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Mycobacterial PE_PGRS62 gene is a virulence factor during tuberculosis.

分枝杆菌PE_PGRS62基因是结核病的毒力因子。

基本信息

批准号：
22590411
负责人：
KIRIKAE Teruo
金额：
$ 2.83万
依托单位：
Research Institute, International Medical Center of Japan
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2012
项目状态：
已结题

项目摘要

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.

PE_PGRS62 被认为是结核病期间的毒力因子。为了确定PE_PGRS62是否是结核分枝杆菌的毒力因子，我们使用分枝杆菌噬菌体系统删除了结核分枝杆菌Erdman (WT)的PE_PGRS62。我们参考Erdman基因组DNA设计了PE_PGRS62/Rv3812的敲除噬菌体。构建了敲除菌株ΔPE_PGRS62，并与插入有PE_PGRS62基因及其启动子的整合质粒pMV306（Δ62Comp）互补。我们还将空 pMV306 作为 Δ62 Comp 的对照菌株转化为 ΔPE_PGRS62（Δ62/模拟）。为了确认这些基因破坏和互补，我们从 WT、Δ62/mock 和 Δ62 Comp 菌株中提取了基因组 DNA。用特异性探针检测经过SmaI处理的片段。接下来，我们检查了 WT、Δ62/mock 和 Δ62Comp 菌株的 PE_PGRS62 表达。这些菌株在7H9/ADC培养基中培养并提取它们的RNA。 PE_PGRS62 的转录水平通过特定引物的 qRT-PCR 测定。我们观察到 Δ62Comp 菌株中 PE_PGRS62 表达的恢复。我们检查了 WT、Δ62/mock 和 Δ62Comp 之间的差异。尽管我们确认在 7H9/ADC 培养基中生长的这些菌株之间没有差异，但 Δ62/模拟突变菌株显示 J774 细胞中胞内分枝杆菌的存活率降低。为了评估ΔPE_PGRS62对小鼠的毒力衰减情况，我们将WT或ΔPE_PGRS62菌株注射到小鼠尾静脉中。在 BALB/c 和 SCID 小鼠中，相对于 WT 菌株，感染 ΔPE_PGRS62 突变菌株的存活率显着延迟。这些结果表明 PE_PGRS62 是结核分枝杆菌毒力所必需的。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A New line probe assay kit for detection of drug-resistant Mycobacterium tuberculosis.

用于检测耐药结核分枝杆菌的新型探针检测试剂盒。

DOI：
发表时间：
2012
期刊：
影响因子：
0
作者：
菊地正;岩附研子;安達英輔;古賀道子;中村仁美;立川愛;鯉渕智彦;藤井毅;河岡義裕;岩本愛吉.;立川(川名)愛;Kirikae T
通讯作者：
Kirikae T

薬剤耐性結核菌迅速検出ラインプローブ法の開発

快速检测耐药结核杆菌的线性探针方法的建立

DOI：
发表时间：
2013
期刊：
影响因子：
0
作者：
Kikuchi T;Iwabu Y;Kawana-Tachikawa A;Koga M;Nomura S;Hosoya N;Brumme ZL;Heiko J;Kelleher AD;Markowitz M;Pereyra F;Trocha A;Walker BD;Iwamoto A;Tokunaga K;Miura T.;切替照雄
通讯作者：
切替照雄

Genetic diagnosis of multidrug resistant tuberculosis

耐多药结核病的基因诊断