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Evolution of the M. tuberculosis Complex

As mentioned above, the M. tuberculosis complex represents a genetically homogeneous group of bacteria that cause tuberculosis in various mammalian species. The members of the complex share greater than 99.9% identity at the DNA level. However, some particular phenotypic characteristics, including different host pre ferences, have led researchers to retain the traditional species names of these bacteria. In a recent study, based on the presence or absence of RD regions, the evolutionary pathway of the M. tuberculosis complex has been redefined (Brosch et al. 2002). For example, region RD9 was found to be present in all M. tuberculosis and M. canettii strains, whereas this 2-kbp genomic segment was absent from M. afri-canum, M. microti, M. bovis, and M. bovis BCG. The segment is predicted to encode two complete genes, Rv2073c and Rv2074, as well as the 5' end of cobL. The interruption of cobL indicates that the RD9 polymorphism is due to the deletion of a 2-kbp fragment from the common ancestor of M. africanum, M. microti, M. bovis, and M. bovis BCG rather than the insertion of these genes into M. tuberculosis. This was confirmed by sequence analysis of the interruption sites of cobL in these species, which showed that the junction sequences of the RD9 region were identical. These findings were of particular importance for the interpretation of the evolution for the members of the M. tuberculosis complex, as they allowed the definition of a direction to the evolutionary processes that have shaped the various lineages (Brosch et al. 2001).

As the deletions have interrupted genes that are still intact in M. tuberculosis and M. canettii, it was possible to propose a new evolutionary scheme for the members of the M. tuberculosis complex. This scheme, which was based on the presence or absence of 20 variable regions in a representative set of 100 strains from the M. tuberculosis complex (Brosch et al. 2002), was confirmed in a separate study that also employed RD markers on a different set of strains (Mostowy et al. 2002), and has been complemented by data on selected single nucleotide polymorphism and microdeletions (Marmiesse et al. 2004). Moreover, a study employing multi-locus sequence typing found similar phylogenetic relationships among strains of the M. tuberculosis complex to that proposed by deletion-based analysis (Gutacker et al. 2002). Altogether these studies suggest that, contrary to previous ideas (Stead et al. 1995), the agent of bovine tuberculosis, M. bovis, is not the ancestor of the human tuberculosis agent M. tuberculosis. From these genomic studies it appears that the common ancestor of the M. tuberculosis complex resembled M. tuberculosis or M. canettii more than M. bovis. Final confirmation that the genome of M. bovis has indeed undergone several deletion processes relative to M. tuberculosis came from the complete genome sequence of M. bovis (Garnier et al. 2003), showing that the genome of M. bovis AF2122/97 is 66kbp smaller than the genome of M. tuberculosis H37Rv and with one exception (M. tuberculosis specific deleted region 1 - TbD1) does not contain any "extra" DNA to that present in M. tuberculosis strains. This genetic approach for differentiation can now be used to replace the often confusing traditional division of the members of the M. tuber-

Fig. 10.1 Use ofthe evolutionary scheme (after Brosch et al. 2002) for the rapid differentiation of the members of the M. tuberculosis complex. (A) Identification of "modern" M. tuberculosis strains, which represent the large majority of clinical isolates;

(B) identification of members of the M. africanum—M. bovis lineage; (C) identification of classical M. bovis strains, which represent the large majority of cattle isolates; (D) identification of attenuated strains.

218 10 Pathogenomics: Insights into Tuberculosis and Related Mycobacterial Diseases (A) Rap id identification of "modern" M. tuberculosis

(C) Identification of M. bovis, naturally resistant to pyrazinamide

culosis complex into rigidly defined subspecies and for rapid identification of clinical specimens (Fig. 10.1).

A molecular study on a collection of tuberculosis clinical isolates from East Africa that showed a smooth, M. canettii-like colony morphology (van Soolingen et al. 1997) revealed that this geographically restricted population had much greater genetic variability than did other extant M. tuberculosis complex strains, suggesting that they represent ancestral lineages of the complex. These findings are consistent with an early emergence of tubercle bacilli in East Africa, perhaps contemporaneously with early hominids three to six million years ago. The present global M. tuberculosis population may therefore represent the clonal expansion of a small subset of smooth tubercle bacilli along the waves of human migration out of Africa (Gutierrez et al. 2005).

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