Regulation Dynamics of Leishmania Differentiation: Deconvoluting Signals and Identifying Phosphorylation Trends

Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of diseases in humans, cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosomes of mammalian macrophages. Although many of the molecular mechanisms of development inside macrophages remain a mystery, the development of a host-free system that simulates phagolysosome conditions (37 °C and pH 5.5) has provided new insights into these processes. The time course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0–5 h after exposure); II, movement cessation and aggregation (5–10 h); III, amastigote morphogenesis (10–24 h); and IV, maturation (24–120 h). Transcriptomic and proteomic analyses have indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes and identified stage-specific phosphorylation motifs. We hypothesized that the differentiation signal activates a phosphorylation pathway that initiates Leishmania transformation, and here we used isobaric tags for relative and absolute quantitation to interrogate the dynamics of changes in the phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominated during phases I and III, whereas phases II and IV were characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signal-specific; 37 °C and pH 5.5), but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa, revealing the outline of a signaling pathway during Leishmania differentiation.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (identifier PXD000671). Data can be viewed using ProteinPilot™ software. Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of diseases in humans, cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosomes of mammalian macrophages. Although many of the molecular mechanisms of development inside macrophages remain a mystery, the development of a host-free system that simulates phagolysosome conditions (37 °C and pH 5.5) has provided new insights into these processes. The time course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0–5 h after exposure); II, movement cessation and aggregation (5–10 h); III, amastigote morphogenesis (10–24 h); and IV, maturation (24–120 h). Transcriptomic and proteomic analyses have indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes and identified stage-specific phosphorylation motifs. We hypothesized that the differentiation signal activates a phosphorylation pathway that initiates Leishmania transformation, and here we used isobaric tags for relative and absolute quantitation to interrogate the dynamics of changes in the phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominated during phases I and III, whereas phases II and IV were characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signal-specific; 37 °C and pH 5.5), but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa, revealing the outline of a signaling pathway during Leishmania differentiation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (identifier PXD000671). Data can be viewed using ProteinPilot™ software. Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis in humans, which can manifest as hepatosplenomegaly, ulcerative skin lesions, or destructive mucosal inflammation and damage to the mucosal tissues (1.Herwaldt B.L. Leishmaniasis.Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1375) Google Scholar, 2.Murray H.W. Berman J.D. Davies C.R. Saravia N.G. Advances in leishmaniasis.Lancet. 2005; 366: 1561-1577Abstract Full Text Full Text PDF PubMed Scopus (1361) Google Scholar). The parasites cycle between two major forms in two distinct environments, starting out as flagellated extracellular promastigotes in the alimentary tract of the female sand fly and subsequently differentiating into immotile intracellular amastigotes within the phagolysosomes of mammalian macrophages (1.Herwaldt B.L. Leishmaniasis.Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1375) Google Scholar). While cycling between these two forms, parasites encounter two distinct environments to which they must quickly adapt. As extracellular promastigotes, parasites are surrounded by the sugar-rich, slightly alkaline environment of the fly's mid-gut, which has a mean temperature of 26 °C. Intracellular amastigotes encounter the sugar-poor, fatty-acid- and amino-acid-rich acidic environment of the phagolysosome at the elevated temperatures of the skin and viscera (3.McConville M.J. Naderer T. Metabolic pathways required for the intracellular survival of.Leishmania. Annu. Rev. Microbiol. 2011; 65: 543-561Crossref PubMed Scopus (103) Google Scholar, 4.Zilberstein D. Physiological and biochemical aspects of Leishmania development.in: Myler P.J. Fasel N. Leishmania After the Genome: Biology and Control. Horizon Scientific Press and Caiser Academic Press, New York2008: 107-122Google Scholar). To enable molecular insight into Leishmania development, an axenic host-free system that simulates Leishmania differentiation by exposing promastigotes to a lysosome-like environment was developed (5.Saar Y. Ransford A. Waldman E. Mazareb S. Amin-Spector S. Plumblee J. Turco S.J. Zilberstein D. Characterization of developmentally-regulated activities in axenic amastigotes of Leishmania donovani.Mol. Biochem. Parasitol. 1998; 95: 9-20Crossref PubMed Scopus (147) Google Scholar, 6.Debrabant A. Joshi M.B. Pimenta P.F. Dwyer D.M. Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics.Int. J. Parasitol. 2004; 34: 205-217Crossref PubMed Scopus (185) Google Scholar, 7.Bates P.A. Robertson C.D. Tetley L. Coombs G.H. Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms.Parasitology. 1992; 105: 193-202Crossref PubMed Scopus (179) Google Scholar). These studies indicated that concomitant exposure to acidic pH levels (usually pH 5.5) and high temperatures (33 °C and 37 °C for cutaneous and visceral strains, respectively) provides the signals that lead promastigotes to start differentiation into amastigotes, a process that is completed within 5 days (8.Barak E. Amin-Spector S. Gerliak E. Goyard S. Holland N. Zilberstein D. Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response.Mol. Biochem. Parasitol. 2005; 141: 99-108Crossref PubMed Scopus (132) Google Scholar). Time-course analyses carried out in Leishmania donovani indicated that differentiation is a highly regulated and coordinated process. Based on cell morphology, Barak et al. divided differentiation into four phases: phase I (the first 5 h after exposure of promastigotes to the differentiation signal), which is dedicated to signal perception; phase II (5–10 h after signal exposure), during which the parasites cease movement and start to aggregate; phase III (10–24 h), when cells undergo morphological change into amastigote-shaped cells; and phase IV (24–120 h), during which the amastigotes undergo maturation (8.Barak E. Amin-Spector S. Gerliak E. Goyard S. Holland N. Zilberstein D. Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response.Mol. Biochem. Parasitol. 2005; 141: 99-108Crossref PubMed Scopus (132) Google Scholar). Significant changes in mRNA (9.Saxena A. Lahav T. Holland N. Aggarwal G. Anupama A. Huang Y. Volpin H. Myler P.J. Zilberstein D. Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in during Biochem. Parasitol. PubMed Scopus Google Scholar, T. D. Volpin H. A. Holland N. Zilberstein D. Myler P.J. levels of differentiation of the intracellular J. 2011; PubMed Scopus Google and protein D. D. S. Zilberstein D. Leishmania sand fly to J. PubMed Scopus Google as as in the of S. N. phosphorylation during the Leishmania differentiation PubMed Scopus Google the parasites to to an amastigote by their to fatty-acid- and the is by an in protein and of as an pathway in amastigotes D. D. S. Zilberstein D. Leishmania sand fly to J. PubMed Scopus Google Scholar, Coombs G.H. Leishmania activities of amastigotes and promastigotes and their by and Parasitol. PubMed Scopus Google Scholar, T. J. E. M.J. of Leishmania major in macrophages and the PubMed Scopus Google Scholar). These biochemical changes are in the of the phase of parasites undergo morphogenesis P.F. Zilberstein D. has proteomics Leishmania PubMed Scopus Google and regulated by changes in T. D. Volpin H. A. Holland N. Zilberstein D. Myler P.J. levels of differentiation of the intracellular J. 2011; PubMed Scopus Google Scholar). at phases of promastigote-to-amastigote differentiation. These protein and phosphorylation D. D. Myler P.J. Zilberstein D. of proteins during Leishmania donovani PubMed Scopus Google Scholar). changes in protein at the of differentiation are regulated by and are a of transient changes in mRNA and protein T. D. Volpin H. A. Holland N. Zilberstein D. Myler P.J. levels of differentiation of the intracellular J. 2011; PubMed Scopus Google Scholar). protein phosphorylation is to of the and is a major regulatory that many J. G. G. H. J. H. G. A. N. S.J. L. M.J. P.F. analysis of protein phosphorylation in 2005; PubMed Scopus Google Scholar). Although the Leishmania has not been to is to that a of the is the of identified to are regulated between promastigotes and amastigotes, with phosphorylation in the P.F. Zilberstein D. analysis of differentiating Leishmania parasites reveals a stage-specific phosphorylation PubMed Scopus Google Scholar, A. analysis of Leishmania donovani and amastigote PubMed Scopus Google Scholar, S. H. and in Leishmania and PubMed Scopus Google Scholar, J. G. J. S. A. dynamics protein to the Leishmania donovani PubMed Scopus Google Scholar). This the that protein phosphorylation an during Leishmania differentiation and is by protein kinases and is that a of phosphorylation and constitutes a regulatory pathway Leishmania differentiation. To protein phosphorylation during differentiation in to phosphorylation that be of the signaling pathway that initiates we proteomics used isobaric tags for relative and absolute used isobaric tags for relative and absolute to protein phosphorylation dynamics during axenic L. donovani promastigote-to-amastigote differentiation. These analyses revealed a of changes in protein phosphorylation as as phase I, of which when promastigotes were to the complete differentiation signal (i.e. 37 °C and pH This work constitutes the first analysis of the phosphorylation of Leishmania promastigote-to-amastigote differentiation and the protein pathway that process in L. of L. donovani was used in and amastigotes were as (8.Barak E. Amin-Spector S. Gerliak E. Goyard S. Holland N. Zilberstein D. Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response.Mol. Biochem. Parasitol. 2005; 141: 99-108Crossref PubMed Scopus (132) Google Scholar). differentiation in axenic was carried out as by Barak et al. (8.Barak E. Amin-Spector S. Gerliak E. Goyard S. Holland N. Zilberstein D. Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response.Mol. Biochem. Parasitol. 2005; 141: 99-108Crossref PubMed Scopus (132) Google Scholar). promastigotes were at 26 °C and pH to amastigote at 37 pH and after the of cells were in amastigote phase promastigotes to were in and in Leishmania at time in differentiation and amastigote cell to were in at pH and in the phosphoproteomic the that was to the signal (i.e. temperature and and the that was to pH were with at pH and the that was to 37 °C was with that were used for were with 5 and cell were and proteins were as in P.F. Zilberstein D. analysis of differentiating Leishmania parasites reveals a stage-specific phosphorylation PubMed Scopus Google were and was as in P.F. 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