External lysis of Escherichia coli by a bacteriophage endolysin modified with hydrophobic amino acids

Abstract

Drug-resistant bacteria are a serious threat to global public health. Gram-positive bacterial endolysin preparations have been successfully used to fight Gram-positive bacteria as a novel antimicrobial replacement strategy. However, Gram-negative bacterial phage endolysins cannot be applied directly to destroy Gram-negative strains due to the externally inaccessible peptidoglycan layer of the cell wall; this has seriously hampered the development of endolysin-like antibiotics against Gram-negative bacteria. In this study, 3–12 hydrophobic amino acids were successively added to the C-terminus of Escherichia coli phage endolysin Lysep3 to create five different hydrophobic-modified endolysins. Compared with endogenous Lysep3, endolysins modified with hydrophobic amino acids surprisingly could kill E. coli from outside of the cell at the appropriate pH and endolysin concentration. The lysis ability of modified endolysins were enhanced with increasing numbers of hydrophobic amino acids at the C-terminus of endolysin. Thus, these findings demonstrate that the enhancement of hydrophobicity at the C-terminus enables the endolysin to act upon E. coli from the outside, representing a novel method of lysing Gram-negative antibiotic-resistant bacteria.

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

“…These engineered lysins act rapidly upon contact, are highly bactericidal, are active against persisters, show no cross-resistance with existing resistance mechanisms (including colistin) and do not elicit resistance development upon serial exposure to subinhibitory doses [35,36,37,38]. Such engineered lysins have been reported against the three critical priority pathogens listed by the WHO [1], that is, P. aeruginosa, A. baumannii, and E. coli [35][36][37][38][39][40][41][42][43] using polycationic [35,40,41], hydrophobic [35,42], amphipathic [35] or antimicrobial peptides [36,43]. Nevertheless, the mere addition of an OMP peptide to the N-terminus or C-terminus of lysin does not automatically result in the best variants and often only moderate activities are observed.…”

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 ].…”

Engineered Bacteriophages for Practical Applications

“…26) A recent preliminary report, using a similar strategy, generated a fusion between a phage receptor binding protein and an endolysin, allowing passage through the outer membrane and killing Escherichia coli. 27) Other studies, also looking to bypass the external membrane of Gram-negative pathogens, relied on the modification of specific amino acids in an endolysin to enable it to lyse E. coli from the outside of the cell, 28) or in the fusion of endolysins with membrane-destabilizing peptides to generate molecules termed artilysins, active against antibiotic-resistant A. baumannii and E. coli. 29,30) Antibodies have also been harnessed to work alongside lysins through the generation of hybrid molecules termed lysibodies.…”