Engineering a lysin with intrinsic antibacterial activity (LysMK34) with cecropin A 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 eLysMK34. The engineered lysin has an improved antibacterial activity compared to the parental lysin LysMK34 in terms of minimum inhibitory concentration (0.45 - 1.2 μM), killing rate and killing extent. eLysMK34 has an at least two-fold increased activity against stationary-phase cells and the bactericidal effect becomes less dependent on the intracellular osmotic pressure. Particularly 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. Importance Phage lysins are a new class of enzyme-based antibiotics that increasingly gain interest. Lysins kill cells through rapid degradation of the peptidoglycan layer, resulting in sudden osmotic lysis. Whereas Gram-positive bacteria are readily susceptible to the action of lysins, Gram-negative bacteria are naturally resistant as the outer membrane protects their peptidoglycan layer. This work reveals that fusing an outer membrane permeabilizing peptide to a lysin with intrinsic antibacterial activity results in a superior lysin that shows improved robustness in its antibacterial activity, including against the most worrisome colistin-resistant strains A. baumannii.