Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.
Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis.



After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway.



This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected by this protein in the parasite. Finally, these findings may be helpful for the development of alternative anti-leishmanial drugs that target purine pathway.
Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
Juliana Ide
JI
Aoki
Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.|Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil.
Adriano Cappellazzo
AC
Coelho
Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
Sandra Marcia
SM
Muxel
Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
Ricardo Andrade
RA
Zampieri
Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, Brazil.
Eduardo Milton Ramos
EM
Sanchez
Department of Clinical Science, University of Bergen, Bergen, Norway.
Audun Helge
AH
Nerland
Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
Lucile Maria
LM
Floeter-Winter
Instituto de Medicina Tropical, Departamento de Moléstias Infecciosas e Parasitárias, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.
Paulo Cesar
PC
Cotrim
D000595:Amino Acid Sequence; D000818:Animals; D000977:Antiparasitic Agents / Q000627:therapeutic use*; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000235:genetics*; D018320:Leishmania major / Q000187:drug effects / Q000235:genetics*; D007896:Leishmaniasis / Q000188:drug therapy*; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000235:genetics; D014372:Tubercidin / Q000627:therapeutic use*
D000977:Antiparasitic Agents; C110928:NupM1 protein, Leishmania mexicana; D014157:Transcription Factors; D014372:Tubercidin