Date of Award

8-20-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Siegfried Detke

Abstract

Leishmania species are parasitic protozoa of the order Kinetoplastida that cause leishmaniasis. Treatment of leishmaniasis with antimonials is difficult and resistance is increasing. Alternative compounds have been investigated, one group is the toxic purine analogs. Resistance to purine analogs, potential anti-Leishmania agents, is brought about by TOR (TOxic nucleoside Resistance protein). TOR is an atypical, multidrug resistance protein that was originally discovered in Leishmania. It renders the cells less sensitive to toxic nucleosides by curtailing the ability of the nucleoside permeases to transport purines across the plasma membrane. Targets of TOR (TOT's) are proteins inside Leishmania that interact with and/or are affected by TOR's activity. One of these is the mitochondrial ADP/ATP translocase (ANT). The goal of my research was to identify the extra-membrane loop(s) of ANT that was recognized by TOR i.e., had TOT activity and to determine the subcellular distribution of ANT as well as its possible site(s) of interaction with TOR inside the cell. To identify the extra-membrane loop(s) of ANT that were recognized by TOR, the seven loops of the enzyme were PCR amplified, cloned into expression vector, pALT-Neo and transformed into Leishmania by electroporation. Transfected Leishmania were cultured and selected using G418. TOT activity was assayed by counting the transformed cells with the different ANT loops under different concentrations of the toxic adenosine analog, tubercidin. The ED50 for each construct was calculated and compared to pALT-Neo transformed Leishmania as a control. Peptides that had TOT activity impaired TOR's inhibitory effects on the other purine nucleoside permeases and the cells become more sensitive to tubercidin. Loop I and V showed TOT activity. The potential TOT activity for the different subparts of loop V was assayed as described above. None of loop V subregions showed TOT activity. To co-localize the interaction of ANT with TOR in Leishmania , ANT polyclonal antibody was raised against a synthetic peptide for loop IV and Leishmania subcellular fractions were isolated. Western blotting, co-immunoprecipitation and immunofluorescence enabled the determination of ANT subcellular distribution as well as its possible site(s) of interaction with TOR within Leishmania.

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