1992 Fiscal Year Final Research Report Summary
MOLECULAR AND GENETICAL STUDIES ON HEMOLYTIC SUBSTANCE PRODUCED BY Pseudomonas cepacia
| Project/Area Number |
03670216
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
細菌学
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| Research Institution | YAMAGUCHI UNIVERSITY |
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Principal Investigator |
NAKAZAWA Teruko YAMAGUCHI UNIVERSITY,SCHOOL OF MEDICINE,PROFESSOR, 医学部, 教授 (40053053)
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| Co-Investigator(Kenkyū-buntansha) |
ABE Mitsuko YAMAGUCHI UNIVERSITY,SCHOOL OF MEDICINE,ASSISTANT, 医学部, 助手 (60107737)
TSUDA Masataka YAMAGUCHI UNIVERSITY,SCHOOL OF MEDICINE,ASSISTANT PROFESSOR, 医学部, 講師 (90172022)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Pseudomonas cepacia / small hemolytic substance / cepalycin / antifunfal activity / transposon mutagenesis / cepalycin-producing genes / DNA fingerprinting / nosocomial infection |
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| Research Abstract |
1)We established the method for purification of heat-stable hemolytic substance from P.cepacia JN106. Cell-washed supernatant was adsorbed to Amberlite XAD2,eluted with 70% ethanol containing 1mM HCI, and applied on reverse-phase HPLC. Two hemolytic substances,designated cepalycin I and cepalycin II, were isolated. 2)Cepalycins showed the same UV spectra,but the specific activity of cepalycin II was 4 times higher than that of cepalycin I. By LC-FAB Mass analysis,cepalycin II has the molecular weight of 240 with an OH group.Cepalycins were inactivated by alkaline,but resistant to acid.According to the solubility of various solvents,they appeared to be amphipathic molecules. 3)Cepalycins had antifungal activity comparable to amphotericin B. 4)Doughnut-like structures were observed by electron microscopy on a cepalycin-treated membrane of sheep erythrocytes,suggesting the pore-forming activity of the molecule. 5)Four types of non-hemolytic mutants of the strain JN106 were obtained by transposon mutagenesis. DNA fragments of JN106 were cloned that conferred the hemolytic activity to the mutants. 6)Distribution of cepalycin-producing genes were screened among the clinical and environmental isolates of P.cepacia by using the DNA fragment adjacent to the transposon of each mutant as a probe. One of the genes were found to be specific and exclusively for hemolytic strains. Thus ceplaycin is widely distributed among hemolytic strains of P.cepacia. 7)We have carried out DNA fingerprinting of P.cepacia to analyze a nosocomial outbreak of respiratory infection,and proved that the infection was associated with contaminated nebulizer devices.
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