Exploiting phage receptor binding proteins to enable endolysins to kill Gram-negative bacteria

Athina Zampara,Lone Brøndsted,Valeria Bortolaia,Fabio Antenucci,Yves Briers,Dennis Grimon,Amira Ruslanovna Vitt,Martine C Holst Sørensen

doi:10.1038/s41598-020-68983-3

Athina Zampara, Lone Brøndsted + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-68983-3

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Abstract

Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since the outer membrane prevents access to the peptidoglycan. Here, we present Innolysins, an innovative concept for engineering endolysins to exert antibacterial activity against Gram-negative bacteria. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As proof-of-concept, we constructed 12 Innolysins by fusing phage T5 endolysin and RBP Pb5 in different configurations. One of these, Innolysin Ec6 displayed antibacterial activity against Escherichia coli only in the presence of Pb5 receptor FhuA, leading to 1.22 ± 0.12 log reduction in cell counts. Accordingly, other bacterial species carrying FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa were sensitive to Innolysin Ec6. To enhance the antibacterial activity, we further constructed 228 novel Innolysins by fusing 23 endolysins with Pb5. High-throughput screening allowed to select Innolysin Ec21 as the best antibacterial candidate, leading to 2.20 ± 0.09 log reduction in E. coli counts. Interestingly, Innolysin Ec21 also displayed bactericidal activity against E. coli resistant to third-generation cephalosporins, reaching a 3.31 ± 0.53 log reduction in cell counts. Overall, the Innolysin approach expands previous endolysin-engineering strategies, allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.

Highlights

Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria
We anticipated that antibacterial activity of an Innolysin Ec requires both that Pb5 is able to bind to the outer membrane protein FhuA, and that the fused phage T5 endolysin remains active to degrade the peptidoglycan
To improve the antibacterial activity of Innolysins, we redundantly screened a library of 228 novel Innolysins each consisting of one out of 23 different endolysins fused with Pb5 in four distinct configurations

Summary

Introduction

Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. The molecular tools developed during phage evolution may be exploited to develop novel phagebased antibacterials that are able to pass the outer membrane and to kill Gram-negative bacteria Phages recognize their host bacteria by binding to specific surface receptors that may be outer membrane proteins, lipopolysaccharides or components of bacterial capsules, pili and flagella[8,9,10]. Fusion of the E. coli phage endolysin Lysep[3] with the translocation and receptor-binding domain of another bacteriocin, colicin A, targeting the outer membrane protein BtuB exerted antibacterial activity against E. coli[24]. The translocation and receptor-binding domain of P. aeruginosa bacteriocin pyocin S2 (PyS2) was recently fused with GN4 lysin to construct lysocins that could kill P. aeruginosa[25] These studies have demonstrated that endolysins can be engineered to overcome the outer membrane barrier and subsequently kill Gram-negative bacteria

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