title: Genetically enhanced lysozyme evades a pathogen derived inhibitory protein.
abstract: 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.
authors.affiliation: †Thayer School of Engineering at Dartmouth College, 14 Engineering Drive, Hanover, New Hampshire 03755, United States.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Dostal
authors.affiliation: †Thayer School of Engineering at Dartmouth College, 14 Engineering Drive, Hanover, New Hampshire 03755, United States.
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Fang
authors.affiliation: †Thayer School of Engineering at Dartmouth College, 14 Engineering Drive, Hanover, New Hampshire 03755, United States.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Guerrette
authors.affiliation: †Thayer School of Engineering at Dartmouth College, 14 Engineering Drive, Hanover, New Hampshire 03755, United States.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Scanlon
authors.affiliation: †Thayer School of Engineering at Dartmouth College, 14 Engineering Drive, Hanover, New Hampshire 03755, United States.|‡Program in Molecular and Cellular Biology, Dartmouth College, Hanover, New Hampshire 03755, United States.
authors.forename: Karl E
authors.initials: KE
authors.lastname: Griswold
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009113:Muramidase / Q000235:genetics* / Q000378:metabolism*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C429555:Ivy protein, E coli; C515605:MliC protein, E coli; D019151:Peptide Library; D011994:Recombinant Proteins; D009113:Muramidase