Art-175 Is a Highly Efficient Antibacterial against Multidrug-Resistant Strains and Persisters of Pseudomonas aeruginosa

Abstract

e Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophageencoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (ϳ5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to >4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections.

Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolates

“…We have not found such residues in CfP1 lysin, and therefore, its antibacterial mechanism remains unknown. Covalent modification of lysins with LPS-destabilizing peptides has been also a recent approach to increase the lysin activity against Gramnegative pathogens (Briers et al 2014a, b;Thandar et al 2016). These chimeric proteins have additional peptides of polycationic, hydrophobic, or amphipathic nature, known to interfere with the lipopolysaccharide-stabilizing forces, assisting the lysins to spontaneously penetrate the Gramnegative outer membrane barrier and become active antimicrobials.…”

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…SMAP-29-assisted uptake of Art-175 induced cell lysis of P. aeruginosa, in contrast to SMAP-29, which did not cause cell lysis. 51 Unfortunately, fusion of SMAP-29 to an endolysin abolished the AMP bactericidal effect. The loss of membrane permeabilization activity might be beneficial since SMAP-29 is highly cytotoxic to human red blood cells, 53 and Art-175 failed to show cytotoxicity in a mouse fibroblast cell line.…”

“…The loss of membrane permeabilization activity might be beneficial since SMAP-29 is highly cytotoxic to human red blood cells, 53 and Art-175 failed to show cytotoxicity in a mouse fibroblast cell line. 51 Briers et al also constructed other Artilysins by fusing Gram-negative phage endolysins with different LPS-destabilizing peptides [polycationic peptide (PCNP), hydrophobic pentapeptide (HPP), Parasin (Pa1), and lycotoxin] as single-or mixed double-peptide fusion constructs. The bacteriolytic effect on Gram-negative cells was most prominent for the PCNP fused enzymes at the N-terminus.…”

“…96 Protein engineering has allowed endolysin-based enzymes to translocate across the outer membrane barrier of Gram-negative bacteria and control P. aeruginosa infection in animal. [50][51][52] (For details see Engineering novel endolysin constructs) Briers et al conducted a Caenorhabditis elegans nematode gut P. aeruginosa infection model to evaluate the efficacy of a polycationic nonapeptide (PCNP)-fused endolysin called Artilysin LoGT-008. 52 Grazing on P. aeruginosa lawns for one day resulted in a 90% C. elegans death rate within 5 d. When infected nematodes were transferred to a buffer containing 50 ml of the (PCNP)-fused endolysin and 50 ml of EDTA, survival rate improved 63% compared to 45% from a ciprofloxacin treatment.…”

“…50 Aritlysins is a term coined by Briers et al that describes a group of endolysin-based enzymes designed to penetrate the outer-membrane of Gram-negative species, such as Pseudomonas aeruginosa, E. coli, and Salmonella enterica. 51,52 For example, Artilysin Art-175 covalently combines the P. aeruginosa phage KZ144 endolysin with the sheep myeloid antimicrobial peptide of 29 amino acids (SMAP-29). 51 SMAP-29 is an a-helical cathelicidin that acts rapidly to permeabilize membranes of susceptible organisms, making it a potent antimicrobial peptide (AMP) with a broad spectrum of activity against bacteria, fungi, and viruses.…”