GENE EXPRESSION/DRUG RESISTANCE IN TRICHOMONAS

本研究的长期目标是了解对阴道毛滴虫基因表达至关重要的基本特性，研究基因表达改变在甲硝唑耐药寄生虫中的作用，并确定耐药机制。阴道绦虫是美国人最常见的寄生虫之一。这种寄生虫的分离物对甲硝唑（唯一被许可用于治疗的药物）的治疗是难治的，经常被报道。我们之前的研究已经提供了证据，证明耐药菌株已经改变了编码蛋白质的基因的转录，这些蛋白质是将这种前药激活为细胞毒性形式所必需的。通过对阴道绦虫转录调控机制的研究，以及这些机制如何被改变从而产生耐药性，我们发现了一个高度保守的基本启动子元件（Inr），它是所有阴道绦虫蛋白编码基因转录所必需的。我们建议确定该元件及其相互作用蛋白在转录起始中的作用。可能的新机制用于翻译阴道绦虫蛋白也将进行检查。由于我们的研究重点是寄生虫与人类宿主的区别，所获得的信息可能会导致鉴定新的治疗靶点。我们建议通过以下途径来扩展基因表达和耐药性的研究：(1)通过纯化蛋白质并克隆编码该蛋白质的基因来鉴定特异性结合Inr启动子元件的蛋白质；(2)检测不寻常的mrna 5'非翻译区域在阴道滴虫基因表达中的作用；(3)确定基因表达改变是否与滴虫耐药有关。这些研究将大大提高我们对早期进化的真核生物基因表达的理解。此外，更好地了解滴虫基因表达的基本特性将有助于深入了解寄生原生生物基因调控机制的进化，以及产生耐药性的生化机制。

The long term objectives of this research are to understand the basic properties that are critical for the expression of genes in the parasite Trichomonas vaginalis, to examine the role of altered gene expression in metronidazole resistant parasites and to determine the mechanism underlying drug resistance. T. vaginalis is one of the most common parasites encountered by Americans. Isolates of this parasite that are refractory to treatment with metronidazole, the only drug licensed for therapy, are frequently reported. Our previous research has provided evidence that drug resistant strains have altered transcription of genes encoding proteins that are necessary for activation of this prodrug to its cytotoxic form. Investigation of the mechanisms that govern transcription in T. vaginalis and how these mechanisms can be altered to give rise to drug resistance has lead to our discovery of a highly conserved, essential promoter element (Inr) that is necessary for transcription of all T. vaginalis protein-coding genes. We propose to define the role of this element and its interacting proteins in initiation of transcription. Possible novel mechanisms used for the translation of T. vaginalis proteins will also be examined. As our studies focus on properties of the parasite that distinguish it from its human host, the information gained may lead to identification of novel, therapeutic targets. We propose to extend our research on gene expression and drug resistance by (1) characterizing a protein that specifically binds to the essential Inr promoter element by purifying the protein and cloning the gene encoding this protein, (2) examining the role of the unusual 5' untranslated regions of mRNAs in expression of T. vaginalis genes and (3) determining whether altered gene expression is responsible for drug resistance in trichomonads. These studies will greatly enhance our understanding of gene expression in early-evolving eukaryotes. Moreover, a better understanding of the basic properties underlying gene expression in trichomonads will provide insights into the evolution of gene regulatory mechanisms in parasitic protists, as well as biochemical mechanisms that give rise to drug resistance.