Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein

The accelerating spread of drug-resistant
bacteria is creating
demand for novel antibiotics. Bactericidal enzymes, such as human
lysozyme (hLYZ), are interesting drug candidates due to their inherent
catalytic nature and lack of susceptibility to the resistance mechanisms
typically directed toward chemotherapeutics. However, natural antibacterial
enzymes have their own limitations. For example, hLYZ is susceptible
to pathogen derived inhibitory proteins, such as Escherichia
coli Ivy. Here, we describe proof of concept studies demonstrating
that hLYZ can be effectively redesigned to evade this potent lysozyme
inhibitor. Large combinatorial libraries of hLYZ were analyzed using
an innovative screening platform based on microbial coculture in hydrogel
microdroplets. Isolated hLYZ variants were orders of magnitude less
susceptible to E. coli Ivy yet retained high catalytic
proficiency and inherent antibacterial activity. Interestingly, the
engineered escape variants showed a disadvantageous increase in susceptibility
to the related Ivy ortholog from Pseudomonas aeruginosa as well as an unrelated E. coli inhibitory protein,
MliC. Thus, while we have achieved our original objective with respect
to escaping E. coli Ivy, engineering hLYZ for broad-spectrum
evasion of proteinaceous inhibitors will require consideration of
the complex and varied determinants that underlie molecular recognition
by these emerging virulence factors.