The hemolysin(HS) produced by V. vulnificus was purified and its pathophysiological activities of were studied in an attempt to understand the pathomechanism of V. vulnificus septicemia. When about 1.0×10E10 CFU of V. vulnificus CDC C7184 strain
were
inoculated in 2.5%-NaCl heart infusion (HI) broth and shaking incubated by 220rpm at 35℃, HS could be detected in the culture supernatant in 3 hours and increased in titer until 8 hours. When 1 mM or 5 mM of iron specific chelator ethylene-N,
N'-diacetic acid(EDDA) was added to the media, which reproduces the relatively iron restricted in vivo environment of the host, multiplication of the bacteria was hampered slightly but production of HS per unit bacterial population was
significantly
increased. HS with 17, 143 HU of specific activity was purified by about 188 fold through ammonium sulfate precipitaion, Sephadex G-100 gel filtration, and DEAE-Sephadex A-25 ion exchange chromatography. Purified HS exhibited a major band with a
molecular weight of 51 KDa in SDS polyaciylamide gel electrophoresis. Partially purified HS(PPHS) from Sephadex G-100 gel filtration showed cytotoxicity to the Chinese hamster ovary cell line CHO-K1 and the bovine pulmonary artery endothelium
cell
line
CPAE in a time and dose dependent manner. CHO-K1 cells were more susceptible than CPAE cells to the cytotoxicity of HS, Effect of doxycycline(KC) and EDTA which were reported to be very effective in treating experimental V. vulnificus septicemia,
and of
normal human serum(NHS) on the activity of HS was evaluated. DC and NHS significantly inhibited the hemolysis of human erythrocytes by HS while EDTA had no effect. NHS also significantly protected CHO-K1 and CPAE cells from the cytotoxiciy of HS.
When
the PPHS was injected to ICR mice intravenously, LD50 was 3HU of PPHS, the LD100, killed mice within 12 hours. When indomethacin, antihistamine hydroxyzine and pheniramine, dexamethasone, DC, or calcium disodium EDTA(CaEDTA) was administered to
mice and
injected with LD100 of PPHS, only dexamethasone showed strong protective acitvity with 50% of the mice saved. Indomethacin, hydroxyzine, pheniramine showed 30%, 20%, and 20% of protection respectively. DC and EDTA showed no protective activity.
When
5LD50 was injected to the mice no drug was protective.
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