Merging bacteriophage biology with microbial glycobiology

PROJECT SUMMARY Bacteriophages, or phages, are considered the most abundant organisms on Earth with an estimated 10+31 particles in the biosphere. These viruses are in a perpetual arms race with bacteria and arguably the major force driving bacterial evolution. Similarly, phages continue to develop novel mechanisms to infect their host and avoid detection, and carbohydrates are central to all these processes. As a microbial glycobiologist, my research originally focused on understanding the mechanisms behind bacterial glycoconjugate biosynthesis where we discovered that bacteria are capable of N-glycosylating proteins and campylobacters synthesize capsules rather than repetitive lipopolysaccharides. These studies evolved into better understanding the selective pressures that promote selection for glycoconjugate variants and discovering that a single strain of C. jejuni can express over 1000 different capsule structures and many of these variations can be observed when combining the strain with different phages. Our curiosity has now expanded toward understanding the importance of other glycostructures involved in phage-host interactions beyond C. jejuni and beyond surface polysaccharides. Using the knowledge we gained in studying microbial glycoconjugate synthesis and phage-host interactions over the last two decades, current studies will focus on exciting new discoveries describing other phage glycosylated macromolecules. 1) We are eager to explore the two mechanisms used by C. jejuni phages to replace canonical DNA bases with non-canonical bases with 100% efficiency. This will involve investigating the activity of a new deoxyribosyltranferase and how it couples to a transglycosyltransferase that efficiently removes nucleobases post-replicatively. CryoEM structures of this transglycosyltranferase in comparison with less efficient enzymes will not only help us to understand the transfer mechanism, but can also lead to the development of new biotechnological tools for nucleic acid biosynthesis and vaccine development. 2) Recently a set of glycosyltransferases (GTs) responsible for glycan addition to capsids and tail tubes of phages infecting Mycobacterium species were identified, but these modifications showed no benefit to phage infectivity. We hypothesize that these GTs could also modify host proteins, including phage receptors that subsequently prevent phage superinfection. We propose to use our methods in recombinant bacterial glycoengineering as well as GT expression in the native host to better understand the impact of phage GT expression on the microbe as well as expand the toolbox of novel characterized enzymes available for therapeutic development. 3) All cystoviruses isolated to date infect Pseudomonas species, but we recently isolated an enveloped virus infecting a multidrug resistant strain of Acinetobacter radioresistens. These viruses enclose themselves with bacterial inner membranes that also contain glycoconjugate biosynthetic machinery. We aim to verify their composition and better understand their biogenesis for possible use as maleable self-assembling nanoparticles.