Investigating the Metabolic Role of the Redox Cofactor F420 in Mycobacterium tuberculosis Hypoxic Persistence

/ ABSTRACT Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), the leading cause of death by a pathogen worldwide, despite being a treatable and often curable disease. Mtb infection in the lungs triggers the recruitment of immune cells to seclude the bacterium and infected macrophages from the rest of the body, resulting in the formation of a granuloma. Biochemical stressors within the granuloma, such as hypoxia, can cause Mtb cells to enter a non-replicating persistent (NRP) state which is thought to play an important role in immune evasion and drug tolerance. As a result, understanding how Mtb supports its metabolism under hypoxic conditions could reveal novel druggable targets for NRP Mtb. This proposal focuses on the redox cofactor F420, which is involved in the activation of nitroimidazole prodrugs, including pretomanid. Due to its low redox potential (E0’ – 340 mV) compared to that of flavins (E0’ – 220 mV) and nicotinamides (E0’ – 320 mV), it has been suggested to play an important role in survival under hypoxia. It has been shown that mycobacterial strains with disrupted F420 biosynthesis are more susceptible to host relevant stresses such as reactive oxygen species. In addition, all mycobacterial species produce F420 and Mtb is predicted to contain 28 putative F420-dependent proteins. Its role in Mtb physiology, however, is not yet understood. First, we are interested in identifying metabolic pathways that are affected by the loss of F420. This could identify 1) F420-depdendent pathways, which will provide insight into the effects of inhibiting F420 and 2) compensatory pathways that could serve as targets for clinical isolates that develop mutations in F420 biosynthesis pathway (and are subsequently resistant to drugs like pretomanid). In AIM 1, we will examine how knocking out two genes in the F420 biosynthesis pathway affects the metabolomic and transcriptomic profiles of Mtb. We will use these data to determine key pathways that are needed for survival under hypoxic conditions and perform follow-up survival experiments with related knockdown mutants. Second, we are interested in gauging the potential of F420 as a drug target within granulomas. Specifically, we want to determine how important F420 is for Mtb in hypoxic lesions. In AIM 2, we will use C3HeB/FeJ mice that form granuloma-like hypoxic lesions to gauge the extent to which F420 is needed. Disease readouts will include mouse survival, organ bacterial burden, and organ pathology to assess the in vivo effects of knocking out F420. Project Number: 1F30AI194700-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Alexander Chong | Institution: ALBERT EINSTEIN COLLEGE OF MEDICINE, BRONX, NY | Award Amount: $54,538 | Activity Code: F30 | Study Section: Special Emphasis Panel[ZRG1 F07A-W (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F30AI19470001