Chemical Biology Approaches to Dissect Microbial Pathogenesis

This talk gives an overview of the current projects in the Lupoli group, which seek to answer questions that fall under two main categories related to microbial pathogenesis: (1) regulation of bacterial chaperones; (2) assembly of bacterial surface glycans. The initial project focuses on the “skin”, or surface, of bacteria called the cell envelope, which mediates infection of the host and protects bacteria from host immune defense tactics. While Gram-negative bacteria contain a protective outer membrane layer absent in most Gram-positives, almost all bacteria contain polymers composed of unique monosaccharides that extend from the cell surface. Gram-negative bacteria typically contain lipopolysaccharide (LPS) in the outer leaflet of the outer membrane with attached polysaccharides called O-antigens that help mediate interactions with the environment. O-antigens are composed of repeating oligosaccharides that define particular bacterial serotypes, which distinguishes bacterial strains within a single species. Foundational chemical biology work has contributed to our understanding of eukaryotic cell surface composition. However, we still lack a clear understanding of assembly of bacterial surface glycan polymers that contain prokaryote-specific or “rare” sugars. In this talk, we describe synthetic and chemoenzymatic methods to construct rare nucleotide sugars to study substrate recognition by bacterial glycosyltransferases that build O-antigens. We identify key regions in sugar substrates that are required for substrate binding and activity, and we use this knowledge to design chemical probes that will be used for the construction of synthetic O-antigens and small molecule inhibitors that will stall O-antigen synthesis. This work will expand our understanding of cellular mechanisms underlying bacterial polysaccharide synthesis, and will teach us about the roles that rare sugars play in bacterial cellular interactions.

The latter project focuses on DnaK, the bacterial homolog of Hsp70, an ATP-dependent chaperone that functions in concert with cofactor proteins to catalyze nascent protein folding and salvage misfolded proteins. In the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), DnaK and its cofactor proteins DnaJ1, DnaJ2 and GrpE are proposed drug targets. Despite the importance of chaperone function in human cancers and infectious disease, there are limited chemical probes or inhibitors that enable in vivo studies on the function of individual chaperones/cofactors. In this talk, we describe the discovery of small molecule inhibitors of mycobacterial DnaK, most notably a peptidomimetic called telaprevir, which is able to inhibit chaperone function through interactions with the peptide-binding cleft of DnaK. Binding of telaprevir leads to allosteric conformational changes that prevent ATP hydrolysis in a distal domain. We find that telaprevir also inhibits E. coli DnaK and human Hsc70 chaperones due to high conservation of Hsp70 sequences. Using in vitro and in vivo chaperone assays, we demonstrate that telaprevir modulates the function of mycobacterial DnaK and its cofactor protein DnaJ2 in cells, which disrupts cellular proteostasis. Co-treatment of mycobacteria with telaprevir and aminoglycosides, which further stress the proteome, enhances the potency of these antibiotics. In addition, telaprevir combats mycobacterial resistance to the frontline TB drug rifampin, as DnaK-DnaJ2 function is required for stabilization of protein mutants that confer drug tolerance. This work sets the stage for our current work on the design of peptide-based inhibitors with higher selectivity for bacterial chaperones to probe chaperone-protein interactions and explore resulting synergy with different classes of antibiotics.