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

Abstract

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 ...

Developing Innolysins Against Campylobacter jejuni Using a Novel Prophage Receptor-Binding Protein

“…To screen Innolysins Cj for muralytic activity, proteins were expressed in E. coli BL21-CodonPlus-(DE3)-RIL cells, as previously described ( Zampara et al, 2020 ). Briefly, freshly transformed colonies were re-suspended in 500 μl of auto-induction medium [93% ZY medium, 0.05% 2M MgSO 4 , 2% 50 × 5052 (0.5% glycerol, 0.05% glucose, and 0.2% α-lactose in 20 ml water), 5% 20 × NPS (50 mM Na 2 HPO 4 , 50 mM KH 2 PO 4 , and 25 mM (NH 4 ) 2 SO 4 to 50 mL of water)].…”

“…These artificial endolysins, known as Artilysins ® , are under commercial development and with reported antibacterial activities against Pseudomonas aeruginosa , Acinetobacter baumannii , and colistin-resistant E. coli ( Briers et al, 2014 ; Defraine et al, 2016 ; Schirmeier et al, 2018 ). Recently, we provided a proof of concept for the use of a phage receptor binding protein (RBP), enabling the fused endolysin to exert antibacterial activity and kill E. coli ( Zampara et al, 2020 ). In our previous work, we constructed hundreds of RBP-endolysin hybrids (Innolysins) by fusing 24 different endolysins with Pb5 (phage T5 RBP) in different configurations.…”

“…Screening of these Innolysin variants for antibacterial activity identified Innolysin Ec21, which was able to reduce E. coli resistant to third-generation cephalosporins by 3.31 ± 0.53 log. The antibacterial activity of Innolysins Ec was proved to be dependent on the Pb5 cognate receptor, FhuA ( Zampara et al, 2020 ). Thus, the phage RBP provides specificity to the fused endolysin of the Innolysin Ec.…”

Lysins breaking down the walls of Gram-negative bacteria, no longer a no-go

“…could be increased to 3.3 log. Innolysins do not have protein domains for translocation and their mode of action is currently unknown [48 ]. Because receptor binding by lysocins or innolysins may be easily inhibited by point mutations giving rise to resistance, OM proteins essential for virulence should be selected as target to achieve an optimal biocontrol of the infection.…”

Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis

“…Phage-derived lytic enzymes such as endolysins are being studied as potential antimicrobial agents to combat infections caused by different gram-negative and -positive pathogens [ 34 , 109 ], with progresses made optimizing lysins through bioengineering [ 106 , 110 , 111 ]. Indeed, a recent study describes the novel concept of “innolysins”, an engineering approach that allows customization of endolysins by combining them with phage Receptor Binding Proteins (RBPs) to target specific bacteria [ 112 ]. Thus, “enzybiotic” is defined as a new and promising class of drugs derived from phage endolysins, characterized by a rapid mode-of-action, high specificity, and less probability to induce bacterial resistance when compared to traditional drugs [ 113 ].…”