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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
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Genetically enhanced lysozyme evades a pathogen derived inhibitory protein.

Sarah M Dostal1, Yongliang Fang1, Jonathan C Guerrette1

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Researchers engineered human lysozyme (hLYZ) to resist bacterial inhibitors like E. coli Ivy. This novel approach enhances antibacterial enzyme efficacy against drug-resistant bacteria.

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Area of Science:

  • Biochemistry
  • Microbiology
  • Drug Discovery

Background:

  • Rising antibiotic resistance necessitates new therapeutic strategies.
  • Bactericidal enzymes like human lysozyme (hLYZ) offer a resistance-proof alternative to traditional antibiotics.
  • Pathogen-derived inhibitors, such as E. coli Ivy, can neutralize natural antibacterial enzymes like hLYZ.

Purpose of the Study:

  • To engineer human lysozyme (hLYZ) variants capable of evading inhibition by E. coli Ivy.
  • To assess the retained catalytic activity and antibacterial efficacy of engineered hLYZ variants.
  • To investigate the cross-reactivity of engineered hLYZ variants against other proteinaceous inhibitors.

Main Methods:

  • Construction and screening of large combinatorial libraries of hLYZ variants.
  • Utilized a novel microbial coculture screening platform in hydrogel microdroplets.
  • Assessed susceptibility to E. coli Ivy, catalytic proficiency, and antibacterial activity of isolated variants.

Main Results:

  • Engineered hLYZ variants demonstrated significantly reduced susceptibility to E. coli Ivy.
  • Variants maintained high catalytic proficiency and inherent antibacterial activity.
  • Engineered variants exhibited increased susceptibility to Pseudomonas aeruginosa Ivy and E. coli MliC.

Conclusions:

  • Successful engineering of hLYZ to evade a specific bacterial inhibitor (E. coli Ivy) was achieved.
  • Broad-spectrum evasion of diverse proteinaceous inhibitors requires addressing complex molecular recognition determinants.
  • Further research is needed to develop hLYZ variants with resistance to a wider range of bacterial virulence factors.