Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine

Crowther, Roger S.; Roomi, Nusrath; Fahim, Raafat; Forstner, Janet F.

doi:10.1016/0304-4165(87)90153-x

Cited by 43 publications

(29 citation statements)

References 28 publications

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“…For example, the V. cholerae HA/protease (Hap) has been shown to nick and activate the A subunit of cholera enterotoxin (5), and the V. vulnificus Vvp metalloprotease causes a hemorrhagic reaction by degrading type IV collagen in basement membranes (17). The LasB elastase of P. aeruginosa is required for tissue destruction during opportunistic infections (18).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

et al. 2008

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The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ϳ10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.Vibrio anguillarum, a marine bacterium, is the causative agent of vibriosis, a systemic disease of both wild and cultured marine fish characterized by hemorrhagic septicemia (2). Outbreaks of vibriosis result in high mortalities among infected fish, and this disease continues to be a major obstacle for the aquaculture industry (2). V. anguillarum enters its fish host through the gastrointestinal (GI) tract and quickly colonizes this nutrient-rich environment (21,22). Garcia et al. (9) have shown that V. anguillarum grows extremely well in salmon intestinal mucus and that mucus-grown cells specifically express a number of different proteins, including several outer membrane proteins (9) and the extracellular metalloprotease EmpA (7).The zinc metalloprotease EmpA has been identified as a virulence factor for V. anguillarum and is important for virulence during infection of the GI tract of Atlantic salmon (Salmo salar) (7,16,19). In V. anguillarum wild-type strain M93Sm, EmpA is expressed during stationary phase when cells are incubated in Atlantic salmon GI mucus (6, 7). The creation of an empA null mutant (M99) by insertional mutagenesis showed that EmpA is responsible for the protease activity observed for wild-type strain M93Sm (7). However, EmpA activity is dependent on successful secretion and processing of the nascent protein to a...

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Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

et al. 2008

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“…To evaluate Pic activity on intestinal mucin in vivo, the rat ligated ileal loop model was used (9,42,48). Sprague-Dawley rats (n ϭ 4) of either sex and between 70 and 100 g in weight were used.…”

Section: Methodsmentioning

confidence: 99%

Pic, an Autotransporter Protein Secreted by Different Pathogens in the Enterobacteriaceae Family, Is a Potent Mucus Secretagogue

et al. 2010

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A hallmark of enteroaggregative Escherichia coli (EAEC) infection is a formation of biofilm, which comprises a mucus layer with immersed bacteria in the intestines of patients. While studying the mucinolytic activity of Pic in an in vivo system, rat ileal loops, we surprisingly found that EAEC induced hypersecretion of mucus, which was accompanied by an increase in the number of mucus-containing goblet cells. Interestingly, an isogenic pic mutant (EAEC ⌬pic) was unable to cause this mucus hypersecretion. Furthermore, purified Pic was also able to induce intestinal mucus hypersecretion, and this effect was abolished when Pic was heat denatured. Site-directed mutagenesis of the serine protease catalytic residue of Pic showed that, unlike the mucinolytic activity, secretagogue activity did not depend on this catalytic serine protease motif. Other pathogens harboring the pic gene, such as Shigella flexneri and uropathogenic E. coli (UPEC), also showed results similar to those for EAEC, and construction of isogenic pic mutants of S. flexneri and UPEC confirmed this secretagogue activity. Thus, Pic mucinase is responsible for one of the pathophysiologic features of the diarrhea mediated by EAEC and the mucoid diarrhea induced by S. flexneri.

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“…For example, it has been shown that the V. cholerae metalloprotease cleavage activity is essential for activating the A subunit of the cholera enterotoxin (12), as well as for degrading intestinal mucin and facilitating the action of cholera toxin (7). In the case of V. vulnificus infection, a metalloprotease has been shown to cause a hemorrhagic reaction by degrading type IV collagen in basement membranes (44).…”

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confidence: 99%

Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

Roux

Binesse

Saulnier

et al. 2007

Appl Environ Microbiol

234

264

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Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the P BAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.Vibrio splendidus is a dominant culturable Vibrio in coastal marine sediments, seawater, and bivalves, including oysters (23). This organism has long been considered to be an environmental organism without any pathogenic significance. However, over the last few years, different strains phenotypically related to this species have been associated with mortality mainly in mollusks, shrimps, gorgonians, and fish (for a review, see reference 35). Compared to human pathogen species, little is known about Vibrio pathogenesis in marine animals, and despite descriptions of invasiveness and extracellular product (ECP) toxicity, no data are available for a group related to V. splendidus (26,37,48,56).The different types of enzymatic activities that have been shown to play a role in the virulence of a variety of pathogenic bacteria include extracellular proteases; for example, such proteases have been described for Vibrio cholerae (7), Vibrio vulnificus (33), and Vibrio anguillarum (42), although a direct role of these proteases in virulence has not been demonstrated. For example, it has been shown that the V. cholerae metalloprotease cleavage activity is essential for activating the A subunit of the cholera enterotoxin (12), as well as for degrading intestinal mucin and facilitating the action of cholera toxin (7). In the case of V. vulnificus infection, a metalloprotease has been shown to cause a hemorrhagic reaction by degrading type IV collagen in basement membranes (44). Finally, the empA-encoded metalloprotease of V. anguillarum has been shown to be involved in the invasive mechanism of this fish pathogen (49).We recently completed sequencing of the genome of V.splendidus strain LGP32 in...

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Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine

Cited by 43 publications

References 28 publications

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

Pic, an Autotransporter Protein Secreted by Different Pathogens in the Enterobacteriaceae Family, Is a Potent Mucus Secretagogue

Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

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