Exploiting phage receptor binding proteins to enable endolysins to kill Gram-negative bacteria

Area of Science:

• Microbiology
• Biotechnology
• Drug Discovery

Background:

• Bacteriophage endolysins show promise as antibacterials against resistant bacteria.
• The outer membrane of Gram-negative bacteria hinders endolysin access to peptidoglycan, limiting their efficacy.
• Engineering strategies are needed to enable endolysins to target Gram-negative pathogens.

Purpose of the Study:

• To introduce and validate the Innolysin concept for engineering endolysins against Gram-negative bacteria.
• To develop novel Innolysins by fusing endolysins with phage receptor binding proteins (RBPs).
• To assess the antibacterial activity of Innolysins against Gram-negative bacteria, including antibiotic-resistant strains.

Main Methods:

• Constructed Innolysins by fusing phage T5 endolysin with RBP Pb5 in various configurations.
• Evaluated the antibacterial activity of Innolysins against Escherichia coli and other Gram-negative bacteria.
• Performed high-throughput screening to identify optimal Innolysin candidates.
• Tested Innolysin efficacy against third-generation cephalosporin-resistant E. coli.

Main Results:

• Innolysin Ec6 demonstrated antibacterial activity against E. coli dependent on the FhuA receptor.
• Innolysin Ec6 showed efficacy against Shigella sonnei and Pseudomonas aeruginosa strains possessing FhuA homologs.
• High-throughput screening identified Innolysin Ec21 as a potent candidate, achieving significant E. coli cell count reduction.
• Innolysin Ec21 exhibited significant bactericidal activity against antibiotic-resistant E. coli.

Conclusions:

• The Innolysin strategy effectively engineers endolysins for targeting Gram-negative bacteria.
• Combining endolysins with RBPs provides a versatile platform for developing novel antibacterial agents.
• Innolysins represent a promising advancement in combating antibiotic-resistant Gram-negative infections.