Chemical and Cellular Defenses against Foreign Pathogens

Abstract

Bacterial and viral infections affect billions of people each year. While bacterial infections are treated by the use of antibiotics, viral infections are eradicated by the immune system. This thesis comprises two parts. Part I presents the reconstitution of enzymes required for the synthesis of the minimal pharmacophore of moenomycin A (MmA), a molecule with antibacterial activity. Part II details single-particle electron microscopy studies of MDA5 alone and in complex with double-stranded RNA (dsRNA). MmA is a natural product antibiotic from Streptomyces ghanaensis that possesses remarkable potency against clinically relevant Gram-positive bacteria. MmA exerts its antibacterial activity by binding directly to peptidoglycan glycosyltransferases, enzymes involved in the synthesis of the glycan strands of peptidoglycan. The genes responsible for MmA biosynthesis have been identified, and a complete biosynthetic pathway has been proposed. Part I of this thesis describes the reconstitution of enzymes required for the synthesis of two trisaccharide scaffolds of MmA that retain antibacterial activity. It also describes the synthesis of unnatural phosphoglycerate lipid acceptors and UDP-amino sugars that can be used to probe the substrate tolerances of key glycosyltransferases in MmA biosynthesis. This work lays the foundation for the synthesis of unnatural MmA analogs that may possess better pharmacokinetic properties than the parent molecule. MDA5 is a helicase that detects viral dsRNA in the cytoplasm of vertebrate cells. Upon dsRNA recognition, MDA5 initiates a series of signal transduction events that activate the innate immune response. Part II of this thesis presents the structures of apo MDA5 protein and the MDA5-dsRNA complex obtained by using single-particle electron microscopy. Two-dimensional averages of apo MDA5 revealed that the protein is very flexible and can adopt multiple conformations. This finding suggests that MDA5 does not adopt an autorepressed conformation in the absence of viral dsRNA. When MDA5 is incubated with dsRNA, the protein assembles onto the dsRNA to form a linear oligomer. Two-dimensional averages and a three-dimensional reconstruction reveal the complex to consist of seven to eight stacked discs per strand of 112 base pair dsRNA. This work lays the foundation for further structural studies aimed at elucidating the mechanism by which MDA5 is activated.