| Project/Area Number |
11680599
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | National Institute of Infectious Diseases |
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Principal Investigator |
HOTTA Kunimoto National Institute of Infectious Diseases, Department of Bioactive Molecules, Laboratory Head, 生物活性物質部, 厚生技官 (90165564)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIKAWA Jun National Institute of Infectious Diseases, Department of Bioactive Molecules, Senior researcher, 厚生技官 (40202957)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Antibiotic resistance gene / AAC(3) / AAC(2') / AAC(6') / APH(2'') / Aminoglycoside antibiotics / diacetylation / Doble stage activity |
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| Research Abstract |
(1)Based on the discovery of that an aminoglycoside antibiotic(AG), arbekacin(ABK), approved as an anti-MRSA (Methicillin-resistant Staphylococcus aureus) retains antibiotic activity even after acetylated, we examined ABK and other AGs for their diacetylation as well as inactivation by the combination action of two aminoglycoside acetylatransferases(AACs). The following results were obtainable.
1)The combination of AAC(2') and AAC(3) resulted in the exclusive formation of 2'-N-acetylABK with antibiotic activity and no substantial formation of diacetylated compound. Therefore, S.lividans TK21 containing the cloned AAC(2') and AAC(3) genes was sensitive to ABK.
2)The combination of AAC(6') and AAC(2') or AAC(3) resulted in the formation of diacetylated ABK derivatives with no antibiotic activity as well as monoacetyalted derivatives retaining Antibiotic activity. Therefore, S.lividans TK21 containing both of the cloned AAC(6') gene and AAC(3) or AAC(2') gene showed weak ABK resistance leve
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l as similar as the strain containing only AAC(6') gene.
(2)Among 14 different AGs, some of their acetylated derivatives such as 6' -N-Acetylated neomycin(NM) also showed antibiotic activity, indicating double stage activity.
(3)The activation mechanism of aac(3) gene was demonstarated to be due to the activation of the promoter activity resulting from the one base substitution (C→T) at the -12 letter of the promoter sequence of the gene. The sequence analysis of AAC(6') gene revealed its similarity at the amino acid level with known AAC(6') genes.
(4)Genetic characterization by colony PCR revealed that 50% of MRSA strains contained the bifunctional enzyme gene, aac(6')/aph(2''), conferring multiple AG resistance. However, the presence of this gene was not always satisfactory for ABK resistance phenotype since this enzyme was not capable of phosphorylating at 2"-O-phosphorylation but acetylating 6'-N-acetylation which does not result in total ABK inactivation.
Based on these, it was conclusive that development of amioglycosides with double stage activity like ABK should be advantageous for prevention of the emergence of new AAC-dependent AG resistance. Less
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