Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani

Abstract

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.

Leishmania Exosomes Modulate Innate and Adaptive Immune Responses through Effects on Monocytes and Dendritic Cells

“…Only consistently overrepresented spots were further analyzed. The proteins in the gel plugs were destained, digested with trypsin (Promega), extracted, purified on a C18 reverse-phase matrix, and eluted in 8 ml 60% acetonitrile and 0.1% trifluor-acetic acid (24). A total of 2.5 ml of the eluted peptides were spotted onto a 378-well, 400-mm Anchor Plate (Bruker Daltonics, Bremen Germany).…”

“…The sediment was resuspended in 50 ml PBS, and 150 ml rehydration buffer (8 M urea, 2 M thiourea, 4% CHAPS, and 2.4% aminosulfobetaine-14) was added. Two-dimensional protein electrophoresis was performed essentially as described previously (24).…”

Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in gene expression during differentiation

“…These axenic amastigotes resemble animal-derived amastigotes and have been widely used for investigating parasites activities without the complication of host cell material [20][21][22][23]. Time-course analysis of L. donovani differentiation showed that promastigotes expressed the amastigote-specific A2 protein family within an hour of exposure to the intralysosomal environment and at five hours they start to transform to amastigote-shaped cells [19].…”

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

“…To understand the differences during transformation and between the different life-cycle stages on the molecular level several groups resolved the proteome of L. infantum, L. donovani and L. mexicana promastigotes and amastigotes [19][20][21] and promastigotes upon reception of the transformation signal 22 or metacylics 23 .…”