Development of a Novel Chimeric Endolysin, Lys109 With Enhanced Lytic Activity Against Staphylococcus aureus

Abstract

As the incidence of antibiotic-resistant bacteria has become increased, phage endolysins are believed as one of the promising alternatives to antibiotics. However, the discovery of potent endolysin is still challenging because it is labor intensive and difficult to obtain a soluble form with high lytic activity. In this respect, the modular structures of Gram-positive endolysins can provide an opportunity to develop novel endolysins by domain rearrangement. In this study, a random domain swapping library of four different endolysins from phages infecting Staphylococcus aureus was constructed and screened to obtain engineered endolysins. The novel chimeric endolysin, Lys109 was selected and characterized for its staphylolytic activity. Lys109 exhibited greater bacterial cell lytic activity than its parental endolysins against staphylococcal planktonic cells and biofilms, showing highly improved activity in eliminating S. aureus from milk and on the surface of stainless steel. These results demonstrate that a novel chimeric endolysin with higher activity and solubility can be developed by random domain swapping and that this chimeric endolysin has a great potential as an antimicrobial agent.

Keywords: Staphylococcus aureus; antimicrobial agent; domain swapping; endolysin; screening.

FIGURE 1

Development of screening system on a 96-well microplate. (A) Scheme of the random domain screening method. (B) EGFP released from SPN1S-induced lysis in host E. coli cells. Cells without induction IPTG for EGFP expression and arabinose for SPN1S lysRz expression) were used as a control. (C) Clones for positive and negative controls were cultured with 0.2% arabinose overnight on the lawn of autoclaved S. aureus RN4220.

FIGURE 2

Lytic activity comparison of Lys109 with parental endolysins. (A) Comparison of the relative lytic activity of four other chimeric endolysins and LysSA12 with Lys109 (300 nM each) against S. aureus CCARM 3090. (B) Schematic representation of Lys109, LysSA12 and LysSA97. (C) SDS-PAGE analysis of Lys109, LysSA12, and LysSA97. (D) Relative lytic activity of Lys109, LysSA12 and LysSA97 against S. aureus CCARM 3090 at different concentrations. (E) Relative lytic activity of Lys109, LysSA12 and LysSA97 (300 nM each) against various S. aureus strains. (F) MIC values of Lys109, LysSA12 and LysSA97 against S. aureus CCARM 3090.

FIGURE 3

Biofilm reduction activity of Lys109 and LysSA12. Biofilms formed by (A) S. aureus CCARM 3090 and (B) S. aureus RN4220 were treated with various concentrations of Lys109 and LysSA12 and visualized by staining biofilms with crystal violet. Dark staining indicates the biofilm that was maintained after treatment with the endolysin. Light or no staining indicates successful removal of the biofilm. Control indicates the sample treated with buffer without endolysins.

FIGURE 4

The effects of temperature and pH on the lytic activity of Lys109 and LysSA12. The optimum (A) temperature and (B) pH of Lys109 and LysSA12 were examined by incubation of the enzymes (300 nM each) with target cells at different temperatures and in different pH buffers, respectively. The relative lytic activities were calculated using the activity of Lys109 assayed in a buffer at pH 6.5 and 25°C, which showed maximal activity.

FIGURE 5

Antibacterial activity of Lys109 and LysSA12 against S. aureus CCARM 3090 in milk. (A) LysSA12 and (B) Lys109 were added to milk artificially contaminated with S. aureus CCARM 3090 at different concentrations. Bacterial cells were counted every 15 min for 1 h. (C) The number of S. aureus CCARM 3090 cells in milk was counted after 1 h of treatment with Lys109 and LysSA12 at concentrations of 0 nM (negative control), 30, 300, 900, and 1500 nM. ND, not detected. Asterisks indicate significant differences (***P < 0.001, **P < 0.01, *P < 0.05).

FIGURE 6

Antibacterial activity of Lys109 and LysSA12 against S. aureus CCARM 3090 on stainless steel. The number of S. aureus CCARM 3090 cells was counted after treatment with Lys109 and LysSA12 at concentrations of 0 nM (negative control), 50 and 100 nM. ND, not detected. Asterisks indicate significant differences (**P < 0.01).