Comparative analyses of nonpathogenic, opportunistic, and totally pathogenic mycobacteria reveal genomic and biochemical variabilities and highlight the survival attributes of Mycobacterium tuberculosis

Abstract

Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size--their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets.

Importance: The complete sequence analysis of Mycobacterium indicus pranii, a novel species of Mycobacterium shown earlier to have strong immunomodulatory properties and currently in use for the treatment of leprosy, places it evolutionarily at the point of transition to pathogenicity. With the purpose of establishing the importance of M. indicus pranii in providing insight into the virulence mechanism of tuberculous and nontuberculous mycobacteria, we carried out comparative genomic and proteomic analyses of 44 mycobacterial species representing nonpathogenic (NP), opportunistic (OP), and totally pathogenic (TP) mycobacteria. Our results clearly placed M. indicus pranii as an ancestor of the M. avium complex. Analyses of comparative metabolic pathways between M. indicus pranii (NP), M. tuberculosis (TP), and M. intracellulare (OP) pointed to the presence of novel alternative pathways in M. tuberculosis with implications for pathogenesis and survival in the human host and identification of new drug targets.

FIG 1

Comparative plot for annotation of M. indicus pranii (MIP) based on annotations in COG and InterPro/Pfam.

FIG 2

The genome size of NP mycobacteria is larger than that of OP and TP mycobacteria. The green box plot represents the NP genome sizes, the orange box represents OP genomes, and TP genomes are represented by the pink box, which is very tight. The red line is the regression line, and the blue line is the lowess line. The small circles denote outliers. The TP genomes were on average smaller yet variable in size. The genome plasticity for TP mycobacteria possibly highlights their abilities to evolve and survive under various environmental conditions.

FIG 3

(A) Homologs of M. indicus pranii proteins in 12 other Mycobacterium species were determined to be functionally related using InterPro/Pfam domain knowledge. (B) The ROC points are in the upper left corner, suggesting the findings are statistically significant. MIP, M. indicus pranii.

FIG 4

Comparative genomics of selected mycobacterial genomes. The genomes of Mycobacterium indicus pranii (shown in green; an NP), Mycobacterium intracellulare ATCC 13950 (orange; an OP), and Mycobacterium tuberculosis H37Rv (pink; a TP) were selected for comparative genomic analyses. We used BLASTp, with a cutoff of 20% identity and e value of 10e–4, to determine the number of homologous protein-coding genes common between them (shown as edge labels between the nodes). The arrowhead represents the query genome, whereas the arrow tail represents the subject genome.

FIG 5

The enzymatic similarities between M. indicus pranii (MIP), M. intracellulare ATCC 13950 (MIA), M. avium 104 (MYCA1), and M. tuberculosis H37Rv (MYCTU) highlight interesting enzymatic plasticity properties. M. intracellulare ATCC 13950 (OP; orange) shares three enzymes (EC 1.8.7.1, sulfite reductase [ferredoxin]; EC 2.7.1.6, galactokinase [phosphorylating]; EC 5.4.2.8, phosphor mannose mutase) with M. avium 104 (OP; blue) and M. tuberculosis H37Rv (TP; red), which are absent in M. indicus pranii (NP; green). M. intracellulare ATCC 13950 and M. indicus pranii share 17 enzymes between them that are absent in M. tuberculosis H37Rv (a), while they share 12 enzymes between them that are absent in M. avium 104 (b).

FIG 6

A comparative metabolic pathway analysis between M. tuberculosis H37Rv, M. intracellulare ATCC 13950, and M. indicus pranii reveals the presence of novel pathways in Mycobacterium tuberculosis that are not present in M. indicus pranii or M. intracellulare ATCC 13950. The comparative analysis of metabolic enzymes present in M. tuberculosis H37Rv, M. intracellulare ATCC 13950, and M. indicus pranii based on KEGG pathways are shown. (a) The unique pathways in M. tuberculosis H37Rv are shown in red. The common pathways between M. intracellulare ATCC 13950 and M. indicus pranii are shown in pink. The common pathways present in these three organisms are shown in black. Note that M. tuberculosis H37Rv has acquired alternate pathways (red) for its survival. There are few common pathways between M. intracellulare ATCC 13950 and M. indicus pranii (pink) that are absent in M. tuberculosis H37Rv. (b) A section (circled) of the lipid biosynthesis subpathway (glycerolipid metabolism) highlights the presence of an alternate enzyme (EC 3.1.1.3, Rv3097c) that performs molecular transformations in M. tuberculosis H37Rv. (c) A section (circled) of the lipid biosynthesis subpathway (fatty acid metabolism) highlights an alternate enzyme (FabH gene) present in M. intracellulare ATCC 13950 and M. indicus pranii but absent in M. tuberculosis H37Rv.