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The biochemistry and evolution of antifolate resistance mutations in Plasmodium and Toxoplasma.
- Format:
- Book
- Thesis/Dissertation
- Author/Creator:
- Reynolds, Mary.
- Language:
- English
- Subjects (All):
- Biochemistry.
- Microbiology.
- Molecular biology.
- 0307.
- 0410.
- 0487.
- Penn dissertations--Parasitology.
- Parasitology--Penn dissertations.
- Local Subjects:
- Penn dissertations--Parasitology.
- Parasitology--Penn dissertations.
- 0307.
- 0410.
- 0487.
- Physical Description:
- 127 pages
- Contained In:
- Dissertation Abstracts International 59-04B.
- System Details:
- Mode of access: World Wide Web.
- text file
- Summary:
- Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a bifunctional, multisubstrate enzyme, of critical importance for folate metabolism, and a crucial drug target for the clinical management of both toxoplasmosis and malaria--diseases caused by the closely-related parasites Toxoplasma gondii and Plasmodium falciparum. Taking advantage of the accessibility of Toxoplasma to molecular genetic manipulation, we generated a series of mutant T. gondii DHFR-TS alleles that mirrors naturally-occurring P. falciparum variants associated with resistance to the parasite DHFR inhibitors pyrimethamine and cycloguanil. In addition, novel pyrimethamine-resistance mutations were produced in the T. gondii DHFR-TS via random mutagenesis. Recombinant shuttle vectors were then engineered to permit examination of these variant DHFR alleles by (i) by transient or stable transformation of T. gondii parasites, (ii) complementation assays in bacteria and yeast, and (iii) biochemical analysis of purified (recombinant) DHFR-TS enzyme.
- Analysis of T. gondii DHFR-TS variants harboring P. falciparum mutations reveals that the commonly-occurring Asn-108 mutation confers only low-level pyrimethamine-resistance, but does not substantially compromise enzyme catalytic function. In contrast, the laboratory mutation Phe-223 confers moderate-level pyrimethamine resistance, but results in diminished enzyme function and temperature-sensitivity in parasites, suggesting a barrier to the establishment of this mutation in the field. Detailed examination of other mutations, alone and in combination, highlights the importance of Val-16 in cycloguanil resistance, and suggests a model for the evolution of high-level antifolate resistance in the field. This study also demonstrates that pyrimethamine resistance in Toxoplasma can be acquired through single moderately potent mutations, in contrast to the multiple substitutions that produce high-level resistance in malaria.
- Our results suggest that selection for drug-resistance mutations in parasites is sufficiently acute to favor not merely resistance, but also metabolic efficiency. Understanding the molecular mechanisms of resistance and the associated physiological costs to the parasite should provide insights into the dynamics of drug-resistant disease dissemination, and may permit the development of strategies to minimize its impact.
- Notes:
- Thesis (Ph.D. in Parasitology) -- University of Pennsylvania, 1998.
- Source: Dissertation Abstracts International, Volume: 59-04, Section: B, page: 1505.
- Advisers: David S. Roos; Jay P. Farrell.
- Local Notes:
- School code: 0175.
- ISBN:
- 9780591828085
- Access Restriction:
- Restricted for use by site license.
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