Investigating the Link Between Fluoroquinolone-Induced Alterations in the Cell Envelope of Mycobacterium tuberculosis and Broad-Spectrum Drug Tolerance - ABSTRACT Tuberculosis (TB) is the leading cause of human mortality from a single infectious disease. Mycobacterium tuberculosis (M.tb), the etiologic agent of TB, can undergo rapid changes in response to anti-TB drugs to promote survival, even without resistance-conferring mutations (RCMs). This property, known as drug tolerance, is a well- studied characteristic of other bacterial pathogens, but the mechanisms employed by M.tb are poorly understood. Importantly, these mechanisms can reduce M.tb sensitivity to a broad spectrum of antimicrobials, making them attractive targets for the development of novel therapeutics, studying how drug resistance develops, and informing more effective stewardship practices. Despite this, little is known about the drug-specific tolerance mechanisms M.tb utilizes in response to second-line anti-TB drugs, including the fluoroquinolones (FQ), a class of potent antimicrobials which target DNA gyrase. Here, we include preliminary data that indicates exposure to FQ decreases M.tb cell envelope permeability and thus decreases sensitivity to numerous anti-TB drugs. Our current results are supported by studies in other prokaryotes showing that exposure to subinhibitory concentrations of FQ can significantly increase the frequency of developing RCMs to non-FQ antimicrobials, a phenomenon termed quinolone-induced antibiotic resistance (QIAR). Other studies have shown that bacterial pathogens with FQ RCMs in gyrA have global changes in DNA supercoiling, transcription, and decreased sensitivity to a broad spectrum of non-FQ antimicrobials. We hypothesize that exposure and/or resistance to FQ decreases drug susceptibility, increases drug tolerance, and promotes the acquisition of RCMs to non-FQ anti- TB drugs due to upregulation of cell envelope peripheral lipids which decrease cell envelope permeability. To prove our hypothesis, we propose: Aim 1. Validate the effects of FQ exposure on drug tolerance and the acquisition of RCMs to non-FQ anti-TB drugs in vitro. Aim 2. Define the mechanism(s) underlying FQ-induced cell surface remodeling in M.tb focusing on, but not limited to: alterations in DNA supercoiling, transcription, and the effect on cell envelope permeability. At the end of these proposed studies, we will characterize the DNA supercoiling topology of the M.tb chromosome and its potential role in a novel antimicrobial stress response pathway. Additionally, we will elucidate the effects of FQ exposure and resistance on cell envelope composition, permeability, and the development of RCMs to non-FQ antimicrobials via broad spectrum drug tolerance. The research proposed here will be conducted at The Ohio State University Wexner Medical Center, a leading academic medical institution dedicated to advancing patient care, education, and biomedical research. The training plan detailed in this proposal will allow the applicant to accomplish 3 primary goals: i) Learn new skills and develop a deeper understanding of TB biology, drug resistance, and microbial genetics; ii) Conduct research in the lab’s area of expertise while also allowing for the applicant to investigate new subject areas; and iii) Strengthen professional skills like scientific writing for grants and manuscripts, oral presentation, and mentorship.
