Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus

Lee, Jonghee; Kim, Gu-Taek; Lee, Jong‐Young; Jun, Hong-Ki; Yu, Ju-Hyun; Kong, In‐Soo

doi:10.1016/s0167-4838(97)00205-7

Cited by 30 publications

(19 citation statements)

References 25 publications

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“…In addition, two FAXWXXT motifs, FDQWAQS and FSAWLDT (conserved residues are in boldface), which are implicated in collagen binding (32), and two bacterial prepeptidase C-terminal (PPC) domains also were identified at amino acids 537 to 543, 555 to 561, 612 to 686, and 731 to 804, respectively. A comparison of the amino acid sequences of class II metalloproteases in different Vibrio species showed that VC1650 displays the highest similarity (over 70%) to the 61-kDa collagenase VMC from V. mimicus and 63-kDa PrtVp protease (VPPRT) from V. parahaemolyticus 93 (31)(32)(33)(34).…”

Section: Resultsmentioning

confidence: 99%

A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

et al. 2015

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bVibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program. Members of the genus Vibrio are important inhabitants of the marine coastal waters, estuaries, and ocean sediments, with some pathogenic species also causing wound infections, primary septicemia, gastroenteritis, and diarrhea in humans (1, 2). Among the more than 200 serogroups of V. cholerae, only O1 and O139 are recognized as agents of cholera, a potentially life-threatening diarrheal disease. Cholera remains a significant public health problem by affecting millions of people annually, predominantly in developing countries that have limited clean water supplies and poor sanitation (3). Infection of the human host occurs upon ingestion of water or food contaminated with the bacterium. Following colonization of the small intestine, V. cholerae utilizes a multicomponent secretory pathway, the type II secretion system (T2SS), to release cholera toxin. Cholera toxin induces acute diarrhea in people infected with cholera, enabling the bacteria to escape from the host into the aquatic reservoir (4). During interepidemic periods, V. cholerae persists freely or in association with various aquatic organisms, including copepods, insect egg masses, shellfish, and vertebrate fish (5-7). Extracellular proteins, including those secreted by the T2SS, have been implicated in facilitating the fitness of V. cholerae in both the human host and aquatic niches (4,6,8). The array of extrace...

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Section: Resultsmentioning

confidence: 99%

A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

et al. 2015

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“…NT, nontreated; TEP, tetraethylene pentamine; OPA, 1,10-phenanthroline; PMSF, phenylmethylsulfonyl fluoride; PPA, pepstatin A; CHOL, cholesterol. (23,31). VtpA possesses a zinc binding motif (HEXXH) at residues 341H to 345H as well as another critical ligand, 365E, located 18 amino acid residues from the HEXXH motif (Fig.…”

Section: Fig 3 Effects Of Protease and Hemolysin Inhibitors On Protmentioning

confidence: 99%

The Extracellular Metalloprotease ofVibrio tubiashiiIs a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae

Hasegawa

Lind

Boin

et al. 2008

Appl Environ Microbiol

131

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Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.Vibriosis caused by marine Vibrio species is considered one of the most serious diseases of hatchery-reared oyster larvae (10,11,17,47,52). The disease is characterized by a rapid and dramatic reduction in larval motility, detached vela, and necrotic soft tissue, which lead to high mortality rates, exceeding 90% within 1 day of infection (45). Pathogenic agents that cause larval bivalve vibriosis have intermittently and severely curtailed shellfish hatchery production on the Atlantic and Pacific coasts of the United States, causing substantial losses in the industry (3, 10, 13). Vibrio tubiashii, a bacterial species first reported by Tubiash et al. (51), was identified as a causative agent of vibriosis (originally referred to as bacillary necrosis) in larval and juvenile bivalves of the hard clam (Mercenaria mercenaria) and Eastern oyster (Crassostrea virginica). Estes et al. (14) characterized a number of pathogenic and nonpathogenic bacterial strains from diseased Pacific oysters (Crassostrea gigas) at shellfish hatcheries on the Pacific coast of North America and described some of the highly pathogenic bacterial isolates as V. tubiashii.The genus Vibrio is the largest member of the family Vibrionaceae, which includes gram-negative and curved rod-shaped facultative anaerobes. The genus consists of at least 30 known species, which are w...

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“…into two distinct categories according to the amino acid sequences. Class I Vibrio metalloproteases included Hap, VVP, VVF, and NprV, and class II Vibrio metalloproteases included VMC, VPPRT, and VAC [6] The untagged 33-amino acid peptide from Leu529 to Thr561, VMCBD peptide, was generated by digestion of GSTCBD-C with thrombin. The digested mixture was then incubated with collagen.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, an atypical strain of V. cholerae, V. mimicus, produces two metalloproteases, the 31-kDa VMP protein and the 61-kDa VMC protein [6,19]. Previously, we showed that the intact VMC protein (VMC61) has a specific collagenase activity and that its amino acid sequence is similar to those of VCC of V. cholerae, VAC of V. alginolyticus, and VPPRT of V. parahaemolyticus [20].…”

Section: Introductionmentioning

confidence: 99%

The FAXWXXT motif in the carboxyl terminus of Vibrio mimicus metalloprotease is involved in binding to collagen

Lee¹,

Ahn²,

Lee³

et al. 2005

FEBS Letters

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We have shown previously that the C-terminal region of the extracellular metalloprotease of Vibrio mimicus (VMC) is essential for collagenase activity. Here, we demonstrate that deletion of 100 amino acids, but not 67 amino acids, from the C-terminus of the intact VMC protein (VMC61) abolished the collagenase activity. The intervening 33-amino acid region contains a repeated FAXWXXT motif that is essential for insoluble type I collagen binding; the isolated 33-amino acid peptide bound to insoluble type I collagen, while a peptide containing only the first FAXWXXT motif did not. Compared to the VMC61, the 33-amino acid peptide corresponding to the C-terminus exhibited a similar binding affinity and a lower binding capacity.

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Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus

Cited by 30 publications

References 25 publications

A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

The Extracellular Metalloprotease ofVibrio tubiashiiIs a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae

The FAXWXXT motif in the carboxyl terminus of Vibrio mimicus metalloprotease is involved in binding to collagen

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