Molecular genetic and biochemical analysis of purine salvage pathways in toxoplasma GONDII / John Darling.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Darling, John.

Contributor:

Roos, David S., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Biology.

Biology--Penn dissertations.

Biology.

Academic Dissertations as Topic.

Medical Subjects:

Biology.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Biology.

Biology--Penn dissertations.

Physical Description:

xi, 136 pages : illustrations (some color) ; 29 cm

Production:

1999.

Summary:

The obligate intracellular parasite Toxoplasma gondii is recognized as both a serious cause of congenital human birth defects and a common opportunistic infection of immunocompromised individuals such as those diagnosed with AIDS. Novel chemotherapeutic approaches to treatment of this pathogen are mandated by the acknowledged shortcomings of conventional treatment strategies. The development of such novel approaches will require a detailed understanding of the biochemical relationship between parasite and host, most specifically of those pathways which represent potentially exploitable targets for the rational design of antiparasitic drugs.

The inability of T. gondii to conduct de novo purine synthesis makes this parasite entirely dependent upon host purines, thus implicating those biochemical activities responsible for purine salvage as ideal targets for specific chemotherapeutic interventions. The work described in this dissertation exploits a number of complementary molecular and biochemical techniques to demonstrate that T. gondii relies solely upon its hypoxanthine/guanine/xanthine phosphoribosyltransferase (HGXPRT) and adenosine kinase (AK) enzymes for its obligate salvage of purines from the host. Although both HGXPRT and AK have been knocked out independently, the failure of attempts to knockout the endogenous, genomic loci for both salvage enzymes in a single parasite indicate that T. gondii requires at least one of these activities in order to survive. These results are consistent with biochemical evidence suggesting that T. gondii lacks a functional adenine phosphoribosylotransferase (APRT) activity as a supplemental purine salvage pathway. The discovery that T. gondii is dependent entirely upon HGXPRT and AK to acquire host purines further validates investigation of these purine salvage pathways as potential drug targets.

These findings have prompted a more detailed examination of the recently cloned T. gondii AK. Recombinant enzyme has been expressed and purified to apparent homogeneity by standard biochemical methods. Here we report kinetic analysis of the T. gondii AK with respect to both its native substrates and a number of previously recognized inhibitors which have now been shown to act as subversive substrates of the parasite enzyme. Combining these data with recent structural evidence provides the basis for the rational development and/or design of compounds that specifically target parasite purine salvage enzymes.

Notes:

Supervisor: David S. Roos.

Thesis (Ph.D. in Biology) -- University of Pennsylvania, 1999.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 99-53519.

OCLC:

187483620