The riboflavin biosynthetic pathway and the MHC class I related protein 1 (MR1) ligandome of mycobacteria

Abstract

Tuberculosis (TB) serves as the major cause of mortality due to an infectious disease even after over 100 years of identifying Mycobacteria tuberculosis (Mtb), the causative organism. We have been in a continuous arms race with Mtb on several fronts: the bacilli has developed resistance towards most if not all the antimycobacterial agents available; the only approved vaccine for TB, BCG, confers limited protection and does not prevent transmission; the diagnosis of the different clinical and subclinical stages of TB has proven to be difficult due to the lack of robust biomarkers. To overcome these challenges, novel strategies and approaches are needed in developing pharmacotherapeutics, immunotherapeutic and vaccines. To develop these novel tools, a comprehensive understanding of the essential physiological functions of Mtb, its interaction with the host immune system, and its molecular components that may serve as biomarkers are needed. The riboflavin biosynthetic pathway (RBP) in Mtb serves as means of tackling two of these challenges. The RBP is an essential pathway as it is required to provide cofactors, FMN and FAD required for a litany of enzymatic reactions in Mtb. Also, the RBP serves as the source of antigens recognized by MHC Class I related protein 1 (MR1) restricted T-cells (MR1T cells); a subset of T cells that recognize metabolites presented by the non-polymorphic MR1 molecule. The research in this dissertation explores the essentiality of the RBP in Mtb, how the RBP (enzymes and metabolic intermediates) contribute to the production of MR1 antigens and strategies to identify MR1 antigens from Mtb. This dissertation also explores mechanisms through which mycobacteria ensures flavin homeostasis and the impact of flavin induced changes to the global proteome of mycobacteria. In Chapter 3, we investigate the role of the RBP in the production of MR1 antigens. Using knockout and complemented mutants of the RBP in Mycolicibacterium smegmatis (Msm) and Mtb, in combination with a multiomics approach, we determined the impact of the stepwise loss of the enzymes in the RBP on MR1 antigenicity. We uncover key differences in the RBP of Msm and Mtb as well as the centrality of 5-A-RU to the production of MR1 antigens in both organisms. In Chapter 4, we investigate the impact of elevated intracellular riboflavin on the proteome of Msm and Mtb. We initially observed that elevated intracellular riboflavin did not translate into elevated levels of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in mycobacteria. This finding clearly indicated the presence of a mechanism that controls the level of available FMN and FAD. We then show that elevated riboflavin led to an increased abundance of known flavin sequesters and the induction of the DosR regulon. We also showed distinctions and similarities in the response to high riboflavin between Msm and Mtb especially the impact of elevated riboflavin on isoniazid inducible genes suggesting a potential interaction between intracellular riboflavin levels and INH susceptibility in Mtb. Finally, Chapter 5 details mass spectrometry and purification science approaches to identify and isolate MR1 antigens from Mtb. Through this work, we determined the absence of known MR1 antigens produced by Msm, Photolumazine I and III, in Mtb. Using a 5-A-RU null background, the ribA2 mutant of Mtb, we identified additional molecules that are potential MR1 antigen candidates from Mtb. Finally, we were able to develop a high-pressure liquid chromatography (HPLC) method to enrich for MR1 antigens from Mtb, revealing a diverse array of MR1 antigen species. Together, this dissertation contributes to our understanding of the RBP of Mtb. We provide useful tools to better understand MR1-mediated immune response and flavin homeostasis in Mtb. The result presented in this dissertation provides a path for further studies to better understand the RBP with the goal of developing novel strategies to control the global health burden imposed by Mtb.