title: Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage.
abstract: Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.|Laboratory of Molecular Microbiology and Biotechnology, Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Guerrini
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Selvakumar
authors.initials: S
authors.lastname: Subbian
authors.affiliation: Aix-Marseille Univ, Centre National de la Recherche Scientifique, Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, Marseille, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Santucci
authors.affiliation: Aix-Marseille Univ, Centre National de la Recherche Scientifique, Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, Marseille, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Canaan
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Maria Laura
authors.identifier: http://orcid.org/0000-0003-4801-3567
authors.initials: ML
authors.lastname: Gennaro
authors.affiliation: Laboratory of Molecular Microbiology and Biotechnology, Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors.forename: Gianni
authors.initials: G
authors.lastname: Pozzi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D019143:Evolution, Molecular; D005260:Female; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D059645:Mutation Rate; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D012641:Selection, Genetic; D012692:Serial Passage; D010738:Type C Phospholipases / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010738:Type C Phospholipases