title: Molecular characterization of lipoamide dehydrogenase gene in Trypanosoma cruzi populations susceptible and resistant to benznidazole.
abstract: Lipoamide dehydrogenase (LipDH) is a flavin-containing disulfide oxidoreductase from the same group of thioredoxin reductase, glutathione reductase and trypanothione reductase. This enzyme is found in the mitochondria of all aerobic organisms where it takes part in at least three important multienzyme complexes from the citric acid cycle. In this study, we performed a phylogenetic analysis comparing the amino acid sequence of the LipDH from Trypanosoma cruzi (TcLipDH) with the LipDH from other organisms. Subsequently, the copy number of the TcLipDH gene, the mRNA and protein levels, and the enzymatic activity of the LipDH were determined in populations and strains of T. cruzi that were either resistant or susceptible to benznidazole (BZ). In silico analysis showed the presence of two TcLipDH alleles in the T. cruzi genome. It also showed that TcLipDH protein has less than 55% of identity in comparison to the human LipDH, but the active site is conserved in both of them. Southern blot results suggest that the TcLipDH is a single copy gene in the genome of the T. cruzi samples analyzed. Northern blot assays showed one transcript of 2.4 kb in all T. cruzi populations. Northern blot and Real Time RT-PCR data revealed that the TcLipDH mRNA levels were 2-fold more expressed in the BZ-resistant T. cruzi population (17LER) than in its susceptible pair (17WTS). Western blot results revealed that the TcLipDH protein level is 2-fold higher in 17LER sample in comparison to 17WTS sample. In addition, LipDH activity was higher in the 17LER population than in the 17WTS. Sequencing analysis revealed that the amino acid sequences of the TcLipDH from 17WTS and 17LER populations are identical. Our findings show that one of the mechanisms associated with in vitro-induced BZ resistance to T. cruzi correlates with upregulation of LipDH enzyme.
authors: Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil.
authors: Paula F
authors: PF
authors: Dos Santos
authors: Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil.
authors: Douglas S
authors: DS
authors: Moreira
authors: Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil.
authors: Elio H
authors: EH
authors: Baba
authors: Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil.
authors: Caroline M O
authors: CMO
authors: Volpe
authors: Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil.
authors: Jerônimo C
authors: JC
authors: Ruiz
authors: Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil.
authors: Alvaro J
authors: AJ
authors: Romanha
authors: Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil. Electronic address: silvane@cpqrr.fiocruz.br.
authors: Silvane M F
authors: SMF
authors: Murta
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D018628:Gene Dosage; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D009593:Nitroimidazoles / Q000494:pharmacology*; D010802:Phylogeny; D012333:RNA, Messenger / Q000378:metabolism; D016053:RNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics
chemical_list: D016054:DNA, Protozoan; D009593:Nitroimidazoles; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D014344:Trypanocidal Agents; D008058:Dihydrolipoamide Dehydrogenase; C009999:benzonidazole
keywords: Benznidazole; Drug resistance; Lipoamide dehydrogenase; Trypanosoma cruzi