Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs

Autor: Aimara da Costa Pinheiro, Gabriel Andrés Tafur Gomez, Rodrigo Saar Gomes, Munira Muhammad Abdel Baqui, Gustavo Costa Bressan, Abelardo Silva-Júnior, Lucas Borges-Pereira, Anna Cláudia Alves de Souza, Matheus Silva e Bastos, Ronny Francisco de Souza, Luís Carlos Crocco Afonso, Márcia Rogéria de Almeida, Rafaela de Cássia Firmino, Christiane Mariotini-Moura, Claudia Miranda de Oliveira, Tiago D. Serafim, Talles Eduardo Ferreira Maciel, Felipe Freitas de Castro, Raphael de Souza Vasconcellos, Juliana Lopes Rangel Fietto
Rok vydání: 2014
Předmět:
Protozoan Proteins
Biochemistry
Mice
chemistry.chemical_compound
0302 clinical medicine
Leishmania infantum
Phylogeny
0303 health sciences
biology
lcsh:Public aspects of medicine
Apyrase
Recombinant Proteins
3. Good health
Infectious Diseases
Nucleoside triphosphate
Leishmaniasis
Visceral
Female
Rabbits
Research Article
lcsh:Arctic medicine. Tropical medicine
lcsh:RC955-962
Molecular Sequence Data
030231 tropical medicine
Cell Line
Microbiology
03 medical and health sciences
Dogs
parasitic diseases
medicine
Animals
Triphosphate diphosphohydrolase-2
Amino Acid Sequence
Trypanosoma cruzi
Amastigote
Molecular Biology
030304 developmental biology
Macrophages
Public Health
Environmental and Occupational Health
Biology and Life Sciences
lcsh:RA1-1270
Leishmaniasis
biology.organism_classification
medicine.disease
Leishmania
Virology
Visceral leishmaniasis
chemistry
MODELOS ANIMAIS
Parasitology
Lymph Nodes
Sequence Alignment
Zdroj: Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual)
Universidade de São Paulo (USP)
instacron:USP
PLoS Neglected Tropical Diseases
LOCUS Repositório Institucional da UFV
Universidade Federal de Viçosa (UFV)
instacron:UFV
PLoS Neglected Tropical Diseases, Vol 8, Iss 11, p e3309 (2014)
Popis: Background Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection. Methodology/Principal Findings We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP = UDP> ADP> UTP = ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis. Conclusions/Significance In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design.
Author Summary Visceral leishmaniasis is a dangerous and important, but neglected, tropical disease that affects millions of people, mainly in underdeveloped and developing countries. Presently, there are no vaccines against Leishmaniasis, and the few drugs with which the disease is treated have low efficacy and high side effects. The pathogenic agent of this disease in the New World is Leishmania infantum chagasi. In this work, we studied a protein from this parasite named ENTPDase-2. We expressed it in a bacterial system, purified it and characterized it as a genuine nucleotidase of the ENTPDase family. This protein seems to be localized at the surface of the parasite and in other intracellular locations. ENTPDase seems to facilitate in vitro infection because its blockade leads to lower levels of infection of macrophages. In addition, the protein is found in naturally infected dogs. A previous study demonstrated that ENTPDase-2 from L. infantum chagasi is a good antigen for immunodiagnosis of canine visceral leishmaniasis. We have now studied this protein in greater depth and suggest that it may be a good target for drug development.
Databáze: OpenAIRE
Externí odkaz: