Identification of Trichomonas vaginalis resistance targets to inform future drug development

项目摘要/摘要 NIH F31-分集拨款申请的目的是为PI、Keonte Graves、 在他的生物学博士研究生培训计划中进行指导研究和职业发展活动，以 增强他成为学术医学成功研究科学家的潜力。该项目的目标 是以Keonte在他的生物学研究和硕士学位期间获得的基本实验室培训为基础 掌握先进实验室技术的新技能(例如，RNA测序的工作流程和 定量聚合酶链式反应、引物设计和基因敲除/敲除的细胞转染)以及学习生物信息学 以及生物统计学方法来分析大型转录组数据集。 这项研究提案的主要目标是识别和验证候选基因。 用于阴道毛滴虫对甲硝唑(MTZ)的耐药性。这种耐药性在美国的真实流行。 阴道毛滴虫目前还不是一种可报告的疾病，然而，一些研究表明它在 5-10%，而且可能还会上升。MTZ是FDA批准用于治疗的唯一一类药物的成员 滴虫病在美国(即5-硝基咪唑)。它于20世纪60年代初首次用于治疗阴道毛滴虫。 到1962年，感染和耐药性迅速发展。这表明甲氨蝶呤耐药的可能性是 编码在阴道毛滴虫基因组中。MTZ利用阴道毛滴虫的代谢途径来激活药物。 来自两个实验的初步数据显示，使用少量分离物(n=3和n=8)的特异性 与MTZ抗性相关的基因在MTZ抗性和敏感T. 阴道分离株。在这项提议中，PI将对更多的人进行RNA-Seq和生物信息学分析 对MTZ耐药和敏感的阴道毛滴虫分离株进行评估，以确定候选的MTZ耐药基因(目标1a)。MTZ- 将在AIM 1b中进行药敏试验和定量聚合酶链式反应，以确认和扩展RNA-Seq数据。基因 AIM 2中候选基因的敲除/敲除将随后测试它们在MTZ抗性中的作用 机械装置。该项目的长期目标是更好地了解5-硝基咪唑的作用机理。 阴道毛滴虫的耐药性，导致快速耐药性检测的发展和改进的治疗 耐药感染患者的选择。 国际和平组织的研究项目导师克里斯蒂娜·穆兹尼博士和共同导师简·诺瓦克博士赞助了 建议的培训计划。培训计划将涉及三个主要重点领域：(1)获取知识 与阴道毛滴虫实验室研究相关的先进实验室技术；(2)开发生物信息学和 具备分析基因组数据的生物统计学技能；以及(3)以团队的形式提供专业发展 交流，研究结果的陈述，摘要和手稿的准备，以及赠款写作的技巧。 这一培训计划的总体目标是为私人投资提供坚实的基础，以提高他 转变为性健康研究领域的一名独立资助的研究员。

PROJECT SUMMARY/ABSTRACT The purpose of this NIH F31-Diversity grant application is to provide support for the PI, Keonte Graves, for mentored research and career development activities within his Biology PhD graduate training program to enhance his potential to become a successful research scientist in academic medicine. The goals of the project are to build upon basic laboratory training that Keonte obtained during his Master’s in Biology research and acquire new skillsets in performing advanced laboratory techniques (i.e., workflows for RNA-Sequencing and qPCR, primer design, and cell transfection for gene knock-down/knock-out) as well as learning bioinformatics and biostatistical approaches to analyze large transcriptomics datasets. The primary objective of this research proposal is to identify and validate candidate genes responsible for metronidazole (MTZ) resistance in Trichomonas vaginalis. The true prevalence of this resistance in the U.S. is not known as T. vaginalis is not currently a reportable disease, however, several studies suggest it is between 5-10% and may be rising. MTZ is a member of the only class of medications that is FDA-approved to treat trichomoniasis in the U.S. (i.e., the 5-nitroimidazoles). It was first introduced in the early 1960s to treat T. vaginalis infections and resistance developed rapidly by 1962. This suggests that the potential for MTZ resistance is encoded in the genome of T. vaginalis. MTZ uses the metabolic pathways of T. vaginalis for drug activation. Preliminary data from two experiments using a small number of isolates (n=3 and n=8) have shown that specific genes involved with MTZ resistance are differentially expressed between MTZ-resistant and sensitive T. vaginalis isolates. In this proposal, the PI will perform RNA-Seq and bioinformatics analyses on a larger number of MTZ-resistant and sensitive T. vaginalis isolates to identify candidate MTZ resistance genes (Aim 1a). MTZ- susceptibility testing and qPCR will be performed in Aim 1b to confirm and extend the RNA-Seq data. Gene knock-down/knock-out of candidate genes in Aim 2 will subsequently test their roles in MTZ resistance mechanisms. The long-term goal of this project is to better understand mechanisms of 5-nitroimidazole resistance in T. vaginalis, leading to development of rapid resistance detection assays and improved treatment options for patients with resistant infections. The PI’s research project mentor, Dr. Christina Muzny, and co-mentor, Dr. Jan Novak, sponsor the proposed training plan. The training plan will address three main areas of focus: (1) Acquiring knowledge in advanced laboratory techniques pertinent to T. vaginalis bench research; (2) Developing bioinformatic and biostatistics skillset to analyze genomic data; and (3) Providing professional development in the form of team communication, presentation of research results, abstract and manuscript preparation, and skills in grant writing. The overall goal of this training plan is to provide the PI with a solid foundation to improve the likelihood that he transitions to becoming an independently funded investigator in the field of sexual health research.