A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region

Abstract

The human- and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), seemingly restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region - involved in the cobalamin/vitamin B12 synthesis - and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle.

Fig. 1. Image of colonies from the Rwandan Lineage 8 strain.

Isolate grown on solid medium, with the typical rough morphotype of Mycobacterium tuberculosis. Colonies grown on Dubos agar medium were visualized by microscopy. The scale bar represents ~2 mm.

Fig. 2. Deeplex-MycTB results identifying a MDR-TB strain from Rwanda with an atypical genotypic profile.

Deeplex-MycTB results identifying a MDR-TB strain from Rwanda with an atypical genotypic profile. Target gene regions are grouped within sectors in a circular map according to the tuberculous drug resistance with which they are associated. The two sectors in red indicate regions where rifampicin and isoniazid resistance-associated mutations are detected. The multiple sectors in blue refer to regions, where as yet uncharacterised mutations are detected, whereas sectors in green indicate regions where no mutation or only mutations not associated with resistance (shown in grey around the map) were detected. Green lines above gene names represent the reference sequences with coverage breadth above 95%. Limits of detection (LOD) of potential heteroresistance (reflected by subpopulations of reads bearing a mutation), depending on the coverage depths over target sequence positions, are represented by grey (LOD 3%) and orange zones (variable LOD > 3–80%; only seen in extremities of a few targets, such as the two rrs regions) above the reference sequences within the sectors. Information on an unrecognised spoligotype, an equivocal SNP-based phylogenetic lineage and on mycobacterial species identification, based on hsp65 sequence best match, are shown in the centre of the circle. AMI amikacin, BDQ bedaquiline, CAP capreomycin, CFZ clofazimine, EMB ethambutol, ETH ethionamide, FQ fluoroquinolones, KAN kanamycin, LIN linezolid, INH isoniazid, PZA pyrazinamide, RIF rifampin, SM streptomycin, SIT spoligotype international type.

Fig. 3. Maximum likelihood phylogeny of 241 MTBC genomes, inferred from 43,442 variable positions.

Maximum likelihood phylogeny of 241 MTBC genomes, inferred from 43,442 variable positions. The scale bar indicates the number of substitutions per polymorphic site. Branches corresponding to human-adapted strains are coloured and branches corresponding to animal-adapted strains are depicted in black. The phylogeny is rooted on M. canettii and bootstrap values are shown for the most important splits.

Fig. 4. Differential presence of the cobF region in specific Mycobacterium spp. genomes.

Aligned genome segments showing the cobF gene region in M. tuberculosis L8, M. canettii CIPT140010059 (alias STB-A), M. kansasii ATCC12478 and M. marinum M strains, and the corresponding deletion in M. tuberculosis H37Rv, M. bovis AF2122/97 and M. africanum GM041182. Coding sequences of this region are shown in green, and flanking coding sequences in red. Sequences flanking the deletion point in truncated genes in M. tuberculosis, M. africanum and M. bovis, and in the cobF region present in L8, M. canettii, M. kansasii and M. marinum are indicated in red and black, respectively. Dashed lines correspond to missing segment parts relatively to the longest segment found in M. marinum.

Fig. 5. Architecture of the pks8/17 region of specific Mycobacterium spp. genomes.

Aligned genome segments showing the interrupted coding sequences pks8/17 in M. tuberculosis H37Rv and M. bovis AF2122/97, and complete pks8 genes in L8, M. canettii CIPT140010059 (alias STB-A) and M. kansasii ATCC12478. The coding sequences of pks8/17 and pks8 are shown in green, and flanking genes in red. Sequences flanking the 1-nucleotide deletion and resulting in a frameshift in M. tuberculosis complex strains are indicated.