In vitro and in vivo efficacy studies of an engineered endolysin targeting Gram-negative pathogens

Area of Science:

• Microbiology
• Biotechnology
• Infectious Diseases

Background:

• Endolysins are promising antibacterial agents but face challenges targeting Gram-negative bacteria due to their outer membrane.
• Bacteriophage-derived endolysins offer a potential alternative to conventional antibiotics.

Purpose of the Study:

• To engineer an endolysin effective against Gram-negative pathogens.
• To evaluate the antibacterial spectrum, mechanism of action, and resistance potential of the engineered endolysin.
• To assess the in vivo efficacy and synergistic potential of the engineered endolysin.

Main Methods:

• Heterologous expression and engineering of a Pseudomonas aeruginosa bacteriophage endolysin (PBPA90).
• Modification via amino acid substitution and cecropin A fusion to create LNT103.
• Determination of minimum inhibitory concentrations (MICs) against various Gram-negative bacteria.
• Assessment of membrane permeability, synergistic effects with antibiotics, in vitro resistance development, and in vivo efficacy in mouse bacteremia models.

Main Results:

• The engineered endolysin LNT103 exhibited potent activity against P. aeruginosa and other Gram-negative pathogens (A. baumannii, E. coli, K. pneumoniae, K. aerogenes, E. cloacae) with MICs as low as 4 μg/ml.
• LNT103 permeabilized both outer and inner bacterial membranes.
• It showed synergistic effects with colistin and additive effects with carbapenems.
• Minimal to no in vitro resistance developed against LNT103.
• LNT103 demonstrated in vivo efficacy in a mouse model of A. baumannii bacteremia and resensitized resistant bacteria to meropenem.

Conclusions:

• Engineered endolysins like LNT103 represent a viable strategy to combat Gram-negative bacterial infections.
• LNT103 effectively breaches bacterial membranes and exhibits broad-spectrum activity with low resistance potential.
• Combination therapy with LNT103 shows promise for overcoming antibiotic resistance in vivo.