In order to survive extremely different environments, intracellular
parasites require highly adaptable physiologic and metabolic systems. Leishmania
donovani extracellular promastigotes reside in a glucose-rich, slightly
alkaline environment in the sand fly vector alimentary tract. Upon entry into
human macrophage phagolysosomes, promastigotes differentiate into intracellular
amastigotes. These cope with an acidic milieu,
where glucose is scarce, while amino acids are abundant. We used an axenic
differentiation model and a novel high-coverage, comparative proteomic
methodology to analyze in detail protein expression changes throughout the
differentiation process. The analysis identified and quantified 21% of the
parasite proteome across 7 time-points during differentiation. The data reveals
a delayed increase in gluconeogenesis enzymes, coinciding with a decrease in
glycolytic capacity. At the same time, β-oxidation, amino acid catabolism,
tricarboxylic acid cycle, mitochondrial respiration chain and oxidative
phosphorylation capacities were all up-regulated. The results indicate that the
differentiating parasite shifted from glucose to fatty acids and amino acids as
its main energy source. Furthermore, glycerol and amino acids were used as
precursors for sugar synthesis, compensating for lack of exogenous sugars.. The
vast majority of the proteomic changes occurred while promastigotes transformed
morphologically. In addition to the extensive protein expression profiling, the
analysis yielded a wealth of information on posttranslational modifications
that occur in the parasite. iTRAQ detected protein phosphorylation, methylation,
acetylation and glycosylation sites throughout differentiation. Methylations
were detected on arginines, aspartic acids, glutamic acids, asparagines and
histidines. Detected acetylation sites included serines and protein N-terminal
acetylations on methionines, serines, alanines and threonines. Phosphorylations
were detected on serines and threonines, but not tyrosines. iTRAQ identified
novel fucosylation sites, as well as hexoylations. We observed significant quantity
changes in some modifications during differentiation, suggesting a role in L.
donovani intracellular development. This study constitutes the first
high-throughput analysis of an intracellular developmental process and provides
new insights into the adaptations of Leishmania to the extreme
environments it encounters. In addition to the proteomic analysis, a
description is given for the partial purification of a possible
differentiation-modulating factor from a mammal host serum.
