A proteomic approach to identify developmentally regulated proteins in Leishmania infantum

Abstract

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.

Distinct 3′-Untranslated Region Elements Regulate Stage-specific mRNA Accumulation and Translation in Leishmania

“…Promastigotes were cultured at pH 7.0 and 25°C in SDM-79 medium supplemented with 10% heatinactivated fetal calf serum (Multicell, Wisent Inc.) and 5 g/ml hemin. L. infantum promastigote to amastigote differentiation in a host-free culture and the maintenance of axenic amastigotes were performed as previously described (22,27). Briefly, late stationary phase promastigotes (in average 6-day promastigotes) were inoculated in MAA/20 medium in 25-cm 2 -ventilated flasks and grown at 37°C and pH 5.8 with 5% CO 2 for ϳ5 days until they fully differentiated to amastigote-like forms.…”

“…Similar data were obtained with the related parasite Trypanosoma brucei, where only ϳ2% of mRNAs showed more than 2-fold differences in expression (20). However, recent proteomic analyses revealed that ϳ9% of the Leishmania proteins are differentially expressed in the amastigote stage (21,22). 6 This difference between large-scale transcriptomic and proteomic studies further outlines the importance of translational and post-translational control in regulating gene expression in this organism.…”

Using Genomic Information to Understand Leishmania Biology

“…Even so, proteomics have not revealed evident differential expression between cell stages. Only 5% of proteins are differentially expressed between L. (L.) infantum promastigotes and amastigotes [111]. Around 90% of the L. (L.) mexicana proteome is qualitatively unchanged during the life cycle [108].…”

“…Global protein profile analyses have contributed with noteworthy perception on energetic and metabolic requirements of amastigotes. Several hypothetical proteins have been identified by proteomic analysis [111,115] confirming their expression for the first time. Studies have identified several protein isoforms differentially expressed during Leishmania life-cycle through successful enrichment of poorly represented proteins [99,116].…”

Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

“…(Paba, et al, 2004, Parodi-Talice, et al, 2004, van Deursen, et al, 2003, Leishmania sp. (Acestor, et al, 2002, Drummelsmith, et al, 2003, El Fakhry, et al, 2002, Nugent, et al, 2004, Thiel and Bruchhaus, 2001, Toxoplasma gondii and Cryptosporidium sp. there have been major inroads made into our understanding of the parasite.…”