Iñaki Comas

Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved

Mycobacterium tuberculosis is an obligate human pathogen capable of persisting in individual hosts for decades. We sequenced the genomes of 21 strains representative of the global diversity and six major lineages of the M. tuberculosis complex (MTBC) at 40- to 90-fold coverage using Illumina next-generation DNA sequencing. We constructed a genome-wide phylogeny based on these genome sequences. Comparative analyses of the sequences showed, as expected, that essential genes in MTBC were more evolutionarily conserved than nonessential genes. Notably, however, most of the 491 experimentally confirmed human T cell epitopes showed little sequence variation and had a lower ratio of nonsynonymous to synonymous changes than seen in essential and nonessential genes. We confirmed these findings in an additional data set consisting of 16 antigens in 99 MTBC strains. These findings are consistent with strong purifying selection acting on these epitopes, implying that MTBC might benefit from recognition by human T cells.

Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans

Tuberculosis caused 20% of all human deaths in the Western world between the seventeenth and nineteenth centuries and remains a cause of high mortality in developing countries. In analogy to other crowd diseases, the origin of human tuberculosis has been associated with the Neolithic Demographic Transition, but recent studies point to a much earlier origin. We analyzed the whole genomes of 259 M. tuberculosis complex (MTBC) strains and used this data set to characterize global diversity and to reconstruct the evolutionary history of this pathogen. Coalescent analyses indicate that MTBC emerged about 70,000 years ago, accompanied migrations of anatomically modern humans out of Africa and expanded as a consequence of increases in human population density during the Neolithic period. This long coevolutionary history is consistent with MTBC displaying characteristics indicative of adaptation to both low and high host densities.

Genotyping of Genetically Monomorphic Bacteria: DNA Sequencing in Mycobacterium tuberculosis Highlights the Limitations of Current Methodologies

Because genetically monomorphic bacterial pathogens harbour little DNA sequence diversity, most current genotyping techniques used to study the epidemiology of these organisms are based on mobile or repetitive genetic elements. Molecular markers commonly used in these bacteria include Clustered Regulatory Short Palindromic Repeats (CRISPR) and Variable Number Tandem Repeats (VNTR). These methods are also increasingly being applied to phylogenetic and population genetic studies. Using the Mycobacterium tuberculosis complex (MTBC) as a model, we evaluated the phylogenetic accuracy of CRISPR- and VNTR-based genotyping, which in MTBC are known as spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU)-VNTR-typing, respectively. We used as a gold standard the complete DNA sequences of 89 coding genes from a global strain collection. Our results showed that phylogenetic trees derived from these multilocus sequence data were highly congruent and statistically robust, irrespective of the phylogenetic methods used. By contrast, corresponding phylogenies inferred from spoligotyping or 15-loci-MIRU-VNTR were incongruent with respect to the sequence-based trees. Although 24-loci-MIRU-VNTR performed better, it was still unable to detect all strain lineages. The DNA sequence data showed virtually no homoplasy, but the opposite was true for spoligotyping and MIRU-VNTR, which was consistent with high rates of convergent evolution and the low statistical support obtained for phylogenetic groupings defined by these markers. Our results also revealed that the discriminatory power of the standard 24 MIRU-VNTR loci varied by strain lineage. Taken together, our findings suggest strain lineages in MTBC should be defined based on phylogenetically robust markers such as single nucleotide polymorphisms or large sequence polymorphisms, and that for epidemiological purposes, MIRU-VNTR loci should be used in a lineage-dependent manner. Our findings have implications for strain typing in other genetically monomorphic bacteria.

Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes

Epidemics of drug-resistant bacteria emerge worldwide, even as resistant strains frequently have reduced fitness compared to their drug-susceptible counterparts. Data from model systems suggest that the fitness cost of antimicrobial resistance can be reduced by compensatory mutations; however, there is limited evidence that compensatory evolution has any significant role in the success of drug-resistant bacteria in human populations. Here we describe a set of compensatory mutations in the RNA polymerase genes of rifampicin-resistant M. tuberculosis, the etiologic agent of human tuberculosis (TB). M. tuberculosis strains harboring these compensatory mutations showed a high competitive fitness in vitro. Moreover, these mutations were associated with high fitness in vivo, as determined by examining their relative clinical frequency across patient populations. Of note, in countries with the worlds highest incidence of multidrug-resistant (MDR) TB, more than 30% of MDR clinical isolates had this form of mutation. Our findings support a role for compensatory evolution in the global epidemics of MDR TB.

Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis

Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.

Human macrophage responses to clinical isolates from the Mycobacterium tuberculosis complex discriminate between ancient and modern lineages.

The aim of the present study was to determine whether there is a correlation between phylogenetic relationship and inflammatory response amongst a panel of clinical isolates representative of the global diversity of the human Mycobacterium tuberculosis Complex (MTBC). Measurement of cytokines from infected human peripheral blood monocyte-derived macrophages revealed a wide variation in the response to different strains. The same pattern of high or low response to individual strains was observed for different pro-inflammatory cytokines and chemokines, and was conserved across multiple human donors. Although each major phylogenetic lineage of MTBC included strains inducing a range of cytokine responses, we found that overall inflammatory phenotypes differed significantly across lineages. In particular, comparison of evolutionarily modern lineages demonstrated a significant skewing towards lower early inflammatory response. The differential response to ancient and modern lineages observed using GM-CSF derived macrophages was also observed in autologous monocyte-derived dendritic cells and murine bone marrow-derived macrophages, but not in human unfractionated peripheral blood mononuclear cells. We hypothesize that the reduced immune responses to modern lineages contribute to more rapid disease progression and transmission, which might be a selective advantage in the context of expanding human populations. In addition to the lineage effects, the large strain-to-strain variation in innate immune responses elicited by MTBC will need to be considered in tuberculosis vaccine development.

Sequence-based analysis uncovers an abundance of non-coding RNA in the total transcriptome of Mycobacterium tuberculosis.

RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5’ and 3’ untranslated regions, antisense transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses.

The past and future of tuberculosis research.

Renewed efforts in tuberculosis (TB) research have led to important new insights into the biology and epidemiology of this devastating disease. Yet, in the face of the modern epidemics of HIV/AIDS, diabetes, and multidrug resistance—all of which contribute to susceptibility to TB—global control of the disease will remain a formidable challenge for years to come. New high-throughput genomics technologies are already contributing to studies of TBs epidemiology, comparative genomics, evolution, and host–pathogen interaction. We argue here, however, that new multidisciplinary approaches—especially the integration of epidemiology with systems biology in what we call “systems epidemiology”—will be required to eliminate TB.

Genome-wide Mapping of Transcriptional Start Sites Defines an Extensive Leaderless Transcriptome in Mycobacterium tuberculosis

Summary Deciphering physiological changes that mediate transition of Mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an individual host for decades. We have combined RNA sequencing (RNA-seq) of 5′ triphosphate-enriched libraries with regular RNA-seq to characterize the architecture and expression of M. tuberculosis promoters. We identified over 4,000 transcriptional start sites (TSSs). Strikingly, for 26% of the genes with a primary TSS, the site of transcriptional initiation overlapped with the annotated start codon, generating leaderless transcripts lacking a 5′ UTR and, hence, the Shine-Dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria. Genes encoding proteins with active growth functions were markedly depleted from the leaderless transcriptome, and there was a significant increase in the overall representation of leaderless mRNAs in a starvation model of growth arrest. The high percentage of leaderless genes may have particular importance in the physiology of nonreplicating M. tuberculosis.

Evolution of Snake Venom Disintegrins by Positive Darwinian Selection

PII-disintegrins, cysteine-rich polypeptides broadly distributed in the venoms of geographically diverse species of vipers and rattlesnakes, antagonize the adhesive functions of beta(1) and beta(3) integrin receptors. PII-disintegrins evolved in Viperidae by neofunctionalization of disintegrin-like domains of duplicated PIII-snake venom hemorrhagic metalloproteinase (SVMP) genes recruited into the venom proteome before the radiation of the advanced snakes. Minimization of the gene (loss of introns and coding regions) and the protein structures (successive loss of disulfide bonds) underpins the postduplication divergence of disintegrins. However, little is known about the underlying genetic mechanisms that have generated the structural and functional diversity among disintegrins. Phylogenetic inference and maximum likelihood-based codon substitution approaches were used to analyze the evolution of the disintegrin family. The topology of the phylogenetic tree does not parallel that of the species tree. This incongruence is consistent with that expected for a multigene family undergoing a birth-and-death process in which the appearance and disappearance of loci are being driven by selection. Cysteine and buried residues appear to be under strong purifying selection due to their role in maintaining the active conformation of disintegrins. Divergence of disintegrins is strongly influenced by positive Darwinian selection causing accelerated rate of substitution in a substantial proportion of surface-exposed disintegrin residues. Global and lineage-specific sites evolving under diversifying selection were identified. Several sites are located within the integrin-binding loop and the C-terminal tail, two regions that form a conformational functional epitope. Arginine-glycine-aspartic acid (RGD) was inferred to represent the ancestral integrin-recognition motif, which emerged from the subgroup of PIII-SVMPs bearing the RDECD sequence. The most parsimonious nucleotide substitution model required for the emergence of all known disintegrins integrin inhibitory motifs from an ancestral RGD sequence involves a minimum of three mutations. The adaptive advantage of the emergence of motifs targeting beta(1) integrins and the role of positively selected sites located within nonfunctional disintegrin regions appear to be difficult to rationalize in the context of a predator-prey arms race. Perhaps, this represents a consequence of the neofunctionalization potential of the disintegrin domain, a feature that may underlie its recruitment into the venom proteome followed by its successful transformation into a toxin.