Regulation dynamics of Leishmania differentiation: deconvoluting signals and identifying phosphorylation trends

Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of disease in humans; cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosome of mammalian macrophages. While much of the molecular mechanism of development inside macrophages remains a mystery, development of a host-free system that simulates phagolysosome conditions (37ºC 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 hours after exposure); II, movement cessation and aggregation (5-10 hours); III, amastigote morphogenesis (10-24 hours); and IV, maturation (24-120 hours). Transcriptomic and proteomic analyses 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 hypothesize that the differentiation signal activates a phosphorylation pathway, which initiates Leishmania transformation, and here we use Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to interrogate the dynamics of changes in 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 predominating during phase I and III, whereas phase II and IV are characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signalspecific; 37ºC 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: http://www.absciex.com/products/software/proteinpilot-software.