Ancient Origin and Gene Mosaicism of the Progenitor of Mycobacterium tuberculosis

The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success

Génomique comparative chez les mycobacteries

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Bacterial Artificial Chromosome-Based Comparative Genomic Analysis Identifies Mycobacterium microti as a Natural ESAT-6 Deletion Mutant

Mycobacterium microti is a member of the Mycobacterium tuberculosis complex that causes tuberculosis in voles. Most strains of M. microti are harmless for humans, and some have been successfully used as live tuberculosis vaccines. In an attempt to identify putative virulence factors of the tubercle bacilli, genes that are absent from the avirulent M. microti but present in human pathogen M. tuberculosis or Mycobacterium bovis were searched for. A minimal set of 50 bacterial artificial chromosome (BAC) clones that covers almost all of the genome of M. microti OV254 was constructed, and individual BACs were compared to the corresponding BACs from M. bovis AF2122/97 and M. tuberculosis H37Rv. Comparison of pulsed-field gel-separated DNA digests of BAC clones led to the identification of 10 regions of difference (RD) between M. microti OV254 and M. tuberculosis. A 14-kb chromosomal region (RD1(mic)) that partly overlaps the RD1 deletion in the BCG vaccine strain was missing from the genomes of all nine tested M. microti strains. This region covers 13 genes, Rv3864 to Rv3876, in M. tuberculosis, including those encoding the potent ESAT-6 and CFP-10 antigens. In contrast, RD5(mic), a region that contains three phospholipase C genes (plcA to -C), was missing from only the vole isolates and was present in M. microti strains isolated from humans. Apart from RD1(mic) and RD5(mic) other M. microti-specific deleted regions have been identified (MiD1 to MiD3). Deletion of MiD1 has removed parts of the direct repeat region in M. microti and thus contributes to the characteristic spoligotype of M. microti strains

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

To better understand the biology and the virulence determinants of the two major mycobacterial human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, their genome sequences have been determined recently. In silico comparisons revealed that among the 1439 genes common to both M. tuberculosis and M. 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 from Mycobacterium bovis BCG relative to M. tuberculosis. Hybridization of DNA from 40 strains of the M. tuberculosis complex and in silico comparison of these genes with the near-complete genome sequences from Mycobacterium avium, Mycobacterium marinum and Mycobacterium smegmatis were 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 the M. tuberculosis complex. Indeed, the results suggest that, with few exceptions, the 'core' genes conserved among M. tuberculosis H37Rv and M. leprae are 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 the M. tuberculosis complex, and some new specific deletions in Mycobacterium canettii, Mycobacterium microti and 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 synthase pks15/1, these results confirm and further extend the revised phylogenetic model for the M. tuberculosis complex recently presented

Splits Graph of the 17 Concatenated Sequences of the Six Housekeeping Genes

<p>The nodes represent strains and are depicted as small red (smooth tubercle bacilli) or blue (MTBC members) squares. The scale bar represents Hamming distance. Numbers at the edges represent the percent bootstrap support of the splits obtained after 1,000 replicates. The fit was 61.7%. Note that the branching order of MTBC strains is weakly supported, and it should therefore not be seen as contradicting previous evolutionary hypotheses based on deletion patterns [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010005#ppat-0010005-b16" target="_blank">16</a>].</p

Nucleotide Polymorphism Detected in the Six Housekeeping Genes for the 17 Sequenced Strains

<div><p>(A) Location of the genes on the genome of <i>M. tuberculosis</i> H37Rv. Note that <i>gyrB</i> and <i>gyrA</i> are adjacent.</p><p>(B) Pattern of polymorphic sites revealing mosaicism of sequences. Colored blocks correspond to sequence stretches in the smooth strains that are similar or identical to the sequences in the MTBC. Boxes correspond to blocks of consecutive nucleotides in smooth strains that differ by at least three nucleotides from <i>M. tuberculosis</i> H37Rv. The last column indicates the allele number for each gene. Letters N and s indicate nonsynonymous and synonymous substitutions, respectively.</p></div

Phylogenetic Position of the Tubercle Bacilli within the Genus <i>Mycobacterium</i>

<p>The blue triangle corresponds to tubercle bacilli sequences that are identical or differing by a single nucleotide. The sequences of the genus <i>Mycobacterium</i> that matched most closely to those of <i>M. tuberculosis</i> were retrieved from the BIBI database (<a href="http://pbil.univ-lyon.fr/bibi/" target="_blank">http://pbil.univ-lyon.fr/bibi/</a>) and aligned with those obtained for 17 smooth and MTBC strains. The unrooted neighbor-joining tree is based on 1,325 aligned nucleotide positions of the 16S rRNA gene. The scale gives the pairwise distances after Jukes-Cantor correction. Bootstrap support values higher than 90% are indicated at the nodes.</p