טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentPninit Shaked-Mishan
SubjectFunctional Genomics of Amino Acid Permeases in Leishmania:
Gene Cloning and Functional Characterization
DepartmentDepartment of Biology
Supervisors Professor Emeritus Zilberstein Dan
Clinical Professor Ephros Moshe


Abstract

Leishmania donovani are obligatory intracellular parasites that cycle between midgut of sand flies and phagolysosomes of mammalian macrophages. In sand flies they encounter amino acid-rich environment, to which they have adapted by obtaining high level of transport, especially proline and alanine. The permeases that mediate transport create large intracellular pool of free amino acids (approximately 300 mM), which play an important role in parasite response to osmotic changes that often occur in the vector's midgut. To date, none of the Leishmania amino acid permease genes have been cloned. Recently, eight such genes have been cloned in our laboratory, sequenced and shown to be expressed in L. donovani promastigotes. This work focuses on five genes, AAPLD 2,3,8,10,11. Phylogenetic analysis of all cloned L. donovani genes showed that AAPLD 1-3, AAP8LD, AAP11LD, AAP13LD and AAP15LD belong to the ATF-1 super family, as they contain the pfam domain 01490. AAP10LD belong to the APC super family as it contains the pfam domain 00324. Using these sequences as well as known and characterized amino acid permease genes from all kingdoms we revealed two motifs that are specific to the genus Leishmania (L1 and L2 motifs), four to the family trypanosomatids (T 1-4 motifs) and a single motif that is common between trypanosomatids and mammalian A1 and N permeases (N motif). Phylogenic analyses indicated that AAPLD genes that belong to ATF-1 display close phylogenic relationship to mammalian transporters. AAPLD 2,3,8,10,11 were cloned into yeast expression vector, transformed to yeast auxotroph mutants to various amino acids permeases and grown on selective media. Only AAP3LD succeeded to enable growth on low concentrations of citrullin, histidine and lysine in yeast deficient mutants. Transport assays indicated that AAP3LD is a lysine transporter with optimum pH activity at pH 6.