Abstract
Little is known about the survival mechanics of Mycobacterium tuberculosis within the human host. A number of genes are likely to be activated during infection, especially within macrophages, and these potential virulence determinants will be of great importance in the ultimate development of new vaccines and antimycobacterials. In an effort to identify some of these virulence determinants, a M.tuberculosis H37Rv gene promoter-probe library in a BCG host was constructed using an E.coli - mycobacterial shuttle vector with lacZ as the reporter gene. 4800 individual clones were arrayed in a 96 well microtitre(TM) format, enabling the testing of individual clones for promoter activity under a variety of environmental stressing conditions. Two separate in vitro screenings of the arrayed clones identified 53 clones that exhibited up-regulation of lacZ when stressed. 50 of these clones were bi-directionally sequenced to determine the cloned M.tuberculosis H37Rv DNA sequence. Identification of the location of the cloned DNA within the M.tuberculosis H37Rv genome identified 32 clones likely to containing potential promoter sequences. By measuring the levels of lacZ expression post-infection of macrophages, compared to in vitro expression, clones were identified which contained potential promoter sequences up-regulated within the macrophage. Quantitative Reverse Transcriptase-PCR was employed, using cDNA obtained from M.tuberculosis from infected and un-infected macrophages, to measure the levels of expression of 5 M.tuberculosis H37Rv genes identified as potentially up- regulated during infection. The results suggest that two of the five genes, genes Rv1265 and Rv2711, are indeed up-regulated during infection. The protein coded for by the gene Rv1265 has an unknown function whilst the gene Rv2711 encodes the protein IdeR, which is an iron-dependent repressor. Sequence and lacZ expression analysis identified four independently selected and screened clones which contained near identical sequences for the same promoter - that of the gene Rv0440 (groEL2). Gene Rv0440 codes for a 60KDa heat shock protein previously identified as up-regulated during infection. The identification of four independent clones containing the promoter for this gene validates this technology as a means of identifying M.tuberculosis H37Rv genes up-regulated during infection.