Macro-array and bioinformatic analyses reveal mycobacterial ‘core’ genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex

Abstract

To better understand the biology and the virulence determinants of the two major mycobacterial human pathogensMycobacterium tuberculosisandMycobacterium leprae, their genome sequences have been determined recently.In silicocomparisons revealed that among the 1439 genes common to bothM. tuberculosisandM. leprae, 219 genes code for proteins that show no similarity with proteins from other organisms. Therefore, the latter ‘core’ genes could be specific for mycobacteria or even for the intracellular mycobacterial pathogens. To obtain more information as to whether these genes really were mycobacteria-specific, they were included in a focused macro-array, which also contained genes from previously defined regions of difference (RD) known to be absent fromMycobacterium bovisBCG relative toM. tuberculosis. Hybridization of DNA from 40 strains of theM. tuberculosiscomplex andin silicocomparison of these genes with the near-complete genome sequences fromMycobacterium avium,Mycobacterium marinumandMycobacterium smegmatiswere undertaken to answer this question. The results showed that among the 219 conserved genes, very few were not present in all the strains tested. Some of these missing genes code for proteins of the ESAT-6 family, a group of highly immunogenic small proteins whose presence and number is variable among the genomically highly conserved members of theM. tuberculosiscomplex. Indeed, the results suggest that, with few exceptions, the ‘core’ genes conserved amongM. tuberculosisH37Rv andM. lepraeare also highly conserved among other mycobacterial strains, which makes them interesting potential targets for developing new specific anti-mycobacterial drugs. In contrast, the genes from RD regions showed great variability among certain members of theM. tuberculosiscomplex, and some new specific deletions inMycobacterium canettii,Mycobacterium microtiand seal isolates were identified and further characterized during this study. Together with the distribution of a particular 6 or 7 bp micro-deletion in the gene encoding the polyketide synthasepks15/1, these results confirm and further extend the revised phylogenetic model for theM. tuberculosiscomplex recently presented.