Congratulations to the June 2021 funding awards for our Department faculty.
Craig Ellermeier, PhD, (Contact PI) and David Weiss, PhD, (MPI). NIH R01
Title: Cell Envelope Biogenesis in Clostridioides difficile
Project Summary: Clostridioides (Clostridium) difficile infections of the colon strike close to 500,000 people a year in the United States, leading to nearly 30,000 deaths. The CDC has declared this organism an “urgent” threat to public health, the highest threat category. C. difficile infections are difficult to treat in large part because the organism forms dormant spores that survive antibiotic therapy and seed recolonization of the gut when antibiotics are withdrawn. This problem is exacerbated by the fact that the antibiotics used against C. difficile also kill many of the healthy gut bacteria, clearing the way for C. difficile to recolonize when spores germinate. Thus, there is a tremendous need for new drugs that target C. difficile without disrupting the healthy microbiota. The premise of this proposal is that a deeper understanding of cell envelope biogenesis can pave the way towards developing better ways to treat C. difficile infections. The cell envelope is a well-validated target for antibiotics, and in C. difficile the envelope has some unusual features that suggest its assembly requires novel proteins that could be exploited as targets of C. difficile-selective antibiotics. In Aim 1 we will use genetics, biochemistry and microscopy to understand the roles and regulation of enzymes that crosslink the peptidoglycan cell wall. These enzymes captured our attention because in C. difficile the cell wall contains an unusually high percentage of “3-3” crosslinks as compared to the “4-3” crosslinks that predominate in most bacteria. Our experiments will address the following questions: Which enzymes are responsible for 3-3 and 4-3 crosslink formation and do they operate during division, elongation or both? How is the ratio of 3-3 to 4-3 crosslinking regulated? How does C. difficile benefit from using primarily 3-3 crosslinks? In Aim 2 we will leverage a powerful new gene-silencing tool called CRISPR interference (CRISPRi) to assign a set of ~50 putatively essential envelope biogenesis genes to more specific functional pathways. These genes are intrinsically interesting and constitute potential new antibiotic targets. We will also undertake a detailed analysis of a novel transcriptional regulatory system uncovered in a pilot version of our proposed screen. Collectively, the lines of investigation to be pursued here will greatly advance our understanding of C. difficile biology by identifying new proteins involved in assembly of the cell envelope and revealing how their activities are coordinated to accomplish the complex processes of growth and division. PUBLIC HEALTH RELEVANCE: In bacteria the cell envelope protects against lysis and provides the first line of defense against noxious compounds. A better understanding of cell envelope biogenesis in C. difficile should provide new avenues for developing drugs to combat this devasting pathogen.
Li Wu, PhD, (PI), Wendy Maury, PhD, (Co-I), Stanley Perlman, MD, PhD, & Hillel Haim, MD, PhD, (Collaborators) NIH R21
Title: Epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells
Abstract: The COVID-19 pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in global health and economic crises. To develop effective vaccines and antivirals to prevent and control SARS-CoV-2 or other coronavirus infections, it is critical to understand the regulatory mechanisms of SARS-CoV-2 genome replication in host cells. In this R21 project, we will use novel epitranscriptomic technologies to investigate SARS-CoV-2 replication in human lung epithelial cells. N6-methyladenosine (m6A) modifications of many viruses play a major role in epitranscriptomic regulation of viral replication, gene expression, and immune evasion. However, it is unclear whether and how m6A modifications of the SARS-CoV-2 genome and cellular genes affect viral replication and pathogenicity. Our preliminary studies showed that SARS-CoV-2 infection of human lung epithelial cells upregulated m6A levels in cellular RNA. Our bioinformatic analysis of 13,699 full-length SARSCoV- 2 genome sequences predicts multiple m6A modification sites that are highly conserved among SARS-CoV-2 isolates worldwide. Thus, we hypothesize that m6A modifications of the SARS-CoV-2 genome and cellular genes enhance viral replication in cells through epitranscriptomic regulation. We propose two specific aims to test this hypothesis. Aim 1. To investigate the m6A epitranscriptomic profile of SARS-CoV-2-infected lung epithelial cells; Aim 2. To map m6A sites on the SARS-CoV-2 genome and to identify critical sites for viral replication. Defining epitranscriptomic m6A profile of SARS-CoV-2-infected cells has significant implications in understanding the COVID-19 pathogenesis and identifying novel drug targets.
Li Wu, PhD, (PI), Wendy Maury, PhD, Patrick Sinn, PhD, & Lilly Radoshevich, PhD, (Collaborators) New Carver Covid-19 Pilot Grant
Title: The role of SAMHD1 in regulating SARS-CoV-2-induced inflammation
