases mediating haemagglutination by vibrios

Microbiol Immunol

Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: a signaling mechanism involved in associations with higher organisms

Proc Natl Acad Sci USA

Quorum sensing in Vibrio anguillarum: characterization of vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-l-homoserine lactone

J Bacteriol

VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum

J Bacteriol

SmcR-dependent regulation of adaptive phenotypes in Vibrio vulnificus

J Bacteriol

Regulation of metalloprotease gene expression in Vibrio vulnificus by a Vibrio harveyi LuxR homologue

J Bacteriol