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 towards 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 co-culture 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.

Stabilization of a Virus-Like Particle and Its Application as a Nanoreactor at Physiological Conditions

“…Additionally, lysozyme-specific inhibitors exist, which also limits the clinical efficacy of lysozyme. So far, only genetic engineering has been used to address these problems. − In some cases, the engineered variants did indeed show improved properties. Changing the lysozyme genetic sequence, however, has also led to the introduction of new unexpected and unfavorable properties.…”

Engineering of Therapeutic and Detoxifying Enzymes

“…Lysozyme, which performs the β(1,4) glycosidic bond cleavage of the bacterial cell‐wall peptidoglycan, is prone to inhibition by the Escherichia coli ( E. coli) inhibitor of vertebrate lysozyme (Ivyc) and its homologues. To reduce the susceptibility of human lysozyme (hLYZ) to Ivyc, the Griswold lab identified the conserved key residues of hLYZ at the Ivyc‐hLYZ interface, subjected them to combinatorial mutagenesis and screened the libraries using a gel microdroplet‐fluorescence activated cell sorting assay (GMD‐FACS) [33] . The variants were catalytically active and demonstrated decreased inhibition from to E. coli Ivy, however, they were more prone to inhibition from the Pseudomonas aeruginosa Ivy and MliC, another E. coli inhibitory protein.…”

Lysozyme and Its Application as Antibacterial Agent in Food Industry

“…Taking Ivy Ec (PDB 1XS0), for example, a rigid loop protrudes into the HEWL active-site cleft in a key-lock type of interaction, and the central histidine in this loop connects with the two catalytic residues (D52 and E35) via hydrogen bonds. Based on the deciphered interaction mechanism and critical residues, it has been demonstrated possible to genetically engineer lysozymes to evade pathogen-derived inhibitory proteins via gene mutagenesis and an innovative ultrahigh-throughput screening platform [ 88 ].…”