Combination Effect of Engineered Endolysin EC340 With Antibiotics

Abstract

Bacteriophage lysins, also known as endolysins or murein hydrolases, are hydrolytic enzymes produced by bacteriophages during the final stage of the lytic cycle to enable cleavage through the host's cell wall, thus allowing the phages to burst out of their host bacteria after multiplication inside them. When applied externally to Gram-negative bacteria as recombinant proteins, lysins cannot easily reach the cell wall due to the presence of an outer membrane (OM). In this study, endolysin EC340 obtained from phage PBEC131 infecting Escherichia coli was engineered for improved OM permeability and increased activity against Gram-negative bacteria. The engineered endolysin, LNT113, was tested for potential synergistic effects with standard-of-care antibiotics. A synergistic effect was demonstrated with colistin, while an additive effect was seen with meropenem, tigecycline, chloramphenicol, azithromycin, and ciprofloxacin. Neither ceftazidime nor kanamycin showed any synergy or additive effects with the LNT113 endolysin. Moreover, synergy and additive effects could not be generalized by antibiotic class, OM traverse mechanism, molecular weight, or the bactericidal nature of each antibiotic tested.

Keywords: Gram-negative bacteria; antibacterial agent; bacteriophage; cecropin A; colistin; endolysin; synergy.

FIGURE 1

Organization of endolysins used in this study and their enzymatic activities. (A) Domain structure of EC340, mtEC340, and LNT113. It has a predicted lysozyme domain without a cell-wall binding domain (CBD). EC340 from E. coli phage PBEC131 was subjected to several substitutions of amino acids (mtEC340). The locations of substituted amino acids are shown. mtEC340 had cecropin A fused to its N-terminus and was named LNT113. (B) Analysis of purified endolysins, EC340 (19.5 kDa), mtEC340 (18.2 kDa), and LNT113 (23.2 kDa) on SDS-PAGE and subsequent zymogram assay.

FIGURE 2

Antibacterial activities of endolysins. Antibacterial activity of endolysins (2 μM) against E. coli strains (A) and K. pneumoniae strains (B) in 20 mM Tris–HCl buffer (pH 7.5) for 2 h. Tris buffer was used as a negative control. Dotted line denotes the detection limit. In addition to t-tests, two-way ANOVA was performed and is shown as horizontal bars above vertical bars (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 3

Cell penetration and antimicrobial activity of LNT113. (A) The outer membrane permeability of the Gram-negative E. coli ATCC 8739 was determined by NPN assays. CecA-EGFP; cecropin A fused to EGFP, cecA+mtEC340; cecropin A and mtEC340 were separately added to the mixture at 2 μM each, PMB; polymyxin B. (B) LNT113 showed an enhanced antibacterial activity against E. coli compared to Cecropin A and/or mtEC340. All were treated to a final concentration of 0.5 mM. (C) Antibacterial activities of different concentrations of LNT113 (0.1, 0.3, 0.9, or 2.7 μM) against E. coli ATCC 8739. (D) Decrease in bacterial viability after the addition of endolysin (0.5 μM) at different time lengths. Dashed lines show the detection limit. (E) Antibacterial activity of LNT113 against MCR-1 positive E. coli FORC81. The cells were treated with LNT113 or colistin at concentrations of 0.2 or 2 μM for 2 h. Dotted line denotes the detection limit. Asterisks represent statistical differences compared to the control (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 4

Cytotoxicity and hemolytic activity of LNT113. (A) Cytotoxicity assay of LNT113 in a Huh7 cell line. Cells were incubated with different concentrations of LNT113 for 24 or 48 h. Triton X-100 was used as the cytotoxicity control agent. (B) Hemolytic activity of LNT113. RBCs were incubated with PBS or LNT113 at 37°C for 1 h, and hemolysis was determined by measuring the absorbance of the supernatant at 570 nm. 0.1% of Triton X-100 was used as the hemolysis control agent. Statistical difference compared to control ***P < 0.001.

FIGURE 5

Synergistic effect of LNT113 and colistin against three different E. coli strains. The isobologram of LNT113 and colistin in E. coli. FIC indexes (FICIs) represent the sum of Fractional Inhibitory Concentrations (FICs) of LNT113 and colistin.