Innolysins: A novel approach to engineer endolysins to kill Gram-negative bacteria

Bacteriophage-encoded endolysins degrading the essential peptidoglycan of bacteria are promising alternative antimicrobials to handle the global threat of antibiotic resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since their outer membrane prevents access to the peptidoglycan. Here we present Innolysins, a novel concept for engineering endolysins that allows the enzymes to pass through the outer membrane, hydrolyse the peptidoglycan and kill the target bacterium. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As our proof of concept, we used phage T5 endolysin and receptor binding protein Pb5, which binds irreversibly to the phage receptor FhuA involved in ferrichrome transport in Escherichia coli. In total, we constructed twelve Innolysins fusing endolysin with Pb5 or the binding domain of Pb5 with or without flexible linkers in between. While the majority of the Innolysins maintained their muralytic activity, Innolysin#6 also showed bactericidal activity against E. coli reducing the number of bacteria by 1 log, thus overcoming the outer membrane barrier. Using an E. coli fhuA deletion mutant, we demonstrated that FhuA is required for bactericidal activity, supporting that the specific binding of Pb5 to its receptor on E. coli is needed for the endolysin to access the peptidoglycan. Accordingly, Innolysin#6 was able to kill other bacterial species that carry conserved FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa. In summary, the Innolysin approach expands recent protein engineering strategies allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.