Engineering a Lysin with Intrinsic Antibacterial Activity (LysMK34) by Cecropin A Fusion Enhances Its Antibacterial Properties against Acinetobacter baumannii

Bacteriophage-encoded lysins are increasingly reported as alternatives to combat Acinetobacter baumannii infections, for which limited therapeutic options are available. Some lysins, such as LysMK34, have a C-terminal amphipathic helix allowing them to penetrate the otherwise-impermeable outer membrane barrier. Another approach to kill Gram-negative pathogens with lysins relies on fusion of a peptide with outer membrane- permeabilizing properties to the lysin. In this work, we aimed to leverage the intrinsic antibacterial activity of LysMK34 by fusing the peptide cecropin A to its N terminus via a linker of three Ala-Gly repeats, resulting in engineered LysMK34 (eLysMK34). The engineered lysin has an improved antibacterial activity compared to that of the parental lysin, LysMK34, in terms of MICs (0.45 to 1.2 μM), killing rate, and killing extent. eLysMK34 has a ≥2-foldincreased activity against stationary-phase cells, and the bactericidal effect becomes less dependent on the intracellular osmotic pressure. In particular, colistin-resistant strains become highly susceptible to eLysMK34, and enhanced antibacterial activity is observed in complement- deactivated human serum. These observations demonstrate that fusion of a lysin with intrinsic antibacterial activity with a selected outer membrane-permeabilizing peptide is a useful strategy to further improve the in vitro antibacterial properties of such lysins.

K.A. was funded by the Missions Sector, Ministry of Higher Education, Egypt. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. D.G. is supported by the Research Foundation-Flanders (FWO) under grant G066919N. Y.B. is a coinventor of patents related to the Artilysin field. We thank S. Malhotra-Kumar (Laboratory of Medical Microbiology, Vaccine & Infectious Disease, University of Antwerp, Antwerp, Belgium) for providing the colistin-resistant A. baumannii strains (Greek46 and Greek47) and J. P. Pirnay (Laboratory for Molecular and Cellular Technology, Queen Astrid Hospital, Neder-over-Heembeek, Belgium) for providing A. baumannii RUH134 and P. aeruginosa PA14 and Br667.