TY - JOUR

T1 - Exploiting phage receptor binding proteins to enable endolysins to kill Gram-negative bacteria

AU - Zampara, Athina

AU - Sørensen, Martine C.Holst

AU - Grimon, Dennis

AU - Antenucci, Fabio

AU - Vitt, Amira Ruslanovna

AU - Bortolaia, Valeria

AU - Briers, Yves

AU - Brøndsted, Lone

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

U2 - 10.1038/s41598-020-68983-3

DO - 10.1038/s41598-020-68983-3

M3 - Journal article

C2 - 32694655

AN - SCOPUS:85088291409

VL - 10

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 12087

ER -