(78g) Engineering Molecular Escape Artists – Human Lysozyme Variants Evade Pathogen-Derived Inhibitory Proteins

Lysozymes are lytic hydrolases that break the b-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in bacterial peptidoglycan. These enzymes catalytically degrade bacterial cell walls, ultimately causing lysis and death. Lysozymes constitute a central element of innate immunity in the animal kingdom, and their role in protecting against bacterial pathogens has been experimentally confirmed in various animal models. In humans, the conventional or C-type lysozyme is broadly distributed throughout various tissues, secretions, and cell types, and therapeutic administration of recombinant human lysozyme represents an interesting possibility for drug-resistant infections. One barrier to such an approach is the fact that some pathogenic bacteria have evolved resistance mechanisms to this powerful antibacterial agent. Of particular importance to this talk is the recent discovery of proteinaceous lysozyme inhibitors among numerous Gram-negative bacteria. For example, Escherchia coli, Pseudomonas aeruginosa, Burkholderia cepacia and other pathogens produce a periplasmic protein termed “Inhibitor of Vertebrate Lysozyme”, or Ivy. In E. coli, Ivy represents a potent inhibitor of C-type lysozymes having low nanomolar affinity and aiding bacterial survival in biological fluids such as hen egg white and human saliva. Here, we describe our efforts to engineer variants of human lysozyme able to escape the E. coli Ivy inhibitor. We employed a molecular model to identify key lysozyme residues at the binding interface, and we subjected these sites to combinatorial mutagenesis creating millions of mutants. To screen the library, we co-encapsulated yeast expression hosts with bacterial target cells in 50 micron hydrogel droplets. These gel microdroplets were incubated in yeast induction media supplemented with high concentrations of the inhibitory Ivy protein. Hydrogel droplets encasing performance-enhanced yeast clones were selectively stained with a fluorogenic viability probe and then isolated by fluorescence activated cell sorting. Two engineered enzymes were ultimately found to retain high inherent activity while also demonstrating 10- to 100-fold reduced affinity for the E. coli Ivy inhibitor. Our results provide new and unexpected insights into molecular recognition between human lysozyme and pathogen-derived Ivy proteins, and they suggest that further molecular engineering could yield novel lysozymes with clinical utility.