Role of large sequence polymorphisms (LSPs) in generating genomic diversity among clinical isolates of Mycobacterium tuberculosis and the utility of LSPs in phylogenetic analysis

Abstract

Mycobacterium tuberculosis strains contain different genomic insertions or deletions called large sequence polymorphisms (LSPs). Distinguishing between LSPs that occur one time versus ones that occur repeatedly in a genomic region may provide insights into the biological roles of LSPs and identify useful phylogenetic markers. We analyzed 163 clinical M. tuberculosis isolates for 17 LSPs identified in a genomic comparison of M. tuberculosis strains H37Rv and CDC1551. LSPs were mapped onto a single-nucleotide polymorphism (SNP)-based phylogenetic tree created using nine novel SNP markers that were found to reproduce a 212-SNP-based phylogeny. Four LSPs (group A) mapped to a single SNP tree segment. Two LSPs (group B) and 11 LSPs (group C) were inferred to have arisen independently in the same genomic region either two or more than two times, respectively. None of the group A LSPs but one group B LSP and five group C LSPs were flanked by IS6110 sequences in the references strains. Genes encoding members of the proline-glutamic acid or proline-proline-glutamic acid protein families were present only in group B or C LSPs. SNP- versus LSP-based phylogenies were also compared. We classified each isolate into 58 LSP types by using a separate LSP-based phylogenetic analysis and mapped the LSP types onto the SNP tree. LSPs often assigned isolates to the correct phylogenetic lineage; however, significant mistakes occurred for 6/58 (10%) of the LSP types. In conclusion, most LSPs occur in genomic regions that are prone to repeated insertion/deletion events and were responsible for an unexpectedly high degree of genomic variation in clinical M. tuberculosis. Group B and C LSPs may represent polymorphisms that occur due to selective pressure and affect the phenotype of the organism, while group A LSPs are preferable phylogenetic markers.

FIG. 1.

Phylogeny of the M. tuberculosis study isolates. M. tuberculosis isolates were assigned to each SCG or SC subgroup based on SNP alleles at nine loci. The SCG and SC subgroup designations had been defined in a previous work (15). The number of study strains and the number of clinical isolates, as defined by identical RFLP patterns, are shown for each location on the tree. The locations of the three M. tuberculosis reference strains (H37Rv, CDC1551, and 210) and one M. bovis strain (M. bovis AF 2122/97) with sequenced genomes are also shown.

FIG. 2.

Distribution of group A and group B LSPs on the SNP tree. M. tuberculosis strains containing each designated LSP are indicated next to each tree branch. Numbers refer to the total number of strains with the indicated LSP/total number of isolates with the indicated LSP. Thick lines are used to indicate the phylogenetic location of a hypothetical common ancestor in which the LSP first occurred and its progeny. (A) All group A LSPs in the study. (B) All group B LSPs in the study. The locations of the SCG and SC subgroups of these trees as well as the total numbers of strains and isolates present in each SCG and SC subgroup can be found in Fig. 1.

FIG. 3.

Distribution of group C LSPs on the SNP tree. M. tuberculosis strains containing group C LSPs in this study are shown. Numbers refer to the total number of strains with the indicated LSP/total number of isolates with the indicated LSP. Thick lines are used to indicate the phylogenetic location of a hypothetical common ancestor in which the LSP first occurred and its progeny. The locations of the SCG and SC subgroups of these trees as well as the total numbers of strains and isolates present in each SCG and SC subgroup can be found in Fig. 1.

FIG. 4.

Locations of LSP-Ts on the SNP tree. The locations of clinical M. tuberculosis strains identified by LSP-T are shown relative to the location of each SCG and SC subgroup on the SNP tree. Colored LSP-Ts and connecting lines indicate LSP-Ts that are present on multiple SNP tree branches. Tree not drawn to scale.