Mechanistic Dissection and Antibiotic Discovery Targeting Clostridioides difficile RNA Polymerase

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PROJECT SUMMARY With the alarming increase in the incidence of infections caused by antibiotic-resistance bacteria, there is an urgent need to identify new strategies to combat this emerging threat. The development, growth, and survival of all living organisms rely on coordinated gene expression. Central to gene expression is RNA polymerase (RNAP), a multi-subunit protein that transcribes genetic information from DNA to RNA in the complex and highly regulated process of transcription. Transcription has three major stages for creating a nascent RNA: initiation, elongation, and termination, each of which is controlled by protein transcription factors. RNAP is a proven drug target, but RNAP’s mechanistic features and how it is regulated by transcription factors remain poorly understood in pathogenic bacteria. My long-term goal is to understand the mechanisms of action of RNAP and key transcription factors involved in regulating RNAP initiation (CarD), elongation (NusG and NusA) and termination (Rho) in order to improve future antimicrobial development. In this proposed research, I will investigate the biochemical, structural, and genetic basis of the transcriptional machinery of Clostridioides difficile (C. diff.), a life-threatening gut pathogen that is resistant to multiple antibiotics. In Aim 1(K99 phase), I will investigate the functional relationship between two paralogs of the transcription regulator CarD and RNAP through in vitro and in vivo studies to test the hypothesis that the two CarD paralogs compete to bind and regulate RNAP, and the interplay of these factors is critical for coordinated control of transcription initiation in C. diff. In Aim 2 (K99/R00 phase), I will use genomic-scale mapping techniques and genetic assays to interrogate how Rho rewires gene expression by terminating transcription by RNAP. I will also design biochemistry assays to elucidate the mechanisms by which NusA and NusG, two universal elongation factors, modulate Rho-RNAP behavior. In Aim 3 (R00 phase), I will build an in vitro platform using the Broccoli fluorescent RNA aptamer to enable high-throughput screening of inhibitors of C. diff. RNAP. Virtual screening will be conducted to identify novel inhibitors based on our newly obtained cryo-EM structure. The proposed research in the K99 phase will mainly be conducted in the lab of Prof. Robert Landick at the University of Wisconsin-Madison. The key area that I will acquire additional research training is genome-scale mapping techniques and corresponding bioinformatics skills to analyze high-throughput datasets. I will also be guided by an advisory committee including collaborators Prof. Federico Rey (UW-Madison, an expert in microbiome-host interactions) and Prof. Elizabeth Campbell (The Rockefeller Univ., an expert in cryo-EM of RNAP and associated proteins), and consultant Prof. Joseph Sorg (Texas A&M Univ., an expert in C. diff genetics and physiology). I will also benefit from the facilities and abundant resources at UW-Madison. During the mentored phase of this award, I also plan to hone my skills in teaching, leadership and scientific communication, which will facilitate my transition to an independent research career.