טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMazarib Eldar
SubjectAssessment of the role of long N-termini on amino-acid
transport and Isolation of mutants defective
in differentiation of Leishmania
donovani
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,


a disease that affects over 310 million people in 88 countries, with 300,000 new cases


annually. There are three forms of leishmaniasis: cutaneous, mucocutaneous and


visceral, the latter, is fatal if untreated. These organisms cycle between extracellular


promastigotes in the mid-gut of sand flies and intracellular amastigotes in the


phagolysosomes of mammalian macrophages. During their life cycle, the environment


of the organisms changes from relatively alkaline, sugar- and amino acid-rich, to


acidic, fatty acid- and amino acid rich. The abundance of amino acids is a feature of


both environments and, accordingly, the parasites display adaptive mechanisms that


prefer such compounds. In particular, Leishmania maintain a large amino acid pool,


mainly of alanine and proline. Parasites use these amino acids as alternative sources


of carbon and as osmolytes. LdAAP24 is a proline/alanine transporter with a long,


hydrophilic, intracellular N-terminus of 89 amino acids. LdAAP24 is the sole supplier


of cellular pool of proline and contributes to the alanine pool. It is essential for cell


volume regulation after osmotic stress and regulates the transport and homoeostasis of


other amino acids. Long N-terminal tails of amino acid transporters are known to act


as sensors of the internal pool of amino acids and as positive regulators of substrate


flux rate. The N-terminal tail of LdAAP24 regulates alanine transport and determines


substrate specificity of the transporter. In my work, we try to reconstitute the


transporter in liposomes in order to characterize the mechanism of substrate


specificity regulation. Our attempts at reconstitution of LdAAP24 were unsuccessful.


During parasites life cycle, promastigotes differentiate into amastigotes following


exposure to acidic environment of the phagolysosome. This process is mimicked in


vitro by shifting cultured promastigotes (grown at 26°C, pH 7) to a lysosome-like


environment (37°C and pH 5.5; differentiation signal). The differentiation is divided


into four morphologically distinct phases. The first phase is signal perception (0-5


hours); the second phase is movement cessation and aggregation (5-10 hours); the


third phase is amastigote morphogenesis (10-24 hours); and the fourth phase is


amastigote maturation (24-120 hours). Phosphoproteomic analysis of changes in


protein phosphorylation abundance during the course of differentiation using axenic


host free system revealed stage-specific phosphorylation, proteins with multiple


phosphorylation sites and different kinetic trends. This analysis gave information


about several proteins, including protein kinases that were phosphorylated in response


to the differentiation signal. In this work, five proteins were selected for knock-out


attempts to further investigate their role in the differentiation process. We were able to


isolate two null-mutants, one showing no visible phenotype, the second,


LdBPK_191150.1, gave rise to a conditional mutant. This gene encodes a


hypothetical protein of unknown role, its null-mutant promastigotes are not


significantly affected by the knock-out. However, null-mutant mature amastigotes


cannot grow and eventually die. This work constitutes a conditional mutant with a


unique phenotype that is manifested only in mature (phase 4) amastigotes. Further


investigation is required to fully characterize this phenotype and better understand the


function of this gene.