Immune Targeting of Mycobacteria through Cell Surface Glycan Engineering.

Mycobacteria and other organisms in the order Mycobacteriales cause a range of significant human diseases, including tuberculosis, leprosy, diphtheria, Buruli ulcer, and non-tuberculous mycobacterial (NTM) disease. However, the intrinsic drug tolerance engendered by the mycobacterial cell envelope undermines conventional antibiotic treatment and contributes to acquired drug resistance. Motivated by the need to augment antibiotics with novel therapeutic approaches, we developed a strategy to specifically decorate mycobacterial cell surface glycans with antibody-recruiting molecules (ARMs), which flag bacteria for binding to human-endogenous antibodies that enhance macrophage effector functions.

A Bifunctional Chemical Reporter for in Situ Analysis of Cell Envelope Glycan Recycling in Mycobacteria.

In mycobacteria, the glucose-based disaccharide trehalose cycles between the cytoplasm, where it is a stress protectant and carbon source, and the cell envelope, where it is released as a byproduct of outer mycomembrane glycan biosynthesis and turnover. Trehalose recycling via the LpqY-SugABC transporter promotes virulence, antibiotic recalcitrance, and efficient adaptation to nutrient deprivation. The source(s) of trehalose and the regulation of recycling under these and other stressors are unclear.

Chemical Reporters for Bacterial Glycans: Development and Applications.

Bacteria possess an extraordinary repertoire of cell envelope glycans that have critical physiological functions. Pathogenic bacteria have glycans that are essential for growth and virulence but are absent from humans, making them high-priority targets for antibiotic, vaccine, and diagnostic development. The advent of metabolic labeling with bioorthogonal chemical reporters and small-molecule fluorescent reporters has enabled the investigation and targeting of specific bacterial glycans in their native environments.

Photoactivatable Glycolipid Probes for Identifying Mycolate-Protein Interactions in Live Mycobacteria.

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins.

A FRET-Based Fluorogenic Trehalose Dimycolate Analogue for Probing Mycomembrane-Remodeling Enzymes of Mycobacteria.

The mycobacterial outer membrane, or mycomembrane, is essential for the viability and virulence of and related pathogens. The mycomembrane is a dynamic structure, whose chemical composition and biophysical properties can change during stress to give an advantage to the bacterium. However, the mechanisms that govern mycomembrane remodeling and their significance to mycobacterial pathogenesis are still not well characterized.

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues.

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne-modified TMM analogue (O-AlkTMM-C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane.

Chemoenzymatic radiosynthesis of 2-deoxy-2-[F]fluoro-d-trehalose ([F]-2-FDTre): A PET radioprobe for in vivo tracing of trehalose metabolism.

Trehalose analogues bearing fluorescent and click chemistry tags have been developed as probes of bacterial trehalose metabolism, but these tools have limitations with respect to in vivo imaging applications. Here, we report the radiosynthesis of the F-modified trehalose analogue 2-deoxy-2-[F]fluoro-d-trehalose ([F]-2-FDTre), which in principle can be used in conjunction with positron emission tomography (PET) imaging to allow in vivo imaging of trehalose metabolism in various contexts. A chemoenzymatic method employing the thermophilic TreT enzyme from Thermoproteus tenax was used to rapidly (15-20 min), efficiently (70% radiochemical yield; ≥ 95% radiochemical purity), and reproducibly convert the commercially available radiotracer 2-deoxy-2-[F]fluoro-d-glucose ([F]-2-FDG) into the target radioprobe [F]-2-FDTre in a single step; both manual and automated syntheses were performed with similar results.

Delivery of foreign cytotoxic T lymphocyte epitopes to tumor tissues for effective antitumor immunotherapy against pre-established solid tumors in mice.

Cytotoxic T lymphocyte (CTL) can have remarkable abilities to kill tumor cells. However, the establishment of successful CTL-based anticancer therapy has met with many challenges. Within tumor cells, there exist subpopulations with low or no expression of the targeted antigen (termed as antigen-loss variants).

A chemical reporter strategy for detecting and identifying O-mycoloylated proteins in Corynebacterium.

Protein O-mycoloylation is a unique post-translational lipidation that was recently discovered in Corynebacterium. We describe an alkyne-modified trehalose monomycolate chemical reporter that can metabolically tag O-mycoloylated proteins in C. glutamicum, enabling their detection and identification through click chemistry.

Identification of Lectins from Metastatic Cancer Cells through Magnetic Glyconanoparticles.

Cancer cells can have characteristic carbohydrate binding properties. Previously, it was shown that a highly metastatic melanoma cell line B16F10 bound to galacto-side-functionalized nanoparticles much stronger than the corresponding less metastatic B16F1 cells. To better understand the carbohydrate binding properties of cancer cells, herein, we report the isolation and characterization of endogenous galactose binding proteins from B16F10 cells using magnetic glyconanoparticles.

Bioorthogonal Chemical Reporters for Selective In Situ Probing of Mycomembrane Components in Mycobacteria.

The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development.