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, 13 (2), e0192507
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In Vitro Characterization of PlyE146, a Novel Phage Lysin That Targets Gram-negative Bacteria

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In Vitro Characterization of PlyE146, a Novel Phage Lysin That Targets Gram-negative Bacteria

Yu Larpin et al. PLoS One.

Abstract

The recent rise of multidrug-resistant Gram-negative bacteria represents a serious threat to public health and makes the search for novel effective alternatives to antibiotics a compelling need. Bacteriophage (Phage) lysins are enzymes that hydrolyze the cell wall of bacteria and represent a promising alternative to tackle this ever-increasing problem. Despite their use is believed to be restricted to Gram-positive bacteria, recent findings have shown that they can also be used against Gram-negative bacteria. By using a phage genome-based screening approach, we identified and characterized a novel lysin, PlyE146, encoded by an Escherichia coli prophage and with a predicted molecular mass of ca. 17 kDa. PlyE146 is composed of a C-terminal cationic peptide and a N-terminal N-acetylmuramidase domain. Histidine-tagged PlyE146 was overexpressed from a plasmid in Lactococcus lactis NZ9000 and purified by NI-NTA chromatography. PlyE146 exhibited in vitro optimal bactericidal activity against E. coli K12 (3.6 log10 CFU/mL decrease) after 2 h of incubation at 37°C at a concentration of 400 μg/mL in the absence of NaCl and at pH 6.0. Under these conditions, PlyE146 displayed antimicrobial activity towards several other E. coli, Pseudomonas aeruginosa (3 to 3.8-log10 CFU/mL decrease) and Acinetobacter baumannii (4.9 to >5-log10 CFU/mL decrease) strains. Therefore, PlyE146 represents a promising therapeutic agent against E. coli, P. aeruginosa and A. baumannii infections. However, further studies are required to improve the efficacy of PlyE146 under physiological conditions.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structural prediction of the PlyE146 C-terminal domain and sequence alignment with P307.
The C-terminal domain of PlyE146 (P146) assumes a hairpin-like di-alpha helical structure linked by a flexible unstructured region (A) and shares 68% identity with PF307 (B). In blue are conserved amino acids, in yellow positively-charged amino acids and in green conserved, positively-charged amino acids.
Fig 2
Fig 2. Bactericidal activity of PlyE146 on logarithmic growing E. coli K12 bacteria.
E. coli K12 cells (106 to 107 CFU/mL) were exposed to increasing concentrations of PlyE146 in NaPi buffer (pH 6.0) at 37°C for 2 h, serially diluted and plated for colony counts. Results represent the mean ± standard deviation of triplicate experiments.
Fig 3
Fig 3. Effect of pH and NaCl on the antibacterial activity of PlyE146.
To study the effect of pH (A) and NaCl (B) on E. coli K12, logarithmic growing cells were exposed to PlyE146 (400 μg/mL) in NaPi buffer at different pH and NaCl concentrations for 2 h at 37°C, serially diluted and plated for colony counts. Results represent the mean ± standard deviation of triplicate experiments.
Fig 4
Fig 4. Time-kill curve and effect of EDTA on the antibacterial activity of PlyE146.
For the time-kill experiments (A), logarithmic-phase growing E. coli K12 cells were exposed to PlyE146 (400 μg/mL) in NaPi buffer (pH, 6.0) for 4 h at 37°C. For EDTA experiments (B), logarithmic- and stationary-phase growing E. coli K12 cells were exposed to PlyE146 (400 μg/mL) in the absence or the presence of EDTA (5 mM) in NaPi buffer (pH, 6.0) for 2 h at 37°C. Results represent the mean ± standard deviation of triplicate experiments.
Fig 5
Fig 5. Antibacterial activity of PlyE146 against different Gram-negative and Gram-positive bacteria.
The assays were conduct with bacteria in logarithmic growing phase, incubated with 400 μg/mL of PlyE146 in NaPi buffer (pH 6.0) at 37°C for 2 h. The dashed line indicates the limit of detection.
Fig 6
Fig 6. TEM.
Representative TEM images of 50 nm sections of untreated control (left panel) and PlyE146-treated (right panel) A. baumannii (strain FER). TEM images show an evident disruption of the cell wall after exposure to PlyE146 (400 μg/mL) for 1 h.

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Grant support

This work was supported by the Swiss National Science Foundation (grant 310030-125325). There was no additional external funding received for this study.
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