Leveraging metabolic labeling strategies to study mycobacterial glycolipids
Keywords: Chemistry.

Tuberculosis (TB) is a communicable disease spread through the air by the bacillus Mycobacterium tuberculosis (Mtb). TB is a major global health burden and was the leading cause of death from a single infectious agent prior to the coronavirus (COVID-19) pandemic, ranking above HIV/AIDS. Although curable, TB is notoriously difficult to treat due to the very hydrophobic and highly impermeable Mtb cell wall. This mycomembrane is made up of a dense network of lipids and glycolipids that modulate mycobacterial pathogenesis and host immune response. In this work, we describe how bioorthogonal labeling with glycolipid metabolic precursor probes can be harnessed to understand new biological and therapeutic insights. In Chapter 1, we review previous work characterizing the biosynthetic pathways responsible for the construction and maintenance of the mycomembrane. Additionally, we highlight the role mycobacterial glycolipids play in altering the typical immune response to infection and the dynamics of these host-pathogen interactions across space and time. To understand this work more deeply, we describe the recent advantages in bioorthogonal labeling leveraging metabolic pathways that incorporate fluorescent reporters and/or affinity tags to track the dynamics of mycobacterial glycolipids during host infection. In Chapter 2, we study trehalose monomycolate (TMM) during early stages of macrophage infection. These glycolipids are among the most abundant within the mycomembrane and serve an essential role in bacterial survival and TB pathology. We use a previously identified metabolic engineering strategy using 6-azido-6-deoxy-trehalose (6-TreAz) to modify trehalose monomycolate (TMM) with azide functionalization and parse out the role of TMM signaling during macrophage infection. We observe for the first time that TMM spreads to host membranes after phagocytosis, so we develop a series of cross-linking trifunctional probes to identify novel interacting partners between mycobacterial glycolipid and host proteins. This work demonstrates that TMM is not simply an important part of the structural integrity of the mycomembrane, but also has its own role in promoting pathogenesis. In Chapter 3, we turn to another important mycomembrane component, phenolic glycolipid (PGL), which has been understudied despite its prevalence in pathogenic strains of Mtb in part due to the complex biosynthesis where traditional genetic manipulation is untenable. We synthesize and characterize an azide-functionalized PGL metabolic precursor, 3-azido-4-hydroxybenzoic acid (3-azido 4HB). We demonstrate that the metabolic incorporation of 3-azido 4HB in Mycobacterium marinum relies on key PGL biosynthetic machinery and does not alter its growth or infection capabilities, showing its potential to examine PGL dynamics in future studies. In Chapter 4, we repurpose a solvatochromic diagnostic tool recently developed by our lab into a new assay that rapidly screens hundreds of putative antibiotic compounds that target mycomembrane biosynthesis. The environment-sensitive probe, called DMN-Tre, utilizes the Antigen 85 complex (Ag85) to identify metabolically active, live mycobacteria and is inhibited by ebselen. We test a library of ebselen derivatives for antimicrobial potential, which is a crucial step to combat the rising threat of antibiotic resistance strains of Mtb. Chapter 5 concludes by contextualizing this work in the real-world landscape surrounding TB health policy and research. There is a concerted effort by global agencies and philanthropic organizations to fund research toward diagnostic, antibiotic, and vaccine development. The COVID-19 pandemic has reversed several years in recent progress, so it is important that research in these areas continue to innovate to reach international goals of eradicating TB. Work like ours illuminates new avenues for therapeutic and diagnostic efforts targeting the interactions between pathogenic glycolipids and host factors during the lifecycle of infection