Yoko Maehara

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

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.

Differential gene expression and extracellular secretion of the collagenolytic enzymes by the pathogen Vibrio parahaemolyticus

Vibrio parahaemolyticus, a causative agent of wound infections as well as food poisoning, harbors two collagenase genes: vppC and prtV. When cultivated at 26 degrees C in gelatin broth supplemented with 3.0% NaCl, significant collagenolytic activity was detected in the culture supernatant at the early stationary phase. Native polyacrylamide gel electrophoresis analysis revealed a 90-kDa protein, and N-terminal amino acid sequencing showed that this protein was VppC, generated through truncation of 72 N-terminal amino acid residues. Additionally, significant expression of only vppC was observed by reverse transcriptase PCR. By contrast, a vppC-negative mutant constructed through single crossover homologous recombination secreted a 50-kDa-collagenolytic enzyme; however, this enzyme was a serine protease that was reported previously. These results suggest that VppC is a primary extracellular collagenase produced by V. parahaemolyticus.

Modulation of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease

Vibrio mimicus is a causative agent of human gastroenteritis and food poisoning, and this species produces an enterotoxic hemolysin (V. mimicus hemolysin) as a virulence determinant. Vibrio mimicus hemolysin is secreted as an 80 kDa precursor, which is later converted to the 66 kDa mature toxin through removal of an N‐terminal propeptide via cleavage of the Arg151–Ser152 bond. In this article, we investigate the role of the endogenous metalloprotease (V. mimicus protease) in the maturation of V. mimicus hemolysin. In vitro experiments using purified proteins showed that, although it activated the precursor at the early stage via cleavage of the Asn157–Val158 bond, V. mimicus protease finally converted the activated and physiologically maturated toxin to a 51 kDa protein through removal of the C‐terminal polypeptide. This 51 kDa derivative was unable to lyse erythrocytes because of its inability to bind to the erythrocyte membrane. Vibrio mimicus protease‐negative strains were found to produce high levels of V. mimicus hemolysin at the logarithmic phase of bacterial growth and maintained high hemolytic activity even at the stationary phase. These findings indicate that, although it is not directly related to toxin maturation in vivo, V. mimicus protease can modulate the activity of V. mimicus hemolysin and/or its precursor through limited proteolysis.

Defensive Effects of Human Intestinal Antimicrobial Peptides against Infectious Diseases Caused by Vibrio mimicus and V. vulnificus

Of human pathogenic Vibrio species, V. mimicus causes gastroenteritis whereas V. vulnificus causes fatal septicemia after consumption of contaminated seafood. These two pathogens produce hemolytic toxins termed V. mimicus hemolysin (VMH) and V. vulnificus hemolysin (VVH), respectively. These toxins elicit the cytolysis of various eukaryotic cells, as well as erythrocytes. The human intestine secretes cationic antimicrobial peptides (AMPs) to prevent infectious diseases. Paneth cells in the small intestine secrete α-defensin 5 (HD-5) and epithelial cells in the large intestine produce LL-37. In the present study, we examined the bactericidal activities of AMPs against V. mimicus and V. vulnificus. Although HD-5 showed no bactericidal activity, LL-37 revealed significant activity against both Vibrio species, suggesting that neither V. mimicus nor V. vulnificus can multiply in the large intestine. We also tested whether AMPs had the ability to inactivate the hemolytic toxins. Only HD-5 was found to inactivate VMH, but not VVH, in a dose-dependent manner through the direct binding to VMH. Therefore, it is considered that V. mimicus cannot penetrate the small intestinal epithelium because the cytolytic action of VMH is inactivated by HD-5.

Inactivation of Vibrio vulnificus hemolysin through mutation of the N- or C-terminus of the lectin-like domain

Vibrio vulnificus is an etiological agent causing serious systemic infections in the immunocompromised humans or cultured eels. This species commonly produces a hemolytic toxin consisting of the cytolysin domain and the lectin-like domain. For hemolysis, the lectin-like domain specifically binds to cholesterol in the erythrocyte membrane, and to form a hollow oligomer, the toxin is subsequently assembled on the membrane. The cytolysin domain is essential for the process to form the oligomer. Three-dimensional structure model revealed that two domains connected linearly and the C-terminus was located near to the joint of the domains. Insertion of amino acid residues between two domains was found to cause inactivation of the toxin. In the C-terminus, deletion, substitution or addition of an amino acid residue also elicited reduction of the activity. However, the cholesterol-binding ability was not affected by the mutations. These results suggest that mutation of the C- or N-terminus of the lectin-like domain may result in blockage of the toxin assembly.

Antibacterial Activities of Surfactants in the Laundry Detergents and Isolation of the Surfactant Resistant Aquatic Bacteria

　Linear alkylbenzene sulfonate (LAS) and polyoxyethylene lauryl ether (POLE) are major surfactants contained in the laundry detergents. In the present study, the antibacterial activities of the surfactants to aquatic microorganisms were compared. When freshwater samples from a small river in Okayama city were treated with each of the surfactants, only LAS showed the significant antibacterial activity. Several strains, which survived after the treatment with 2.0% LAS, were isolated and identified by sequencing of 16S rDNA. All strains were classified into the family Enterobacteriaceae. However, this family was not a major member of the aquatic microflora, suggesting that the bacteria in Enterobacteriaceae have a common property of LAS-resistance in the river water.

A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin

Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N‐terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin‐like substrate specificity was purified from the bacterial culture supernatant. The N‐terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg151Ser152 bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.