Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

David Stucki, Daniela Brites, Leïla Jeljeli, Mireia Coscolla, Qingyun Liu, Andrej Trauner, Lukas Fenner, Liliana Rutaihwa, Sonia Borrell, Tao Luo, Qian Gao, Midori Kato-Maeda, Marie Ballif, Matthias Egger, Rita Macedo, Helmi Mardassi, Milagros Moreno, Griselda Tudo Vilanova, Janet Fyfe, Maria Globan, Jackson Thomas, Frances Jamieson, Jennifer L Guthrie, Adwoa Asante-Poku, Dorothy Yeboah-Manu, Eddie Wampande, Willy Ssengooba, Moses Joloba, W Henry Boom, Indira Basu, James Bower, Margarida Saraiva, Sidra E G Vaconcellos, Philip Suffys, Anastasia Koch, Robert Wilkinson, Linda Gail-Bekker, Bijaya Malla, Serej D Ley, Hans-Peter Beck, Bouke C de Jong, Kadri Toit, Elisabeth Sanchez-Padilla, Maryline Bonnet, Ana Gil-Brusola, Matthias Frank, Veronique N Penlap Beng, Kathleen Eisenach, Issam Alani, Perpetual Wangui Ndung'u, Gunturu Revathi, Florian Gehre, Suriya Akter, Francine Ntoumi, Lynsey Stewart-Isherwood, Nyanda E Ntinginya, Andrea Rachow, Michael Hoelscher, Daniela Maria Cirillo, Girts Skenders, Sven Hoffner, Daiva Bakonyte, Petras Stakenas, Roland Diel, Valeriu Crudu, Olga Moldovan, Sahal Al-Hajoj, Larissa Otero, Francesca Barletta, E Jane Carter, Lameck Diero, Philip Supply, Iñaki Comas, Stefan Niemann, Sebastien Gagneux, David Stucki, Daniela Brites, Leïla Jeljeli, Mireia Coscolla, Qingyun Liu, Andrej Trauner, Lukas Fenner, Liliana Rutaihwa, Sonia Borrell, Tao Luo, Qian Gao, Midori Kato-Maeda, Marie Ballif, Matthias Egger, Rita Macedo, Helmi Mardassi, Milagros Moreno, Griselda Tudo Vilanova, Janet Fyfe, Maria Globan, Jackson Thomas, Frances Jamieson, Jennifer L Guthrie, Adwoa Asante-Poku, Dorothy Yeboah-Manu, Eddie Wampande, Willy Ssengooba, Moses Joloba, W Henry Boom, Indira Basu, James Bower, Margarida Saraiva, Sidra E G Vaconcellos, Philip Suffys, Anastasia Koch, Robert Wilkinson, Linda Gail-Bekker, Bijaya Malla, Serej D Ley, Hans-Peter Beck, Bouke C de Jong, Kadri Toit, Elisabeth Sanchez-Padilla, Maryline Bonnet, Ana Gil-Brusola, Matthias Frank, Veronique N Penlap Beng, Kathleen Eisenach, Issam Alani, Perpetual Wangui Ndung'u, Gunturu Revathi, Florian Gehre, Suriya Akter, Francine Ntoumi, Lynsey Stewart-Isherwood, Nyanda E Ntinginya, Andrea Rachow, Michael Hoelscher, Daniela Maria Cirillo, Girts Skenders, Sven Hoffner, Daiva Bakonyte, Petras Stakenas, Roland Diel, Valeriu Crudu, Olga Moldovan, Sahal Al-Hajoj, Larissa Otero, Francesca Barletta, E Jane Carter, Lameck Diero, Philip Supply, Iñaki Comas, Stefan Niemann, Sebastien Gagneux

Abstract

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Conflict of interest statement

Statement The authors have no competing interests as defined by Springer Nature, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Figures

Figure 1. Definition and global frequency of…
Figure 1. Definition and global frequency of Lineage 4 sublineages.
(a) We defined 10 sublineages based on the analysis of 72 MTBC Lineage 4 genome sequences published previously,. Sublineages were labeled according to Coll et al. (whenever possible) and previous designations based on spoligotyping (see Supplementary Fig. 1). Black triangles indicate sublineages identified as specialists, black circles indicate generalists. Filled shapes indicate sublineages, for which we performed deep genomic analyses. (b) Global proportion of each sublineage. A total of 3,366 MTBC Lineage 4 isolates were screened for sublineage-specific SNPs. L4.3/LAM was the most frequent sublineage globally.
Figure 2. Global distribution of Lineage 4…
Figure 2. Global distribution of Lineage 4 sublineages.
Pie charts showing proportions of the 10 Lineage 4 sublineages among all MTBC Lineage 4 isolates in each country. Circle sizes correspond to the number of isolates analyzed per country. A total of 3,366 MTBC Lineage 4 isolates were included. Color codes are as in Fig. 1.
Figure 3. Country-specific proportions of sublineages reveal…
Figure 3. Country-specific proportions of sublineages reveal generalists and specialists.
(a) The generalist sublineages L4.1.2/Haarlem, L4.3/LAM and L4.10/PGG3 were found globally at high proportions. (b) The locally restricted specialist sublineages L4.1.3/Ghana, L4.5, L4.6.1/Uganda and L4.6.2/Cameroon occurred at high frequencies in only a few countries and were restricted to certain geographical regions. Intensity of red indicates proportion of the sublineage among all Lineage 4 isolates in each country. Countries with fewer than three isolates in total are shown as “no data” and are filled white. A total of 3,366 Lineage 4 isolates were included in this analysis. The color scale for all sublineages is as indicated in Panel a, except for sublineage L4.1.3/Ghana (separate scale shown).
Figure 4. Pair-wise ratios of rates of…
Figure 4. Pair-wise ratios of rates of nonsynonymous to synonymous substitutions (dN/dS) in generalist and specialist sublineages for different gene categories.
Abbreviations: Epi – experimentally confirmed human T cell epitopes; nEpi – non-epitope regions of T-cell antigens, both obtained from the Immune Epitope Database; Ess – essential genes; nEss – non-essential genes. Wilcoxon rank sum tests: L4.6.1/Uganda (N=203) Epi vs nEpi, W=4952, p<0.001; L4.6.1/Uganda (N=203) Ess vs nEss, W=1415, p<0.001; L4.3/LAM (N=293) Epi vs nEpi, W=74540, p<0.001, L4.3/LAM (n=293) Ess vs nEss W=45067, p-value=0.29; L4.1.2/Haarlem (N=228) Epi vs nEpi, W=6561, p<0.001, L4.1.2/Haarlem (N=228) Ess vs nEss W=13369, p<0.001; L4.10/PGG3 (N=301) Epi vs nEpi, W= 27335, p<0.001, L4.10/PGG3 (N=301) Ess vs nEss W= 3103, p<0.001.
Figure 5. Frequency distribution of the number…
Figure 5. Frequency distribution of the number of epitopes with nonsynonymous variants in generalist and specialist sublineages.
A total of 1,226 T cell epitopes were included in the analysis. The number above each bar corresponds to epitope counts. Generalist sublineages L4.3/LAM, L4.1.2/Haarlem and (L4.10/PGG3. Specialist sublineage L4.6.1/Uganda. Tests: L4.6.1/Uganda vs L4.3/LAM Χ2= 27.04, p<0.001; L4.6.1/Uganda vs L4.1.2/Haarlem Χ2=15.75, p<0.001; L4.6.1/Uganda vs L4.1.2/PGG3 Χ2= 68.24, p<0.001.
Figure 6. Genome-based phylogeny and diversity by…
Figure 6. Genome-based phylogeny and diversity by continent of 293 strains of the L4.3/LAM sublineage.
(a) Bayesian phylogeny with label colors indicating continent of strain origin: blue, Europe/Mediterranean; red, Sub-Saharan Africa; yellow, America; pink, Asia. Numbers on nodes indicate posterior probabilities. Pie charts indicate reconstructed ancestral geographical regions of the internal nodes. The hypothetical L4.3/LAM-ancestor is labeled and a European origin for this ancestor was supported using a Bayesian Method (shown) and a Maximum Parsimony method (Supplementary Fig. 14). The pie colors correspond to the colors of the taxa labels. (b) Boxplot of pairwise genetic distances (number of polymorphisms) of L4.3/LAM strains by continent (p-values from Wilcoxon rank sum test). (c) Nucleotide diversity per site (π), measured by continent. Error bars indicate 95% confidence intervals. MTBC isolates from countries of the continent group “Oceania“ (UN category; including Australia and New Zealand, Melanesia, Micronesia and Polynesia) were excluded for the genetic diversity analysis in panels B and C due the low number of samples.

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