Identification of aMycobacterium tuberculosisGene That Enhances Mycobacterial Survival in Macrophages

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Abstract
Intracellular survival plays a central role in the pathogenesis ofMycobacterium tuberculosis. To identifyM. tuberculosisgenes required for intracellular survival within macrophages, anM. tuberculosisH37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated intoMycobacterium smegmatis1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. BecauseM. smegmatisdoes not readily survive within macrophages, any increased intracellular survival is likely due to clonedM. tuberculosisH37Rv DNA. After six sequential passages ofM. smegmatistransformants 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 ofeisas a probe for Southern analysis,eiswas found in the genomic DNA of variousM. tuberculosisstrains and ofMycobacterium bovisBCG but not in that ofM. smegmatisor 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that allM. 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 ofM. smegmatisp69 in U-937 cells. These results suggest a possible role foreisand its protein product in the intracellular survival ofM. tuberculosis.