Date

September 2012

Document Type

Thesis

Degree Name

M.S.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, which can manifest as a single cutaneous ulcer, chronic cutaneous or mucocutaneous lesions, or visceral disease. Leishmaniasis is endemic in nearly 90 countries, and there are an estimated 12 million people currently infected with Leishmania parasites. Two million new cases are thought to occur annually, including 500,000 cases of visceral leishmaniasis. Because of this burden, leishmaniasis is now considered a disease that impedes socioeconomic development. There is a lack of effective vaccines for preventing parasitic diseases, leaving chemotherapy as the only means for treatment and prevention. However, current drugs used in the treatment of leishmaniasis are far from ideal. There is a high risk of toxicity, some treatments require multiple administrations for extended periods of time, there is an emergence of resistance, and availability is hindered by cost. One of the biochemical pathways of Leishmania that has shown potential as a therapeutic target is the metabolism of cationic polyamines. Significant differences exist between the polyamine biosynthetic pathways of Leishmania and their mammalian hosts, reinforcing this pathway as a potential therapeutic target for leishmaniasis. The polyamine pathway of Leishmania consists of four enzymes: arginase (ARG), ornithine decarboxylase (ODC), spermidine synthase (SPDSYN), and S-adenosylmethionine decarboxylase (ADOMETDC). To characterize this pathway in detail, we employed a targeted gene replacement strategy to create a series of null mutants, each lacking one of the four genes in the pathway. Each of the genetic lesions creates a conditional lethal mutation that can be bypassed by media supplementation with downstream metabolites in vitro, illustrating that an intact polyamine biosynthetic pathway is essential for promastigote survival and growth. Moreover, the Δldarg, Δldodc, Δldadometdc, and Δldspdsyn mutants are all markedly compromised in their abilities to establish infections in mice. The extents of the infectivity deficits, however, vary considerably with the nature of the genetic lesion, which we hypothesize is attributable to the differential availability of amino acid and/or polyamine pools in the mammalian phagolysosome. Furthermore, we have shown that oral administration of putrescine to Δldodc-infected mice can partially restore the virulence defect of Δldodc parasites, whereas oral administration of DFMO, an ODC inhibitor, to mice inoculated with wild-type L. donovani results in a partial decrease in parasite burdens, indicating that the polyamine biosynthetic machinery of L. donovani can be nutritionally or pharmacologically targeted.

Identifier

doi:10.6083/M44F1NRN

School

School of Medicine

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