期刊
JOURNAL OF BACTERIOLOGY
卷 182, 期 2, 页码 377-384出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JB.182.2.377-384.2000
关键词
-
类别
资金
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R21AI045537, R01AI045537] Funding Source: NIH RePORTER
- NIAID NIH HHS [R01 AI045537, AI45537, R21 AI045537] Funding Source: Medline
- Wellcome Trust Funding Source: Medline
Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library tvas constructed by using the shuttle vector pOLYG. This plasmid library mas electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937, Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned nl: tuberculosis H37Rv DNA, After six sequential passages of nil. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype, This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various ill. tuberculosis strains and of Mycobacterium bovis BCG but not in that of ill, smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis, The expression of this 42-kDa protein directly correlated to the enhanced survival of nl. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据