טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRosenzweig Doron
SubjectProteomic Analysis of Leishmania donovani Differentiation
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Zilberstein
Full Thesis textFull thesis text - English Version


Abstract

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.