title: In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1.
abstract: Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore , Republic of Singapore.
authors.forename: Pooja
authors.initials: P
authors.lastname: Gopal
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University , Baltimore, Maryland, United States.
authors.forename: Rokeya
authors.initials: R
authors.lastname: Tasneen
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore , Republic of Singapore.
authors.forename: Michelle
authors.initials: M
authors.lastname: Yee
authors.affiliation: Department of Infectious Diseases, University Hospital of Amiens-Picardie , Amiens, France.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Lanoix
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey , Newark, New Jersey, United States.
authors.forename: Jansy
authors.initials: J
authors.lastname: Sarathy
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey , Newark, New Jersey, United States.
authors.forename: George
authors.initials: G
authors.lastname: Rasic
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey , Newark, New Jersey, United States.
authors.forename: Liping
authors.initials: L
authors.lastname: Li
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey , Newark, New Jersey, United States.
authors.forename: Véronique
authors.initials: V
authors.lastname: Dartois
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins University , Baltimore, Maryland, United States.
authors.forename: Eric
authors.initials: E
authors.lastname: Nuermberger
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore , Republic of Singapore.
authors.forename: Thomas
authors.identifier: 0000-0002-9604-9452
authors.initials: T
authors.lastname: Dick
mesh_terms: D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D004195:Disease Models, Animal; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005260:Female; D015870:Gene Expression; D016680:Genome, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism; D011718:Pyrazinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D014376:Tuberculosis / Q000188:drug therapy / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; C534073:ClpC1 protein, Mycobacterium tuberculosis; D003470:Culture Media; D006360:Heat-Shock Proteins; D011718:Pyrazinamide; C005296:pyrazinoic acid; D048630:Polyketide Synthases; D002262:Carboxy-Lyases; C019747:aspartate 4-decarboxylase
keywords: in vivo; pyrazinamide; pyrazinoic acid; resistance; tuberculosis