| Project/Area Number |
22590411
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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 |
Bacteriology (including Mycology)
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| Research Institution | Research Institute, International Medical Center of Japan |
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Principal Investigator |
KIRIKAE Teruo 独立行政法人国立国際医療研究センター, 研究所, 部長 (50192563)
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| Co-Investigator(Kenkyū-buntansha) |
FUNATOGAWA Keiji 栃木県保健環境センター, 微生物部, 部長 (70536896)
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| Project Period (FY) |
2010 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2012: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2011: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2010: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | PE_PGRS62 / Prdx1 / ペルオキシレドキシン / マクロファージ / 結核 / PE PGRS62 / ペルオキシレドキシ1 / Prx1 / 遺伝子破壊 / 病原因子 / マイトジェン活性 |
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| Research Abstract |
PE_PGRS62 is thought to be a virulence factor during tuberculosis. To determine whether PE_PGRS62 is a virulence factor of M. tuberculosis, we deleted PE_PGRS62 of M. tuberculosis Erdman (WT) using mycobacterial phage system. We designed the knockout phage of PE_PGRS62/Rv3812 refer to Erdman genomic DNA. A knockout strain, ΔPE_PGRS62 was constructed and was complimented with the integrative plasmid pMV306 into which PE_PGRS62 gene and its promoter are inserted (Δ62Comp). We also transformed an empty pMV306 as a control strain of Δ62 Comp to ΔPE_PGRS62 (Δ62/mock). To confirm these gene disruption and complementation, we extracted genomic DNA from WT, Δ62/mock and Δ62 Comp strains. The fragments treated with SmaI are detected with specific probe. Next, we examined expression of PE_PGRS62 of WT, Δ62/mock and Δ62Comp strains. These strains were cultured in 7H9/ADC medium and their RNAs were extracted. The transcription levels of PE_PGRS62 were determined by qRT-PCR with specific primers. We observed the recovery of PE_PGRS62 expression in Δ62Comp strain. We checked any difference among WT, Δ62/mock and Δ62Comp. Although we confirmed no differences among these strains grown in 7H9/ADC culture medium, Δ62/mock mutant strain showed reduced the survival rate of intracellular mycobacteria in J774 cells. To evaluate the virulence attenuation of ΔPE_PGRS62 in mice, we injected the strains of WT or ΔPE_PGRS62 into tail vein of mice. There was a significant delay in survival infected with the ΔPE_PGRS62 mutant strain relative to WT strain in BALB/c and SCID mice. These results demonstrate that PE_PGRS62 is required for M. tuberculosis virulence.
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