Purification and Characterization of an Elastolytic Protease of Vibrio vulnificus

Shimizu

Microbiology and Immunology

et al. 1987

108

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone-yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl-Sephacel column chromatography, Sephacryl S-200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000-fold purification was achieved, with a yield of about 30% . The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.The illnesses caused by Vibrio vulnificus, a marine bacterium, are divided into two groups, wound-infection group and primary septicemia group (2, 3). In the former, edema and erythema are formed around a new wound in contact with sea water. In the latter group, cutaneous lesions, such as bullae, erythema or necrotic ulcer, often appear on one or more extremities.V. vulnificus is known to produce many toxic or pathogenic factors, such as cytolysin (8, 13, 23), protease (11, 13, 25), phospholipase A2 (27), and siderophores (24). However, in the pathogenesis of V. vulnificus infection, the actual role of any product has not been clarified yet. Kreger and Lockwood (13) reported that V. vulnificus cytolysin activity in the culture filtrate decreased in the stationary phase of the growth and the decrease correlated with the appearance of protease in the culture filtrate. We also observed significant excretion of protease from many strains of V. vulnificus isolated from patients. The pathogenic significance of proteases by some bacteria, such as pseudomonal protease (7,10,12) and serratial protease (9,16,19), has been reported. These proteases have vascular permeability-enhancing and hemorrhagic activities which are closely related to the above mentioned cutaneous lesions. We also found that the crude V. vulnificus protease preparation

Section: Amino Acid Analysismentioning

confidence: 99%

“…vulnificus is known to produce many toxic or pathogenic factors, such as cytolysin (8,13,23), protease (11,13,25), phospholipase A2 (27), and siderophores (24). However, in the pathogenesis of V. vulnificus infection, the actual role of any product has not been clarified yet.…”

mentioning

confidence: 99%

Purification and Characterization of Vibrio vulnificus Protease

Shimizu

Microbiology and Immunology

et al. 1987

108

“…have been studied in detail. Vibrio vulnificus, which causes wound infections and septicaemia in humans, produces a neutral metalloprotease with elastolytic activity (Kothary & Kreger, 1987) that has elicited dermonecrosis (Smith & Merkel, 1982). Vibrio cholerae haemagglutinin/protease nicks and activates the A subunit of the cholera enterotoxin (Booth et al, 1984), and shows…”

Section: Introductionmentioning

confidence: 99%

Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli

Yu¹,

Lee²

1999

Microbiology

The prtV gene, encoding a collagenase of Vibrio parahaemolyticus, was expressed in Escherichia coli and purified by affinity chromatography. The transformant E. coli BL2l (DE3)(pPRT2) secreted the recombinant Prtv, and the highest enzyme activity was detected in the culture supernatant after 5 h IPTG induction. The molecular mass o f purified PrtV was 62 kDa as determined by gel filtration, which was similar to that obtained by SDS-PAGE (64 kDa). This suggested that PrtV was a monomer protein having no subunit structure. The isoelectric point o f PrtV was 852. In addition, PrtV contained a 27 amino acid signal peptide, and the amino acid composition of the PrtV showed satisfactory agreement with that predicted from the DNA sequence. The optimum temperature and pH of PrtV were 40 OC and pH 7.5, respectively. The activity of PrtV was inhibited by chelators such as EDTA, EGTA and 1,lOphenanthroline; however, its activity was restored by the addition of various metal ions (Co2+, Mn2+, Ca2+, Cu2+, Ni2+ and Zn2+), indicating that PrtV is a metalloprotease. PrtV degraded both type I collagen and synthetic substrate FALGPA well, showing that PrtV is indeed a collagenase.

Journal of Biological Chemistry

“…An aphA homolog was identified in V. vulnificus (56), and although expression of this gene was not directly affected by Qrrs, it was repressed by SmcR. 3 We speculate that there are other as yet unidentified regulatory proteins that are activated at low cell density in V. vulnificus. Qrrs are expressed at low cell densities and derepressed under iron-limiting conditions, suggesting that they may play a role in the regulation of genes required under these conditions.…”

Section: Discussionmentioning

confidence: 99%

Iron- and Quorum-sensing Signals Converge on Small Quorum-regulatory RNAs for Coordinated Regulation of Virulence Factors in Vibrio vulnificus

Wen

Kim

2016

Vibrio vulnificus is a marine bacterium that causes human infections resulting in high mortality. This pathogen harbors five quorum-regulatory RNAs (Qrr1-5) that affect the expression of pathogenicity genes by modulating the expression of the master regulator SmcR. The qrr genes are activated by phosphorylated LuxO to different degrees; qrr2 is strongly activated; qrr3 and qrr5 are moderately activated, and qrr1 and qrr4 are marginally activated and are the only two that do not respond to cell density-dependent regulation. Qrrs function redundantly to inhibit SmcR at low cell density and fully repress when all five are activated. In this study, we found that iron inhibits qrr expression in three distinct ways. First, the iron-ferric uptake regulator (Fur) complex directly binds to qrr promoter regions, inhibiting LuxO activation by competing with LuxO for cis-acting DNA elements. Second, qrr transcription is repressed by iron independently of Fur. Third, LuxO expression is repressed by iron independently of Fur. We also found that, under ironlimiting conditions, the five Qrrs functioned additively, not redundantly, to repress SmcR, suggesting that cells lacking iron enter a high cell density mode earlier and could thereby modulate expression of virulence factors sooner. This study suggests that iron and quorum sensing, along with their cognate regulatory circuits, are linked together in the coordinated expression of virulence factors.