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...