. 2016 Dec;48(12):1535-1543.

doi: 10.1038/ng.3704. Epub 2016 Oct 31.

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

David Stucki^#^{1

2}, Daniela Brites^#^{1

2}, Leïla Jeljeli^#^{3

4}, Mireia Coscolla^{1

2}, Qingyun Liu⁵, Andrej Trauner^{1

2}, Lukas Fenner^{1

2

6}, Liliana Rutaihwa^{1

2}, Sonia Borrell^{1

2}, Tao Luo⁷, Qian Gao⁵, Midori Kato-Maeda⁸, Marie Ballif^{1

2

6}, Matthias Egger⁶, Rita Macedo⁹, Helmi Mardassi⁴, Milagros Moreno¹⁰, Griselda Tudo Vilanova¹¹, Janet Fyfe¹², Maria Globan¹², Jackson Thomas¹³, Frances Jamieson¹⁴, Jennifer L Guthrie¹⁴, Adwoa Asante-Poku¹⁵, Dorothy Yeboah-Manu¹⁵, Eddie Wampande¹⁶, Willy Ssengooba^{16

17}, Moses Joloba¹⁶, W Henry Boom¹⁸, Indira Basu¹⁹, James Bower¹⁹, Margarida Saraiva^{20

21}, Sidra E G Vaconcellos²², Philip Suffys²², Anastasia Koch²³, Robert Wilkinson^{23

24

25}, Linda Gail-Bekker²³, Bijaya Malla^{1

2}, Serej D Ley^{1

2

26}, Hans-Peter Beck^{1

2}, Bouke C de Jong²⁷, Kadri Toit²⁸, Elisabeth Sanchez-Padilla²⁹, Maryline Bonnet²⁹, Ana Gil-Brusola³⁰, Matthias Frank³¹, Veronique N Penlap Beng³², Kathleen Eisenach³³, Issam Alani³⁴, Perpetual Wangui Ndung'u³⁵, Gunturu Revathi³⁶, Florian Gehre^{27

37}, Suriya Akter²⁷, Francine Ntoumi^{31

38}, Lynsey Stewart-Isherwood³⁹, Nyanda E Ntinginya⁴⁰, Andrea Rachow⁴¹, Michael Hoelscher⁴¹, Daniela Maria Cirillo⁴², Girts Skenders⁴³, Sven Hoffner⁴⁴, Daiva Bakonyte⁴⁵, Petras Stakenas⁴⁵, Roland Diel⁴⁶, Valeriu Crudu⁴⁷, Olga Moldovan⁴⁸, Sahal Al-Hajoj⁴⁹, Larissa Otero⁵⁰, Francesca Barletta⁵⁰, E Jane Carter^{51

52}, Lameck Diero⁵², Philip Supply⁵³, Iñaki Comas^{54

55}, Stefan Niemann^{3

56}, Sebastien Gagneux^{1

2}

Affiliations

¹ Swiss Tropical and Public Health Institute, Basel, Switzerland.
² University of Basel, Switzerland.
³ Forschungszentrum Borstel, Germany.
⁴ Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia.
⁵ The Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institutes of Biomedical Sciences and Institute of Medical Microbiology, School of Basic Medical Science of Fudan University, Shanghai, China.
⁶ Institute for Social and Preventive Medicine, University of Bern, Switzerland.
⁷ Laboratory of Infection and Immunity, School of Basic Medical Science, West China Center of Medical Sciences, Sichuan University, Chengdu, Sichuan 610041, China.
⁸ School of Medicine, University of California, San Francisco, USA.
⁹ Laboratòrio de Saùde Publica, Lisbon, Portugal.
¹⁰ Hospital Nossa Senhora Da Paz, Cubal, Benguela, Angola.
¹¹ Servei de Microbiologia, Hospital Clínic-ISGlobal, Barcelona, Spain.
¹² Victorian Infectious Diseases Reference Laboratory, Victoria, Australia.
¹³ Ifakara Health Institute, Bagamoyo, Tanzania.
¹⁴ Public Health Ontario, Toronto, Canada.
¹⁵ Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana.
¹⁶ Department of Medical Microbiology, Makerere University, Kampala, Uganda.
¹⁷ Department of Global Health, University of Amsterdam, Amsterdam, the Netherlands.
¹⁸ Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, USA.
¹⁹ LabPlus, Auckland City Hospital, Auckland, New Zealand.
²⁰ Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.
²¹ ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
²² Oswaldo Cruz Institute, Brazil.
²³ Institute of Infectious Disease and Molecular Medicine and Department of Clinical Laboratory Sciences, University of Cape Town, South Africa.
²⁴ Department of Medicine, Imperial College London, UK.
²⁵ The Francis Crick Institute Mill Hill Laboratory, London, UK.
²⁶ Papua New Guinea Institute of Medical Research, Goroka, PNG.
²⁷ Insitute of Tropical Medicine, Antwerp, Belgium.
²⁸ Tartu University Hospital United Laboratories, Mycobacteriology, Tartu, Estonia.
²⁹ Clinical Research Department, Epicentre, Paris, France.
³⁰ Department of Microbiology, University Hospital La Fe, Valencia, Spain.
³¹ Institute of Tropical Medicine, University of Tübingen, Tübingen, Germany.
³² Institute Laboratory for Tuberculosis Research (LTR), Biotechnology Center (BTC), University of Yaoundé I, Yaoundé, Cameroon.
³³ Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
³⁴ Department of Medical Laboratory Technology, Faculty of Medical Technology, Baghdad, Iraq.
³⁵ Institute of Tropical Medicine and Infectious Diseases (ITROMID), Jomo Kenyatta University of Agriculture and Technology (JKUAT), Nairobi, Kenya.
³⁶ Department of Pathology, Aga Khan University Hospital (AKUH), Nairobi, Kenya.
³⁷ Medical Research Council, Fajara, the Gambia.
³⁸ Fondation Congolaise pour la Recherche Médicale, Université Marien Gouabi, Brazzaville, Congo.
³⁹ Right to Care and the Clinical HIV Research Unit, University of the Witwatersrand, Johannesburg, South Africa.
⁴⁰ National Institute of Medical Research, Mbeya Medical Research Centre (NIMR-MMRC), Mbeya, Tanzania.
⁴¹ Division of Infectious Diseases and Tropical Medicine, Medical Centre of the University of Munich, Munich, Germany; German Centre for Infection Research (DZIF), partner site Munich, Germany.
⁴² Emerging Bacterial Pathogens Unit, IRCCS, San Raffaele Scientific Institute, Milan, Italy.
⁴³ Riga East University Hospital, Centre of Tuberculosis and Lung Diseases, Riga, Latvia.
⁴⁴ WHO Supranational TB Reference Laboratory, Department of Microbiology, The Public Health Agency of Sweden, Solna, Sweden.
⁴⁵ Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
⁴⁶ Institute for Epidemiology, Schleswig-Holstein University Hospital, Kiel, Germany.
⁴⁷ National Tuberculosis Reference Laboratory, Phthysiopneumology Institute, Chisinau, Republic of Moldova.
⁴⁸ 'Marius Nasta' Pneumophtisiology Institute, Bucharest, Romania.
⁴⁹ Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.
⁵⁰ Instituto de Medicina Tropical Alexander von Humboldt, Molecular Epidemiology Unit-Tuberculosis, Universidad Peruana Cayetano Heredia, Lima, Peru.
⁵¹ Alpert School of Medicine at Brown University, Providence, Rhode Island, USA.
⁵² Moi University School of Medicine, Eldoret, Kenya.
⁵³ Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France.
⁵⁴ Institute of Biomedicine of Valencia (IBV-CSIC), 46010, Valencia, Spain.
⁵⁵ CIBER Epidemiology and Public Health, Madrid, Spain.
⁵⁶ German Center for Infection Research, Borstel Site, Borstel, Germany.

^# Contributed equally.

PMID: 27798628
PMCID: PMC5238942
DOI: 10.1038/ng.3704

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

David Stucki et al. Nat Genet. 2016 Dec.

. 2016 Dec;48(12):1535-1543.

doi: 10.1038/ng.3704. Epub 2016 Oct 31.

Authors

Affiliations

¹ Swiss Tropical and Public Health Institute, Basel, Switzerland.
² University of Basel, Switzerland.
³ Forschungszentrum Borstel, Germany.
⁴ Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia.
⁵ The Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institutes of Biomedical Sciences and Institute of Medical Microbiology, School of Basic Medical Science of Fudan University, Shanghai, China.
⁶ Institute for Social and Preventive Medicine, University of Bern, Switzerland.
⁷ Laboratory of Infection and Immunity, School of Basic Medical Science, West China Center of Medical Sciences, Sichuan University, Chengdu, Sichuan 610041, China.
⁸ School of Medicine, University of California, San Francisco, USA.
⁹ Laboratòrio de Saùde Publica, Lisbon, Portugal.
¹⁰ Hospital Nossa Senhora Da Paz, Cubal, Benguela, Angola.
¹¹ Servei de Microbiologia, Hospital Clínic-ISGlobal, Barcelona, Spain.
¹² Victorian Infectious Diseases Reference Laboratory, Victoria, Australia.
¹³ Ifakara Health Institute, Bagamoyo, Tanzania.
¹⁴ Public Health Ontario, Toronto, Canada.
¹⁵ Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana.
¹⁶ Department of Medical Microbiology, Makerere University, Kampala, Uganda.
¹⁷ Department of Global Health, University of Amsterdam, Amsterdam, the Netherlands.
¹⁸ Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, USA.
¹⁹ LabPlus, Auckland City Hospital, Auckland, New Zealand.
²⁰ Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.
²¹ ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
²² Oswaldo Cruz Institute, Brazil.
²³ Institute of Infectious Disease and Molecular Medicine and Department of Clinical Laboratory Sciences, University of Cape Town, South Africa.
²⁴ Department of Medicine, Imperial College London, UK.
²⁵ The Francis Crick Institute Mill Hill Laboratory, London, UK.
²⁶ Papua New Guinea Institute of Medical Research, Goroka, PNG.
²⁷ Insitute of Tropical Medicine, Antwerp, Belgium.
²⁸ Tartu University Hospital United Laboratories, Mycobacteriology, Tartu, Estonia.
²⁹ Clinical Research Department, Epicentre, Paris, France.
³⁰ Department of Microbiology, University Hospital La Fe, Valencia, Spain.
³¹ Institute of Tropical Medicine, University of Tübingen, Tübingen, Germany.
³² Institute Laboratory for Tuberculosis Research (LTR), Biotechnology Center (BTC), University of Yaoundé I, Yaoundé, Cameroon.
³³ Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
³⁴ Department of Medical Laboratory Technology, Faculty of Medical Technology, Baghdad, Iraq.
³⁵ Institute of Tropical Medicine and Infectious Diseases (ITROMID), Jomo Kenyatta University of Agriculture and Technology (JKUAT), Nairobi, Kenya.
³⁶ Department of Pathology, Aga Khan University Hospital (AKUH), Nairobi, Kenya.
³⁷ Medical Research Council, Fajara, the Gambia.
³⁸ Fondation Congolaise pour la Recherche Médicale, Université Marien Gouabi, Brazzaville, Congo.
³⁹ Right to Care and the Clinical HIV Research Unit, University of the Witwatersrand, Johannesburg, South Africa.
⁴⁰ National Institute of Medical Research, Mbeya Medical Research Centre (NIMR-MMRC), Mbeya, Tanzania.
⁴¹ Division of Infectious Diseases and Tropical Medicine, Medical Centre of the University of Munich, Munich, Germany; German Centre for Infection Research (DZIF), partner site Munich, Germany.
⁴² Emerging Bacterial Pathogens Unit, IRCCS, San Raffaele Scientific Institute, Milan, Italy.
⁴³ Riga East University Hospital, Centre of Tuberculosis and Lung Diseases, Riga, Latvia.
⁴⁴ WHO Supranational TB Reference Laboratory, Department of Microbiology, The Public Health Agency of Sweden, Solna, Sweden.
⁴⁵ Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
⁴⁶ Institute for Epidemiology, Schleswig-Holstein University Hospital, Kiel, Germany.
⁴⁷ National Tuberculosis Reference Laboratory, Phthysiopneumology Institute, Chisinau, Republic of Moldova.
⁴⁸ 'Marius Nasta' Pneumophtisiology Institute, Bucharest, Romania.
⁴⁹ Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.
⁵⁰ Instituto de Medicina Tropical Alexander von Humboldt, Molecular Epidemiology Unit-Tuberculosis, Universidad Peruana Cayetano Heredia, Lima, Peru.
⁵¹ Alpert School of Medicine at Brown University, Providence, Rhode Island, USA.
⁵² Moi University School of Medicine, Eldoret, Kenya.
⁵³ Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France.
⁵⁴ Institute of Biomedicine of Valencia (IBV-CSIC), 46010, Valencia, Spain.
⁵⁵ CIBER Epidemiology and Public Health, Madrid, Spain.
⁵⁶ German Center for Infection Research, Borstel Site, Borstel, Germany.

^# Contributed equally.

PMID: 27798628
PMCID: PMC5238942
DOI: 10.1038/ng.3704

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.

PubMed Disclaimer

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 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 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 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 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 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 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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Comment in

Human host range of Mycobacterium tuberculosis.
Hershberg R. Hershberg R. Nat Genet. 2016 Nov 29;48(12):1453-1454. doi: 10.1038/ng.3724. Nat Genet. 2016. PMID: 27898082

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Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Affiliations

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Authors

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Abstract

Conflict of interest statement

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