Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens

Abstract

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.

FIG 1

Visual representation of engineered Artilysins. Seven different endolysins (OBPgp279 [YP_004958186.1], PVP-SE1gp146 [YP_004893953.1], phiKZgp144 [NP_803710.1], 201ϕ2-1gp229 [YP_001956952.1], CR8gp3.5 [unpublished], P2gp09 [NP_046765.1], and PsP3gp10 [NP_958065.1) were selected for modification with different peptides. (A) Single N-terminal fusion constructs, (B) double N-terminal fusion constructs, (C) N-terminal extended-linker constructs, (D) C-terminal extended-linker constructs. Abbreviations: PCNP = polycationic peptide, HPP = hydrophobic pentapeptide, Pa1 = Parasin1, Ly1 = lycotoxin1, L = linker unit consisting of GAGA sequence.

FIG 2

Antibacterial activity of the N-terminal fusion variants of OBPgp279 and PVP-SE1gp146. (A) Fourteen Artilysins with N-terminal fusions of seven different peptides (see Table S1 in the supplemental material) to OBPgp279 and PVP-SE1gp146 are compared to the corresponding unmodified endolysins for their antibacterial activity against P. aeruginosa PAO1. This comparison shows the PCNP to be the most effective peptide among the seven peptides tested here. (B) Comparison of the antibacterial activities of two wild-type endolysins (OBPgp279 and PVP-SE1gp146) and their PCNP-tagged Artilysin counterparts (LoGT-001 and LoGT-008, respectively) with and without 0.5 mM EDTA. Activity was tested on P. aeruginosa strains PAO1 and multidrug-resistant Br667, both in the absence (wt endolysin, white bars; Artilysin, light gray bars) and presence (wt endolysin, dark gray bars; Artilysin, black bars) of 0.5 mM EDTA. These data show that the PCNP fusion for the antibacterial activity of the endolysin added value while not compromising its synergy with EDTA. Averages and standard deviations of results of three replicates are shown.

FIG 3

Time-lapse series of the action of LoGT-008 on P. aeruginosa PAO1. Cells were mixed with LoGT-008 in the presence of 0.5 mM EDTA and were subsequently recorded using time-lapse microscopy. Time points are indicated (min:s). Scale bar, 2 µm.

FIG 4

Antibacterial activity of PCNP-Artilysins based on different endolysins. The effect on antibacterial activity of an N-terminal PCNP fusion to three modular (OBPgp279, PVP-SE1gp146, and 201ϕ2-1gp229) and three globular (PsP3gp10, P2gp09, and CR8gp3.5) endolysins is shown for P. aeruginosa PAO1. Averages and standard deviations of results of three replicates are shown. wt, wild type.

FIG 5

Optimization of PCNP-based Artilysins. (A) The effect of an increasing length of the flexible linker between PCNP and OBPgp279 on the antibacterial activity against P. aeruginosa PAO1 is shown. L0, L1, L2, L3, and L4 correspond to intervening amino acid sequences of AGAS, AGAGAS, AGAGAGAGAS, AGAGAGAGAGAGAS, and AGAGAGAGAGAGAGAGAS. (B) The antibacterial activity of Artilysins comprising a tandem of the PCNP peptide and a second peptide as N-terminal fusions to OBPgp279 is compared to that of PCNP-OBPgp279 (LoGT-001) in the absence (light gray) and presence (dark gray) of 0.5 mM EDTA. (C) LoGT-023, comprising a 16-amino-acid flexible linker between the N-terminal PCNP tag and OBPgp279, generally appeared the most effective Artilysin. The antibacterial activity against P. aeruginosa, A. baumannii, E. coli, and S. Typhimurium in the presence of 0.5 mM EDTA is shown. Averages and standard deviations of results of three replicates are given.

FIG 6

Antibacterial effect of PVP-SE1gp146 and PCNP-PVP-SE1gp146 (LoGT-008) in a P. aeruginosa PA14-infected human keratinocyte monolayer. A mixture of 2 µM of PVP-SE1gp146/LoGT-008 and 0.005 mM of EDTA was applied to a 5-day-old confluent keratinocyte monolayer that was infected with 10⁵ CFU/ml (A) or 10⁷ CFU/ml (B) P. aeruginosa PA14 1 h before treatment. Numbers of surviving bacteria (squares) and surviving keratinocytes (bars) were quantified after 4 h of infection. Proportions of surviving bacteria are shown relative to the infected, untreated control results. Significantly different conditions (P < 0.05) are indicated with bold (keratinocyte survival) or italic (bacterial survival) letters.

FIG 7

Survival rates of P. aeruginosa PA14-infected C. elegans SS104 nematodes treated with PVP-SE1gp146 or PCNP-PVP-SE1gp146 (LoGT-008) in combination with EDTA. PVP-SE1gp146 (5× MIC; △) or LoGT-008 (5× MIC; ▲) was added to infected nematodes in the presence of 0.5 mM EDTA. Ciprofloxacin (□; 5× MIC) was used as a positive control for antibacterial activity. Other controls included were untreated PA14-infected nematodes (○), untreated nematodes grown on E. coli OP 50 (■), and EDTA-treated infected nematodes (●). A total of 30 infected worms were used for each condition. Nematode viability, in percentages relative to initial values at day 0, was assessed at different time points after compound addition (day 0). Averages and standard deviations of results of three independent experiments are shown. Significantly different conditions (P < 0.05) are marked.