Hanna Nebenzahl-Guimaraes1,2,3, Arjan van Laarhoven4, Maha R Farhat5, Valerie A C M Koeken4, Jornt J Mandemakers6, Aldert Zomer7,8, Sacha A F T van Hijum7, Mihai G Netea4, Megan Murray9,10, Reinout van Crevel4, Dick van Soolingen1,11. 1. 1 National Institute for Public Health and the Environment, Bilthoven, the Netherlands. 2. 2 Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal. 3. 3 ICVS/3B's Research Group, PT Government Associate Laboratory, Braga/Guimarães, Portugal. 4. 4 Department of Internal Medicine and Radboud Center for Infectious Diseases. 5. 5 Pulmonary and Critical Care Division, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. 6. 6 University of Wageningen, Wageningen, the Netherlands. 7. 7 Center for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, and. 8. 8 Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands. 9. 9 Department of Global Health and Social Medicine, Harvard Medical School, Boston, Massachusetts; and. 10. 10 Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts. 11. 11 Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, the Netherlands.
Abstract
RATIONALE: Successful transmission of tuberculosis depends on the interplay of human behavior, host immune responses, and Mycobacterium tuberculosis virulence factors. Previous studies have been focused on identifying host risk factors associated with increased transmission, but the contribution of specific genetic variations in mycobacterial strains themselves are still unknown. OBJECTIVES: To identify mycobacterial genetic markers associated with increased transmissibility and to examine whether these markers lead to altered in vitro immune responses. METHODS: Using a comprehensive tuberculosis registry (n = 10,389) and strain collection in the Netherlands, we identified a set of 100 M. tuberculosis strains either least or most likely to be transmitted after controlling for host factors. We subjected these strains to whole-genome sequencing and evolutionary convergence analysis, and we repeated this analysis in an independent validation cohort. We then performed immunological experiments to measure in vitro cytokine production and neutrophil responses to a subset of the original strains with or without the identified mutations associated with increased transmissibility. MEASUREMENTS AND MAIN RESULTS: We identified the loci espE, PE-PGRS56, Rv0197, Rv2813-2814c, and Rv2815-2816c as targets of convergent evolution among transmissible strains. We validated four of these regions in an independent set of strains, and we demonstrated that mutations in these targets affected in vitro monocyte and T-cell cytokine production, neutrophil reactive oxygen species release, and apoptosis. CONCLUSIONS: In this study, we identified genetic markers in convergent evolution of M. tuberculosis toward enhanced transmissibility in vivo that are associated with altered immune responses in vitro.
RATIONALE: Successful transmission of tuberculosis depends on the interplay of human behavior, host immune responses, and Mycobacterium tuberculosis virulence factors. Previous studies have been focused on identifying host risk factors associated with increased transmission, but the contribution of specific genetic variations in mycobacterial strains themselves are still unknown. OBJECTIVES: To identify mycobacterial genetic markers associated with increased transmissibility and to examine whether these markers lead to altered in vitro immune responses. METHODS: Using a comprehensive tuberculosis registry (n = 10,389) and strain collection in the Netherlands, we identified a set of 100 M. tuberculosis strains either least or most likely to be transmitted after controlling for host factors. We subjected these strains to whole-genome sequencing and evolutionary convergence analysis, and we repeated this analysis in an independent validation cohort. We then performed immunological experiments to measure in vitro cytokine production and neutrophil responses to a subset of the original strains with or without the identified mutations associated with increased transmissibility. MEASUREMENTS AND MAIN RESULTS: We identified the loci espE, PE-PGRS56, Rv0197, Rv2813-2814c, and Rv2815-2816c as targets of convergent evolution among transmissible strains. We validated four of these regions in an independent set of strains, and we demonstrated that mutations in these targets affected in vitro monocyte and T-cell cytokine production, neutrophil reactive oxygen species release, and apoptosis. CONCLUSIONS: In this study, we identified genetic markers in convergent evolution of M. tuberculosis toward enhanced transmissibility in vivo that are associated with altered immune responses in vitro.
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