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

Abstract

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 ...

Phage Endolysins as Potential Antimicrobials against Multidrug Resistant Vibrio alginolyticus and Vibrio parahaemolyticus: Current Status of Research and Challenges Ahead

“…Zampara and his co-workers constructed twelve Innolysins using phage T5 endolysin and receptor binding protein Pb5, which bind irreversibly to the phage receptor FhuA involved in ferrichrome transport in E coli . It was proved that they pass through the outer membrane and degrade the PG layer, thereby killing the target bacteria [47].…”

Phages and engineered lysins as an effective tool to combat Gram‐negative foodborne pathogens

“…The concept of innolysins focuses on the binding of endolysins with phage receptor‐binding proteins (RBPs) (Zampara et al., 2018). The first reported study (Zampara et al., 2020) described a chimera composed of endolysin and the phage RBP Pb5, both from phage T5.…”

Endolysin, a Promising Solution against Antimicrobial Resistance

“…After 3–12 hydrophobic amino acids were successfully added to the C-terminus of E. coli phage endolysin Lysep3, the modified Lysep3 was able to kill E. coli from outside of the cells [ 51 ]. Fifthly, genetic engineering of endolysins has been applied to make innolysins and artilysins, which can pass through the outer membrane barrier and increase the lytic activity [ 11 , 52 ]. Recently, to treat Helicobacter pylori infection, holin and a section of polypeptides were fused to the endolysin of H. pylori to create a novel artilysin [ 53 ].…”