title: Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages.
abstract: Parasitic protozoa belonging to the order Kinetoplastida contain trypanothione as their major thiol. Trypanothione reductase (TR), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. To investigate further the physiological role of TR in Leishmania, we attempted to create TR-knockout mutants by gene disruption in L. donovani and L. major strains using the selectable markers neomycin and hygromycin phosphotransferases. TR is likely to be an important gene for parasite survival since all our attempts to obtain a TR null mutant in L. donovani failed. Instead, we obtained mutants with a partial trisomy for the TR locus where, despite the successful disruption of two TR alleles by gene targeting, a third TR copy was generated as a result of genomic rearrangements involving the translocation of a TR-containing region to a larger chromosome. Mutants of L. donovani and L. major possessing only one wild-type TR allele express less TR mRNA and have lower TR activity compared with wild-type cells carrying two copies of the TR gene. Significantly, these mutants show attenuated infectivity with a markedly decreased capacity to survive intracellularly within macrophages, provided that the latter are producing reactive oxygen intermediates.
authors.affiliation: Centre de Recherche en Infectiologie du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Quebec, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Dumas
authors.forename: M
authors.initials: M
authors.lastname: Ouellette
authors.forename: J
authors.initials: J
authors.lastname: Tovar
authors.forename: M L
authors.initials: ML
authors.lastname: Cunningham
authors.forename: A H
authors.initials: AH
authors.lastname: Fairlamb
authors.forename: S
authors.initials: S
authors.lastname: Tamar
authors.forename: M
authors.initials: M
authors.lastname: Olivier
authors.forename: B
authors.initials: B
authors.lastname: Papadopoulou
mesh_terms: D000818:Animals; D017125:Genes, Protozoan; D005818:Genetic Engineering; D005819:Genetic Markers; D006801:Humans; D019868:Kanamycin Kinase; D007891:Leishmania / Q000472:pathogenicity*; D007893:Leishmania donovani / Q000472:pathogenicity; D018320:Leishmania major / Q000472:pathogenicity; D008264:Macrophages / Q000378:metabolism / Q000469:parasitology*; D051379:Mice; D016296:Mutagenesis; D009247:NADH, NADPH Oxidoreductases / Q000502:physiology*; D018384:Oxidative Stress; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D012641:Selection, Genetic; D013481:Superoxides / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D019868:Kanamycin Kinase