Development of Chimera AMP-Endolysin with Wider Spectra Against Gram-Negative Bacteria Using High-Throughput Assay.

Bacteriophage-derived endolysins are being developed as an alternative to antimicrobials. The development of endolysins against Gram-negative bacteria requires the discovery of effective endolysins against the target species and the capability to penetrate the outer membrane of bacteria by endolysin. Here, we propose an efficient endolysin development approach that combines a data-driven endolysin search utilizing bacterial genomes with high-throughput laboratory assays. As a proof of concept, we analyzed endolysin genes detected in 273 bacterial genomes of , , and . Firstly, we conducted assays of 192 recombinants of endolysin genes obtained through in silico search from bacterial genomes and identified natural endolysins degrading peptidoglycan of . Then, we performed high-throughput screening against Gram-negative bacteria for hundreds of chimera AMP-endolysins, natural endolysin conjugated with antimicrobial peptide. As a result, we obtained four chimera AMP-endolysins against A. baumannii, which demonstrated the minimum inhibitory concentration ranging from 4 to 8 μg/mL. Moreover, we assessed the antimicrobial spectra of these chimera AMP-endolysins, validating that two endolysins exhibited antimicrobial efficacy against and with <32 μg/mL of concentration. This endolysin development approach can be applied to other Gram-negative bacterial targets and is expected to facilitate the acquisition of effective novel endolysins.