Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain

Abstract

Mycobacterium tuberculosis H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain M. tuberculosis H37Rv. Despite extensive study, the reason for the decreased virulence of M. tuberculosis H37Ra has not been determined. A genomic approach was therefore initiated to identify genetic differences between M. tuberculosis H37Rv and M. tuberculosis H37Ra as a means of pinpointing the attenuating mutation(s). Digestion with the rare-cutting restriction endonuclease DraI revealed two polymorphisms between the strains: a 480-kb fragment in M. tuberculosis H37Rv was replaced by two fragments of 220 and 260 kb in M. tuberculosis H37Ra, while there was a approximately 7.9-kb DraI fragment in M. tuberculosis H37Ra that had no counterpart in M. tuberculosis H37Rv. As the M. tuberculosis insertion sequence IS6110 contains a single DraI restriction site, it was considered possible that these polymorphisms were the result of IS6110 transposition events in M. tuberculosis H37Ra, events that may have inactivated virulence genes. The 7.9-kb polymorphism was found to be due to the presence of the previously described H37Rv RvD2 deletion in M. tuberculosis H37Ra, with sequence analysis suggesting an IS6110-mediated deletion mechanism for loss of RvD2. Three other IS6110-catalyzed deletions from the M. tuberculosis H37Rv chromosome (RvD3 to RvD5) were also identified, suggesting that this mechanism plays an important role in genome plasticity in the tubercle bacilli. Comparative mapping and sequencing revealed that the 480-kb polymorphism was due to an IS6110 insertion in M. tuberculosis H37Ra near oriC. Complementation of M. tuberculosis H37Ra with a 2.9-kb restriction fragment from M. tuberculosis H37Rv that encompassed the IS6110 insertion did not increase the survival of recombinant M. tuberculosis H37Ra in mice. In conclusion, this study describes the presence and mechanisms of genomic variation between M. tuberculosis H37Ra and M. tuberculosis H37Rv, although the role that they play in the attenuation of M. tuberculosis H37Ra is unclear.

FIG. 1

DraI restriction profiles of M. tuberculosis H37Rv and M. tuberculosis H37Ra. (Left panel) Agarose-embedded DNA was digested with DraI and resolved by PFGE as described in Materials and Methods. The 480-kb fragment in M. tuberculosis H37Rv is replaced in M. tuberculosis H37Ra by bands of 260 and 220 kb. (Right panel) Digestion of DNA in solution with DraI and migration through a 0.7% agarose gel, revealing the 7.9-kb fragment present in M. tuberculosis H37Ra.

FIG. 2

(a) PCR products generated with primers that flank the IS6110 insertion at bp 13626. The product from M. tuberculosis H37Ra is 1.3 kb larger than that from M. tuberculosis H37Rv, due to the presence of IS6110. MW, molecular size markers. (b) The IS6110 insertion site in M. tuberculosis H37Ra. The region from ca. kb 10 to 19 of the M. tuberculosis H37Rv genome is shown, with ORFs present in all six reading frames and short vertical bars representing stop codons. The position on the M. tuberculosis H37Rv genome is marked on the middle scale, with the IS6110 insertion at bp 13626 indicated. The 2.9-kb NaeI fragment that was used in complementation experiments is marked. Genes are designated by their Rv designation or by their gene name.

FIG. 3

The gel shown in the right panel of Fig. 1 was blotted and hybridized with a radiolabelled internal RvD2 PCR product (Table 1) generated from M. bovis BCG Pasteur genomic DNA. The probe bound only to the 7.9-kb M. tuberculosis H37Ra DraI fragment and not to M. tuberculosis H37Rv DNA, indicating that the 7.9-kb DraI fragment in M. tuberculosis H37Ra is due to the presence of RvD2. MW, molecular size markers.

FIG. 4

Structures of the RvD2 loci in M. tuberculosis H37Ra and M. tuberculosis H37Rv. ORFs present in all six reading frames are shown, with short vertical bars representing stop codons. The ORFs and inverted repeats (IRs) of IS6110 are shown in black, with the 3-bp flanking DR sequences shown in boldface. Recombination between the IS6110 elements would lead to the deletion of the intevening sequence, generating the RvD2-deleted locus shown in M. tuberculosis H37Rv.

FIG. 5

Growth of bacilli in the spleens (A) and lungs (B) of BALB/c mice. The CFU per gram of infected tissue for wild-type M. tuberculosis H37Ra (●), M. tuberculosis H37Ra carrying the shuttle vector pKINT (■), or M. tuberculosis H37Ra harboring the complementing pSG21 (▴) are shown. Error bars indicate standard deviations.