Sun-Hee Ahn

Characterization of Vibrio mimicus phospholipase A (PhlA) and cytotoxicity on fish cell.

Vibrio mimicus is a typical strain of Vibrio cholerae and produces a phospholipase (PhlA) which shares a highly conserved amino acid sequence with the lecithinase (Lec) of V. cholerae. The recombinant protein (rPhlA) produced from the phlA gene of V. mimicus was expressed in Escherichia coli as His-tag fused protein. The rPhlA was purified by gel filtration and Ni-metal affinity chromatographies. When the action mode was investigated by TLC and GC-MS, the purified rPhlA protein showed a phospholipase A activity, which cleaved the fatty acids at the sn-1 and sn-2 positions of phosphatidylcholine. However, it did not show lysophospholipase, sphingomyelinase, and phospholipase C activities. The rPhlA showed maximum activity at temperature of about 40 degrees C and pH around 8-9. Some divalent cations could affect the activity of PhlA. The addition of Co(2+) increased the activity, whereas Mg(2+) and Zn(2+) did not enhance the enzyme activity. The rPhlA could lyse the erythrocytes obtained from the fish such as rainbow trout and tilapia. A significant cytotoxic activity on a fish cell line, CHSE-214, was observed after 24h exposure to 40 microg rPhlA protein.

Identification of an Iron-Regulated Hemin-Binding Outer Membrane Protein, HupO, in Vibrio fluvialis: Effects on Hemolytic Activity and the Oxidative Stress Response

ABSTRACT In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.

A methoxylated fatty acid isolated from the brown seaweed Ishige okamurae inhibits bacterial phospholipase A2.

A methoxylated fatty acid that inhibits phospholipase A2 (PLA2; EC 3.1.1.4) was purified from the brown seaweed Ishige okamurae. Approximately 8.1 mg of the inhibitory compound, 7‐methoxy‐9‐methylhexadeca‐4,8‐dienoic acid, was isolated from 1 kg of I. okamurae powder. Recombinant PLA2 derived from the pathogenic bacterium Vibrio mimicus was used as the target enzyme. The methoxylated fatty acid compound competitively inhibited PLA2 with a Ki value of 3.9 µg/mL. The concentrations required for 50% inhibition of PLA2, oedema and erythema were 1.0 µg/mL, 3.6 mg/mL and 4.6 mg/mL, respectively. The compound strongly inhibited PLA2 activity in vitro and had potent antiinflammatory activity in vivo. Copyright

Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene

Vibrio anguillarum is a major pathogenic bacterium that causes vibriosis and septicemia in fish and shellfish. In this study, we identified the groESL genes, which encode bacterial chaperonins, from V. anguillarum. The groE gene cluster consisted of a 291-bp groES gene, a 69-bp intergenic spacer region, and a 1,635-bp groEL gene order. Sequence analysis with the groESL gene of Vibrio species exhibited that the groEL gene was more species-specific and suitable than the groES gene for V. anguillarum detection. Owing to the difficulty in distinguishing V. anguillarum from the closely related V. ordalii, we compared the sequences of groEL from V. anguillarum and the groEL homolog hsp60 from V. ordalii, in order to design a primer set based on a region dissimilar between the two. PCR with the groEL primer set produced a clear 195-bp amplicon in six serotypes of V. anguillarum, whereas 23 Vibrio species of 39 samples, including V. ordalii, and 10 species of enteric bacteria gave no bands. PCR using the groEL primers also amplified a unique product from V. anguillarum-infected flounder and oyster tissues. These results demonstrate that the groEL-target PCR assay is a sensitive and species-specific tool for the detection of V. anguillarum infection.

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

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.