טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentInbar Ehud
SubjectFunctional Genomics of Amino Acid Permeases in Leishmania
donovani: The Story of Proline and Lysine
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Zilberstein
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Parasitic protozoa of the genus Leishmania are the causative agents of leishmaniasis in human and mammals. These organisms cycle between two major forms; intracellular amastigotes, the disease causing form that resides in phagolysosomes of macrophages, and extracellular flagellated promastigotes in the digestive tracts of sand flies. These environments are rich in amino acids, especially proline and alanine that are utilized by insects as a source of metabolic energy. Trypanosomes utilize amino acids as alternative source of energy and for osmotic regulation. Cellular pools of amino acid, especially the essentials, depend on uptake from outside via transporters. To date, amino acid transport has been characterized biochemically in Trypanosomatids. However, only one amino acid transporter gene has been identified and characterized, the L. donovani arginine transporter LdAAP3.  Here, we used heterologous expression of L. donovani genes in Saccharomyces cerevisiae amino acid transport auxotroph mutants. Two genes, LdAAP7 (DQ402427) and LdAAP24 (EU391425), that encode lysine and proline transporters, respectively have been identified.  LdAAP7 is a high affinity, low capacity and high specificity lysine transporter. The low capacity of lysine transport in promastigotes may indicate tight regulation of cellular lysine as high levels of lysine were shown to be toxic in other organisms. Over expression of LdAAP7 in promastigotes resulted in limited elevation of lysine transport activity only at low external lysine. When L. donovani promastigotes reached stationary growth phase, lysine transport decreased to almost zero, supporting the idea of tight regulation of lysine transport.  Attempts to delete LdAAP7 from L. donovani genome failed due gene duplication. LdAAP24 encodes for promastigote-specific, cation dependent, low affinity and high capacity proline transport system A. Using polyclonal antibodies raised in rabbits, LdAAP24 protein was localizes to the plasma membrane and the flagella pocket. Deleting the transporter from L. donovani chromosome 10 resulted in a dramatic decrease in proline transport, which correlated with dramatic drop to almost zero in the cellular pool of proline. In addition, the cellular levels of glutamate and alanine as well as their transport activities were also affected. Our analyses indicated that glutamate is not a substrate of LdAAP24, suggesting that this transporter regulate glutamate transport and metabolism. The results of our analyses suggest that LdAAP24 has a central role in regulating amino acid transport and metabolism in promastigotes. This work provides the first molecular characterization of proline and lysine transporter genes in parasitic protozoa.