Identification of developmentally-regulated proteins in Leishmania panamensis by proteome profiling of promastigotes and axenic amastigotes.
We have employed proteomics to identify proteins upregulated in the amastigote life-stage of Leishmaniapanamensis, using axenically-differentiated forms as models of authentic intracellular parasites. Resolution of the soluble proteomes of axenic amastigotes and promastigotes by two-dimensional electrophoresis (2DE) in the neutral pI range (5-7) revealed equivalent numbers of protein spots in both life-stages (644-682 using Coomassie Blue and 851-863 by silver staining). Although representing a relatively low proportion (8.1-10.8%) of the predicted 8000 gene products of Leishmania, these proteome maps enabled the reproducible detection of 75 differentially-regulated protein spots in amastigotes, comprising 24 spots "uniquely" expressed in this life-stage and 51 over-expressed by 1.2-5.7-fold compared to promastigotes. Of the 11 amastigote-specific spots analysed by mass spectrometry (MS), 5 yielded peptide sequences with no orthologues in Leishmania major, and the remaining 6 were identified as 7 distinct proteins (some of which were truncated isoforms) representing several functional classes: carbohydrate/energy metabolism (fructose 1,6-bisphosphate aldolase, glucose 6-phosphate dehydrogenase, pyruvate dehydrogenase), stress response (heat shock protein [HSP] 83), cell membrane/cytoskeleton (beta-tubulin), amino acid metabolism (cysteine synthase) and cell-cycle (ran-binding protein). Four additional over-expressed spots were tentatively identified as HSPs 60 and 70 and HSP 70-related proteins -1 and -4 by positional analogy with these landmark proteins in the Leishmania guyanensis proteome. Our data demonstrate the feasibility of proteomics as an approach to identify novel developmentally-regulated proteins linked to Leishmania differentiation and intracellular survival, while simultaneously pinpointing therapeutic targets. In particular, the amastigote-specific expression of cysteine synthase underlines the importance of de novo cysteine synthesis both as a potential parasite virulence factor and as a major metabolic difference from mammalian host cells.
Walker
John
J
Centro Internacional de Entrenamiento e Investigaciones Medicas (CIDEIM), Avenida 1 Norte No. 3-03, Cali, Colombia. john_walker@cideim.org.co
Vasquez
Juan-José
JJ
Gomez
Maria Adelaida
MA
Drummelsmith
Jolyne
J
Burchmore
Richard
R
Girard
Isabelle
I
Ouellette
Marc
M
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D007891:Leishmania / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D020543:Proteome; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
D020543:Proteome; D015800:Protozoan Proteins