1997 Fiscal Year Final Research Report Summary
Analysis of regulation by environmental factors of toxin production by Clostridium difficile
| Project/Area Number |
08457085
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Kanazawa University |
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Principal Investigator |
NAKAMURA Shinichi Kanazawa Univ., Fac.of Med., Prof., 医学部, 教授 (90019620)
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| Co-Investigator(Kenkyū-buntansha) |
MAEGAWA Tuneo Kanazawa Univ., Fac.of Med., Asst., 医学部, 助手 (50283114)
KARASAWA Tadahiro Kanazawa Univ., Fac.of Med., Asst.Prof., 医学部, 講師 (90251917)
YAMAKAWA Koyotaka Kanazawa Univ., Fac.of Med., Asst.Prof., 医学部, 講師 (20110629)
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| Project Period (FY) |
1996 – 1997
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| Keywords | C.difficile / pseudomembranous colitis / toxin / toxin production / biotin / amino acid / stress protein / gene cloning |
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| Research Abstract |
The regulation by environmental factors of toxin production by Clostridium difficile was analyzed with the defined media, using strain KZ 1647 in the presence of glucose and strain VPI 10467 in the absence of glucose. In the presence of glucose, with biotin-limited conditions under which the toxin production was remarkably increased, leucine and isoleucine were taken up preferentially, during the incubation period when toxin titers increased markedly but pacterial growth was deolining. The enhanced toxin production was inhibited by addition of aspargine, glutamic acid, glutamine and lysine. Bacterial browth was accerelated with the former three amino acids but suppressed with lysine. Consumption of leucine and isoleucine was progressive with the former three amino acids but not with lysine. These findings suggest that an inhibition mechanism of lysine is different from that of asparagine, glutamic acid and glutamine. SDS-PAGE analysis of the cytosolic proteins revealed that at least 130 kDa and 145 kDa proteins were overexpressed under biotin-limited conditions. The N-terminal sequences of the proteins were determined and the molecular cloning is under way to elucidate the role in toxin production. In the absence of glucose, supplementation with a high level (100 mM) of histidine, methionine, valine, isoleucine, proline and leucine, in particular isoleucine, markedly increased toxin production. Increasing the concentration of isoleucine from 20 to 100 mM remarkably increased toxin production, while bacterial growth decreased gradually. During the incubation period when toxin titers increased markedly but bacterial growth was declining, leucine and isoleucine were taken up preferentially as in the case of biotin-limited conditions with glucose. These findings suggest that leucine and isoleucine may play an important role in toxin production by C.difficile.
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