title: Leishmania amazonensis: Increase in ecto-ATPase activity and parasite burden of vinblastine-resistant protozoa.
abstract: Leishmania amazonensis is a protozoan parasite that induces mucocutaneous and diffuse cutaneous lesions upon infection. An important component in treatment failure is the emergence of drug-resistant parasites. It is necessary to clarify the mechanism of resistance that occurs in these parasites to develop effective drugs for leishmaniasis treatment. Promastigote forms of L. amazonensis were selected by gradually increasing concentrations of vinblastine and were maintained under continuous drug pressure (resistant cells). Vinblastine-resistant L. amazonensis proliferated similarly to control parasites. However, resistant cells showed changes in the cell shape, irregular flagella and a decrease in rhodamine 123 accumulation, which are factors associated with the development of resistance, suggesting the MDR phenotype. The Mg-dependent-ecto-ATPase, an enzyme located on cell surface of Leishmania parasites, is involved in the acquisition of purine and participates in the adhesion and infectivity process. We compared control and resistant L. amazonensis ecto-enzymatic activities. The control and resistant Leishmania ecto-ATPase activities were 16.0 ± 1.5 nmol Pi × h(-1) × 10(-7) cells and 40.0 ± 4.4 nmol Pi × h(-1) × 10(-7)cells, respectively. Interestingly, the activity of other ecto-enzymes present on the L. amazonensis cell surface, the ecto-5' and 3'-nucleotidases and ecto-phosphatase, did not increase. The level of ecto-ATPase modulation is related to the degree of resistance of the cell. Cells resistant to 10 μM and 60 μM of vinblastine have ecto-ATPase activities of 22.7 ± 0.4 nmol Pi × h(-1) × 10(-7) cells and 33.8 ± 0.8 nmol Pi × h(-1) × 10(-7)cells, respectively. In vivo experiments showed that both lesion size and parasite burden in mice infected with resistant parasites are greater than those of L. amazonensis control cells. Furthermore, our data established a relationship between the increase in ecto-ATPase activity and greater infectivity and severity of the disease caused by vinblastine-resistant L. amazonensis promastigotes. Taken together, these data suggest that ecto-enzymes could be potential therapeutic targets in the struggle against the spread of leishmaniasis, a neglected world-wide public health problem.
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro - UFRJ, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro - UFRJ, Brazil.
authors.forename: Naira Lígia Lima
authors.initials: NL
authors.lastname: Giarola
authors.affiliation: Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro - UFRJ, Brazil; Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro IFRJ, Brazil.
authors.forename: Thaís Souza
authors.initials: TS
authors.lastname: Silveira
authors.affiliation: Instituto Oswaldo Cruz - Fiocruz, Brazil.
authors.forename: Job Domingos Filho
authors.initials: JD
authors.lastname: Inacio
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro - UFRJ, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro - UFRJ, Brazil.
authors.forename: Lisvane Paes
authors.initials: LP
authors.lastname: Vieira
authors.affiliation: Instituto Oswaldo Cruz - Fiocruz, Brazil.
authors.forename: Elmo Eduardo
authors.initials: EE
authors.lastname: Almeida-Amaral
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro - UFRJ, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro - UFRJ, Brazil. Electronic address: meyer@bioqmed.ufrj.br.
authors.forename: José Roberto
authors.initials: JR
authors.lastname: Meyer-Fernandes
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D006224:Cricetinae; D004351:Drug Resistance; D006801:Humans; D007894:Leishmania mexicana / Q000187:drug effects* / Q000201:enzymology* / Q000648:ultrastructure; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D059208:Parasite Load; D010641:Phenotype; D013047:Specific Pathogen-Free Organisms; D050257:Tubulin Modulators / Q000494:pharmacology*; D014747:Vinblastine / Q000494:pharmacology*
chemical_list: D050257:Tubulin Modulators; D014747:Vinblastine; D000251:Adenosine Triphosphatases; C027201:ectoATPase
keywords: Ecto-ATPase activity; Leishmania amazonensis; Leishmania infection; Vinblastine-resistant Leishmania