Differential protein abundance in promastigotes of nitric oxide-sensitive and resistant Leishmania chagasi strains.
OBJECTIVE
Leishmania chagasi is the causative agent of zoonotic visceral leishmaniasis in Brazil. Domestic and stray dogs are the main reservoirs. The life cycle of the parasite involves two stages. Promastigotes are extracellular and develop within the sand fly gut. Amastigotes survive inside the harsh environment of the phagolysosome of mammalian host phagocytes, which display the nitric oxide defense mechanism. Surprisingly, we were able to isolate promastigotes that are also resistant to NO. This finding may be explained by the preadaptative hypothesis. An insight into the proteome of NO-sensitive and resistant promastigotes is presented herein.


METHODS
Total protein extracts were prepared from promastigote cultures of an NO-sensitive and a resistant strain at early-logarithmic, mid-logarithmic and stationary phase. A population enriched in metacyclic promastigotes was also isolated by Percoll gradient centrifugation. In vitro infectivity of both strains was compared. Differential protein abundance was analyzed by 2DE-MALDI-TOF/TOF. The most striking results were tested at the mRNA level by qRT-PCR. Three biological replicates were performed in all cases.


RESULTS
NO-resistant L. chagasi promastigotes are more infective than NO-sensitive ones. Among the differentially abundant spots, 40 proteins could be successfully identified in the sensitive strain and 38 in resistant promastigotes.


CONCLUSIONS
The increase of G6PD and the decrease of ARG and GPX transcripts and proteins contribute to NO resistance in L. chagasi promastigotes. These proteins may be studied as potential drug targets and/or vaccine candidates in the future.
Department of Molecular Microbiology and Biology of Infections and Service of Proteomics and Genomics, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Madrid, Spain.
Pedro J
PJ
Alcolea
Department of Morphology, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil.
Gabriel I L
GI
Tuñón
Department of Molecular Microbiology and Biology of Infections and Service of Proteomics and Genomics, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Madrid, Spain.
Ana
A
Alonso
Department of Molecular Microbiology and Biology of Infections and Service of Proteomics and Genomics, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Madrid, Spain.
Francisco
F
García-Tabares
Proteomics Unit, Centro Nacional de Biotecnología (Consejo Superior de Investigaciones Científicas), Madrid, Spain.
Sergio
S
Ciordia
Proteomics Unit, Centro Nacional de Biotecnología (Consejo Superior de Investigaciones Científicas), Madrid, Spain.
María C
MC
Mena
Department of Morphology, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil.
Roseane N S
RN
Campos
Department of Medicine, Universidade Federal de Sergipe, Aracaju, Sergipe, Brazil.
Roque P
RP
Almeida
Department of Molecular Microbiology and Biology of Infections and Service of Proteomics and Genomics, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Madrid, Spain.
Vicente
V
Larraga
D000818:Animals; D001119:Arginase / Q000235:genetics / Q000378:metabolism; D001853:Bone Marrow / Q000469:parasitology; D004285:Dogs; D004351:Drug Resistance; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D018314:Leishmania infantum / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D007898:Leishmaniasis, Visceral / Q000469:parasitology / Q000473:pathology / Q000662:veterinary; D008297:Male; D009569:Nitric Oxide / Q000633:toxicity*; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015800:Protozoan Proteins; D009569:Nitric Oxide; D005954:Glucosephosphate Dehydrogenase; D005979:Glutathione Peroxidase; D001119:Arginase
Arginase; Glucose-6-phosphate dehydrogenase; Glutathione peroxidase; Leishmania chagasi; Nitric oxide resistance; Promastigote