Molecular characterization of lipoamide dehydrogenase gene in Trypanosoma cruzi populations susceptible and resistant to benznidazole
| Autor: | Jeronimo C. Ruiz, Alvaro J. Romanha, Douglas de Souza Moreira, Paula Fernandes dos Santos, Elio Hideo Baba, Caroline Maria Oliveira Volpe, Silvane M. F. Murta |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Trypanosoma cruzi Thioredoxin reductase Immunology Population Drug Resistance Gene Dosage Biology Real-Time Polymerase Chain Reaction Gene Expression Regulation Enzymologic Mice 03 medical and health sciences Western blot parasitic diseases medicine Animals Amino Acid Sequence RNA Messenger Northern blot Cloning Molecular education Gene Alleles Phylogeny Dihydrolipoamide Dehydrogenase Southern blot education.field_of_study medicine.diagnostic_test Sequence Analysis DNA General Medicine DNA Protozoan Blotting Northern biology.organism_classification Trypanocidal Agents Molecular biology Recombinant Proteins Mitochondria Blotting Southern 030104 developmental biology Infectious Diseases Biochemistry Nitroimidazoles Benznidazole Parasitology RNA Protozoan medicine.drug |
| Zdroj: | Experimental Parasitology. 170:1-9 |
| ISSN: | 0014-4894 |
| DOI: | 10.1016/j.exppara.2016.08.006 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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