טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSalam Mazareb
SubjectDevelopmentally Regulated Proline Transporters in
leishmania donovani
DepartmentDepartment of Biology
Supervisor Professor Emeritus Zilberstein Dan


Abstract

Leishmania donovani are the causative agents of kala azar in humans. These organisms cycle between proline-rich environment in the sand fly vector (extracellular promastigotes) and sugar-rich condition in the mammalian host (intracellular amastigotes). Parasites have adapted to these changes in proline concentrations: promastigotes utilize proline as a carbon source, whereas amastigotes utilize sugars and fatty acids. In previous work I characterized two transport systems in promastigotes: cation dependent and independent proline transport systems (system A and B, respectively). Systems A have broad specificity to almost all amino acids, and obtain optimum activity at pH 6, system B is more specific to proline as it is inhibited by only five amino acids. In this work I investigated proline transport in axenic L. donovani amastigotes, a single cation-independent transporter was observed (system C) that has broad specificity to almost all amino acids and obtain optimum activity at pH 5, similar to system A. The activity of system A during parasite differentiation was assessed. Its activity down regulated within three days after promastigotes were induced to differentiate into amastigotes. Both elevated temperature and acidic pH suppressed the activity of system A. When amastigotes were induced to differentiate back into promastigotes, system A resumed its activity within 24 h after differentiation was initiated. The toxic analog 3,4-Dihydro-DL-proline (DHP) was used to isolate L. donovani promastigote mutants defective in proline transport. Six different mutants that can grow in the presence of 5mM DHP were isolated and characterized. These mutants were used to clone proline and amino acid transporters genes. In parallel we cloned an  amino acid transporter gene of 1815 bp that encodes for an ORF of 605 aa. This putative protein has some homology to mmammalian a.a. transport genes