Protists of the genus Leishmania are obligatory intracellular parasites that cause a wide range of cutaneous, mucocutaneous and visceral diseases in humans. They cycle between phagolysosomes of mammalian macrophages and the sand fly mid-gut, proliferating as intracellular amastigotes and extracellular promastigotes, respectively. 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, 5% CO2) has provided new insight into these processes. Transcriptomic and proteomic analyses indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Quantitative phosphoproteomics revealed extensive differences in phosphorylation between promastigotes and amastigotes, and identified stage-specific phosphorylation motifs. We used Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to investigate the dynamics of changes in phosphorylation profile during L. donovani promastigote-to-amastigote differentiation. These experiments revealed protein kinases that phosphorylate specifically by the differentiation signal at the beginning of differentiation, but not by either high temperature or acidic pH alone. The results of these analyses have begun to reveal the molecular basis of differentiation, including a role for protein kinase A (PKA) in its regulation. 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.
