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Development of sensitizer peptide-fused endolysin Lys1S-L9P acting against multidrug-resistant gram-negative bacteria.

ABSTRACT: The advent of multidrug-resistant (MDR) bacteria poses a major threat to public health, garnering attention to novel antibiotic replacements. Endolysin, a bacteriophage-derived cell wall-degrading enzyme, is a promising alternative to conventional antibiotics. However, it is challenging to control Gram-negative bacteria due to the presence of the outer membrane that shields the peptidoglycan layer from enzymatic degradation. To overcome this threshold, we constructed the fusion endolysin Lys1S-L9P by combining endolysin LysSPN1S with KL-L9P, a sensitizer peptide known to extend efficacy of antibiotics by perturbing the outer membrane of Gram-negative bacteria. In addition, we established a new endolysin purification procedure that increases solubility allowing a 4-fold increase in production yield of Lys1S-L9P. The sensitizer peptide-fused endolysin Lys1S-L9P exhibited high bactericidal effects against many MDR Gram-negative pathogens and was more effective in eradicating biofilms compared to LysSPN1S. Moreover, Lys1S-L9P showed potential for clinical use, maintaining stability at various storage temperatures without cytotoxicity against human cells. In the in vivo Galleria mellonella model, Lys1S-L9P demonstrated potent antibacterial activity against MDR Gram-negative bacteria without inducing any toxic activity. This study suggest that Lys1S-L9P could be a potential biocontrol agent to combat MDR Gram-negative bacteria.

SUBMITTER: Son SM

PROVIDER: S-EPMC10701683 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Development of sensitizer peptide-fused endolysin Lys1S-L9P acting against multidrug-resistant gram-negative bacteria.

Son Su Min SM Kim Joonbeom J Ryu Sangryeol S

Frontiers in microbiology 20231123

The advent of multidrug-resistant (MDR) bacteria poses a major threat to public health, garnering attention to novel antibiotic replacements. Endolysin, a bacteriophage-derived cell wall-degrading enzyme, is a promising alternative to conventional antibiotics. However, it is challenging to control Gram-negative bacteria due to the presence of the outer membrane that shields the peptidoglycan layer from enzymatic degradation. To overcome this threshold, we constructed the fusion endolysin Lys1S-L ...[more]

PMID: 38075915

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Project description:The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). Importance: Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins--enzymes derived from bacterial viruses--represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.

Project description:Polymyxin is used as a last resort antibiotics for infections caused by multi-drug resistant (MDR) Gram-negative bacteria and is often combined with other antibiotics to improve clinical effectiveness. However, the synergism of colistin and other antibiotics remains obscure. Here, we revealed a notable synergy between colistin and flavomycin, which was traditionally used as an animal growth promoter and has limited activity against Gram-negative bacteria, using checkerboard assay and time-kill curve analyses. The importance of membrane penetration induced by colistin was assessed by examining the intracellular accumulation of flavomycin and its antimicrobial impact on Escherichia coli (E. coli) strains with truncated lipopolysaccharides. Besides, a mutation in the flavomycin binding site was created to confirm its role in the observed synergy. This synergy is manifested as an augmented penetration of the E. coli outer membrane by colistin, leading to increased intracellular accumulation of flavomycin and enhanced cell killing thereafter. The observed synergy was dependent on the antimicrobial activity of flavomycin, as mutation of its binding site abolished the synergy. In vivo studies confirmed the efficacy of colistin combined with flavomycin against MDR E. coli infections. This study is the first to demonstrate the synergistic effect between colistin and flavomycin, shedding light on their respective roles in this synergism. Therefore, we propose flavomycin as an adjuvant to enhance the potency of colistin against MDR Gram-negative bacteria.ImportanceColistin is a critical antibiotic in combating multi-drug resistant Gram-negative bacteria, but the emergence of mobilized colistin resistance (mcr) undermines its effectiveness. Previous studies have found that colistin can synergy with various drugs; however, its exact mechanisms with hydrophobic drugs are still unrevealed. Generally, the membrane destruction of colistin is thought to be the essential trigger for its interactions with its partner drugs. Here, we use clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) for specifically mutating the binding site of one hydrophobic drug (flavomycin) and show that antimicrobial activity of flavomycin is critical for the synergy. Our results first give the evidence that the synergy is set off by colistin's membrane destruction and operated the final antimicrobial function by its partner drugs.

Dataset Information

Development of sensitizer peptide-fused endolysin Lys1S-L9P acting against multidrug-resistant gram-negative bacteria.

Publications

Development of sensitizer peptide-fused endolysin Lys1S-L9P acting against multidrug-resistant gram-negative bacteria.

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