Trichomonas vaginalis, lacking ribonucleotide reductase activity, exclusively depends on host deoxyribonucleotides for their nucleic acid metabolism and survival. To overcome deoxyribonucleotide withholding by the host, a repertoire of trichomonad proteins, including adhesins, cytotoxins, DNases, proteinases and membrane bound phosphotransferases are likely to be required. The objectives of this proposal are to understand on a molecular level the trichomonad adaptive response toward growth in a deoxyribonucleotide restricted environment. The specific hypothesis to be tested are (1) deoxyribonucleotide restricted growth will lead to an adaptive response to assure acquisition of deoxyribonucleotides by the parasite from the environment and that the type and magnitude of the response will be modulated by other host biophysiochemical parameters; (2) The adaptive response will involve the expression of multiple virulence factors; (3) differential expression of genes involved in the acquisition of dexoyribonucleotides is the molecular basis of the adaptive response and (4) Extracellular DNases are a critical link in the adaptive response and inhibition of DNase activity will lead to cell death. To test these hypotheses the research is divided into three related areas.
Specific Aim I will characterize and identify the trichomonad adaptive response proteins in a deoxyribonucleotide restricted environment. This involves the growth of T. vaginalis in chemostat-fermentor system and analysis of protein expression by two-dimensional gel electrophoresis. To further delineate the adaptive response, mutants deficient in acquisition of deoxyribonucleotides from host cells will be generated and analyzed. It is anticipated that this analysis will lead to the identification of the deoxyribonucleotide acquisition pathway.
In specific Aim II extracellular trichomonad DNases will be purified and monoclonal antibodies will be generated toward peptide fragments of the purified DNases. The antibodies will be utilized to demonstrate the relatedness of the trichomonad DNases and to show that inactivation of DNases will interrupt the deoxyribonucleotide acquisition pathway.
In specific Aim III cloning of deoxyribonucleotide-regulated genes and analysis of gene structure will be attempted to order to begin to understand the signal transduction pathway and gene-regulation in this eukaryotic parasite. It is anticipated that adaptive response genes encoding virulence factors will represent bona fide candidates with vaccine potential for future testing for immunologic intervention.
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