Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. However, endolysins have limited use against Gramnegative 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 endolysinengineering strategies, allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria. Development of novel antibacterials against Gram-negative bacteria is challenging because they possess an outer membrane that prevents many compounds from reaching their intracellular targets 1. Bacteriophages (phages), viruses that infect bacteria, have naturally evolved mechanisms to overcome the outer membrane to infect their bacterial hosts 2,3. In the first step of infection, phages bind to host cells and inject their genetic material across the outer and inner membrane of the bacterial cells into the cytoplasm 4,5. Also, during the final stage of the lytic infection cycle, phages produce proteins within the cell, which destroy the bacterial cell wall, leading to cell lysis 6,7. Thus, 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 8-10. The adhesion specificity is mediated by receptor binding proteins (RBPs) that form fibers or spikes at the distal phage tail. A wellcharacterized RBP is the monomeric Pb5, located at the tail tip of the phage T5, which binds irreversibly to the bacterial receptor FhuA during infection of the E. coli host 11,12. FhuA is an outer membrane protein that ...

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Campylobacter contaminated poultry remains the major cause of foodborne gastroenteritis worldwide, calling for novel antibacterials. We previously developed the concept of Innolysin composed of an endolysin fused to a phage receptor binding protein (RBP) and provided the proof-of-concept that Innolysins exert bactericidal activity against Escherichia coli. Here, we have expanded the Innolysin concept to target Campylobacter jejuni. As no C. jejuni phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of C. jejuni CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting C. jejuni (Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse C. jejuni strains after in vitro exposure for 45 min at 20°C, reaching up to 1.30 ± 0.21 log reduction in CAMSA2147 cell counts. Screening of a library of Innolysins Cj composed of distinct endolysins for growth inhibition, allowed us to select Innolysin Cj5 as an additional promising antibacterial candidate. Application of either Innolysin Cj1 or Innolysin Cj5 on chicken skin refrigerated to 5°C and contaminated with C. jejuni CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill C. jejuni in situ. The receptor of Innolysins Cj remains to be identified, however, the RBP component (H-fiber) recognizes a novel receptor compared to lytic phages binding to capsular polysaccharide or flagella. Identification of other unexplored Campylobacter phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against Campylobacter.

13Bacteriophage-encoded endolysins degrading the essential peptidoglycan of bacteria are promising 14 alternative antimicrobials to handle the global threat of antibiotic resistant bacteria. However, 15 endolysins have limited use against Gram-negative bacteria, since their outer membrane prevents 16 access to the peptidoglycan. Here we present Innolysins, a novel concept for engineering endolysins 17 that allows the enzymes to pass through the outer membrane, hydrolyse the peptidoglycan and kill 18 the target bacterium. Innolysins combine the enzymatic activity of endolysins with the binding 19 capacity of phage receptor binding proteins (RBPs). As our proof of concept, we used phage T5 20 endolysin and receptor binding protein Pb5, which binds irreversibly to the phage receptor FhuA 21 involved in ferrichrome transport in Escherichia coli. In total, we constructed twelve Innolysins 22 fusing endolysin with Pb5 or the binding domain of Pb5 with or without flexible linkers in between. 23While the majority of the Innolysins maintained their muralytic activity, Innolysin#6 also showed 24 bactericidal activity against E. coli reducing the number of bacteria by 1 log, thus overcoming the 25 outer membrane barrier. Using an E. coli fhuA deletion mutant, we demonstrated that FhuA is 26 required for bactericidal activity, supporting that the specific binding of Pb5 to its receptor on E. 27 coli is needed for the endolysin to access the peptidoglycan. Accordingly, Innolysin#6 was able to 28 kill other bacterial species that carry conserved FhuA homologs such as Shigella sonnei and 29 Pseudomonas aeruginosa. In summary, the Innolysin approach expands recent protein engineering 30 strategies allowing customization of endolysins by exploiting phage RBPs to specifically target 31Gram-negative bacteria. 32 IMPORTANCE 33The extensive use of antibiotics has led to the emergence of antimicrobial resistant bacteria 34 responsible for infections causing more than 50,000 deaths per year across Europe and the US. In 35 response, the World Health Organization has stressed an urgent need to discover new antimicrobials 36 to control in particular Gram-negative bacterial pathogens, due to their extensive multi-drug 37 resistance. However, the outer membrane of Gram-negative bacteria limits the access of many 38 antibacterial agents to their targets. Here, we developed a new approach, Innolysins that enable 39 endolysins to overcome the outer membrane by exploiting the binding specificity of phage receptor 40 binding proteins. As proof of concept, we constructed Innolysins against E. coli using the endolysin 41 and the receptor binding protein of phage T5. Given the rich diversity of phage receptor binding 42 proteins and their different binding specificities, our proof of concept paves the route for creating an 43 arsenal of pathogen specific alternative antimicrobials. 44 intracellular targets (1). Bacteriophages (phages), viruses that infect bacteria, have naturally 48 evolved mechanisms to overcome the outer membrane to infect their ...

Campylobacter phages are divided into two genera; Fletchervirus and Firehammervirus, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in E. coli, followed by lytic assays. Our results show that Fletchervirus phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast, Firehammervirus phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of E. coli after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that Firehammervirus phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two Campylobacter phage genera control lysis by two distinct mechanisms.