The bacterial strains used in this study are listed in Table 1. Staphylococcal strains were grown in tryptic soy broth (TSB) (Difco, Detroit, MI, United States) at 37°C under aerobic conditions. Baird-Parker agar plates with egg yolk tellurite (BPA; Difco) were used for the selective enumeration of S. aureus. Bacillus cereus, B. subtilis, Listeria monocytogenes, and Streptococcus thermophilus was cultivated in brain heart infusion (BHI) medium (Difco). Luria-Bertani (LB) medium (Difco) was used for the growth of Gram-negative strains. Escherichia coli DH5α and BL21 (DE3) star strains were used to clone and express proteins, respectively.

The gene encoding SPN1S lysRz (SPN1S_0028 and SPN1S_0029) was amplified from Salmonella Typhimurium phage SPN1S (GenBank accession number NC_016761) using the oligonucleotides listed in Supplementary Table S1. The gene fragment was digested with EcoRI-SalI and inserted into the pBAD33 vector (Lim et al., 2012). To confirm the cell lysis efficiency, the pET28a_EGFP vector (Kong and Ryu, 2015) was cotransformed into competent E. coli BL21 (DE3) with pBAD33_SPN1S lysRz. The fluorescence of the released EGFP was measured by using a SpectraMax i3 multimode microplate reader (Molecular Devices, Sunnyvale, CA, United States) with excitation at 485 nm and emission at 535 nm. Four different S. aureus phage endolysins, including LysSA12, LysSA97, LysSA11, and LysSAP4, were used to construct two types of libraries (Chang and Ryu, 2017; Chang et al., 2017a). For the first library, we tried to generate chimeric endolysins containing two catalytic domains. The genes encoding four CHAP domains and those encoding three amidase domains from the four endolysins were amplified and digested with BamHI/XhoI for insertion as the N-terminal domain of the chimeric endolysins. For the central domain of chimeric endolysins, the seven genes were amplified and digested with XhoI/BamHI. All plasmids and primers used in this study are listed in Supplementary Table S1. For the second library, all genes encoding CHAPs and amidase domains were amplified and digested with only BamHI to ensure diversity in the number of catalytic domains to be inserted into the endolysins. All gene fragments were randomly ligated into the BamHI sites of pET28a vectors (Novagen, Madison, WI, United States) containing one of the genes encoding four different cell wall binding domains from the four endolysins. The vector libraries were cotransformed into competent E. coli BL21 (DE3) harboring pBAD33_SPN1S lysRz.

The resulting clones from the random library were screened as described in a previous study with some modifications (Yang et al., 2015). In brief, the clones were picked and grown in 96-well plates with fresh LB broth containing 0.01 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown overnight at 37°C to initiate the expression of the chimeric endolysins. Afterward, 0.2% arabinose was added to the wells to express SPN1S lysRz. These cultures were dotted onto agar plates overlaid with autoclaved S. aureus RN 4220 and incubated for 12 h at 37°C. The clones exhibiting a clear lysis zone against S. aureus were screened and picked for sequencing analysis to identify the cloned chimeric endolysin.

Escherichia coli BL21 (DE3) harboring chimeric endolysins were cultivated at 37°C to an optical density at 600 nm (OD₆₀₀) of 0.7 and the protein expression was induced by addition of 0.5 mM IPTG for 20 h at 18°C. Bacterial cells were suspended in lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride and 30% glycerol; pH 8.0) and disrupted by sonication at a duty cycle of 25% and output control of 5 (Branson Ultrasonics, Danbury, CT, United States). After centrifugation (20,000 × g, 30 min), the supernatant was passed through a Ni-NTA superflow column (Qiagen GmbH, Hilden, Germany), and purification of the recombinant proteins was performed according to the manufacturer’s instructions. The purified protein was stored at −80°C until use after the buffer was changed to storage buffer (50 mM sodium phosphate, 300 mM NaCl and 30% glycerol; pH 8.0) using PD Miditrap G-25 (GE Healthcare, Amersham, Bucks, United Kingdom).

The lytic activity of the chimeric endolysins and their original endolysins was assessed with a turbidity reduction assay (Son et al., 2012). Bacterial cells grown to the exponential phase were resuspended in reaction buffer (50 mM Tris–HCl, pH 6.5). Then, the purified proteins were added to the cell suspension at a final concentration of 300 nM, and the OD reduction of the cells was measured over time by using a SpectraMax i3 multimode microplate reader at 600 nm. The relative lytic activity was calculated after 60 min as follows: [ΔOD₆₀₀ test (endolysin added) - ΔOD₆₀₀ control (buffer only)]/initial OD₆₀₀. The antimicrobial spectrum was tested by a plate lysis assay as previously described (Chang et al., 2017a). In brief, 10 μL of diluted endolysin (167, 16.7, and 1.67 pmol) was spotted onto a freshly prepared bacterial lawn on TSA agar plates. Spotted plates were air-dried in a laminar flow hood for 15 min and incubated overnight at 37°C. The MIC of the endolysins was determined by serial dilution of the endolysins by 1:2 in 96-well plates as described previously (Andrews, 2001; Swift et al., 2015). Exponentially growing S. aureus CCARM 3090 was added to each well at a final concentration of 10⁵ CFU/well, and the plate was incubated at 37°C for 20 h. The MIC was defined as the lowest concentration of endolysin that produced inhibition of visible growth.

The biofilm reduction assay was performed as previously described with some modifications (Wu et al., 2003). Staphylococcal strains incubated in TSB medium supplemented with 0.25% D-glucose (Sigma-Aldrich, St. Louis, MO, United States) were prepared and subcultured in the same media in a 96-well polystyrene microplate. After incubating the microplate for 24 h at 37°C, all wells were washed with PBS. Once the biofilm formed, the experimental group wells were filled with endolysins. After incubation for 2 h at 37°C, each well was washed once with PBS and stained with 1.0% crystal violet. Next, each well was washed three times with PBS, followed by solubilization with 33% acetic acid. The absorbance of the obtained solution was measured at 570 nm, and the sessile biomass was presented as an A₅₇₀ value.

For the temperature stability assay of Lys109, the lytic activity was measured in reaction buffer at 25°C for 60 min after the enzyme was incubated at various temperatures (4–65°C) for 30 min. To study the effects of temperature on Lys109 enzymatic activity, 300 nM Lys109 was added into the target cell suspension, and the mixture was incubated at different temperatures (4–65°C) for 60 min. Then, the lytic activity was measured in reaction buffer at 25°C. To test the effects of pH on the activity of Lys109, 300 nM Lys109 was added to S. aureus CCARM 3090 cells suspended in the following buffers: 50 mM sodium acetate (pH 4.5 and 5.4), 50 mM Tris–HCl (pH 6.5–8.0), 50 mM glycine (pH 9.0), and 50 mM N-cyclohexyl-3-aminopropanesulfonic acid (pH 10.0).

The binding ability of EGFP_LysSA12 amidase plus CBD and EGFP_LysSA97 amidase plus CBD to S. aureus cells was evaluated as previously described (Loessner et al., 2002). Bacterial cells grown to the early exponential phase were harvested and resuspended in PBS. EGFP fusion proteins were incubated with the cells for 5 min at 25°C. The mixture was washed twice with PBS to remove unbound protein and was moved to a 96-well plate to measure the fluorescence using a SpectraMax i3 multimode microplate reader (excitation at 485 nm and emission at 535 nm). The OD₆₀₀ of the cells was measured to normalize the fluorescence by calculating the whole-cell fluorescence per OD₆₀₀.

The lytic activity of Lys109 against the MRSA CCARM 3090 strain was tested in commercial whole-fat pasteurized milk as previously described (Chang et al., 2017a). A milk sample was inoculated with exponentially growing MRSA CCARM 3090 cells (approximately 10⁵ CFU/mL). Before the addition of Lys109 and LysSA12 at 0, 30, 300, 900, and 1500 nM, the milk samples were preincubated with the bacteria at 25°C for 1 h to allow the bacteria to adapt to the milk. Each milk sample was then incubated at 25°C for an additional hour. Viable bacterial cells (CFU/mL) were counted every 15 min after the addition of Lys109 and LysSA12 by plating each sample on a BPA plate and incubation at 37°C for 24 h.

The lytic activity of Lys109 against the MRSA CCARM 3090 strain was tested on stainless steel as previously described (Cha et al., 2019). A stainless steel coupon with a size of 2 × 2 cm³ was sterilized in an autoclave. Exponentially growing MRSA CCARM 3090 bacterial cells were harvested and resuspended in PBS to a final concentration of approximately 10⁵ CFU/mL. Prepared bacterial cells were pipetted onto the stainless steel surface and dried for 1 h on a clean bench. Subsequently, the stainless steel samples were treated with Lys109 (0–100 nM) and left for 60 min at 25°C. For the negative control, PBS was used instead of Lys109 solution. S. aureus cells were detached from the surface by agitation in PBST for 2 min with a bench-top vortex mixer at maximum speed. Cell suspensions were serially diluted and plated onto BPA plates and incubated at 37°C for 24 h.

GraphPad Prism (version 5.01) was used to conduct statistical analysis. One-way analysis of variance (ANOVA) followed by one-way Tukey’s test for all pairwise comparisons (95% confidence interval) was performed. The data are presented as the means with standard deviations. A P-value < 0.05 was considered statistically significant.

The overall scheme of random domain swapping method was presented in Figure 1A. The protocol was established based on a 96 well plate format for the rapid and efficient screening, taking advantage of a S. Typhimurium phage SPN1S lysis cassette composed of an endolysin and Rz/Rz1-like proteins (SPN1S lysRz) (Lim et al., 2012). SPN1S lysRz has been reported to cause host cell lysis and release viral progeny at the end of phage life cycle. To evaluate the cell lysis efficiency of SPN1S lysRz, the amount of EGFP protein released from lysed E. coli was measured. Fluorescence significantly increased after induction with arabinose (Figure 1B), indicating that SPN1S lysRz can cause the rapid lysis of E. coli cells from within, thereby releasing accumulated proteins in the cytosol. To identify an active chimeric endolysin from a large random library, the lytic efficacy of the released proteins was evaluated by their ability to form a clear zone on an agar plate overlaid with heat-killed S. aureus cells. As positive controls, four different S. aureus phage endolysins (LysSA11, LysSA97, LysSAP4, and LysSA12) were expressed in the presence of pBAD33_SPN1S lysRz. The clear zones were visualized on the agar plate depending on their activities, whereas clones without pBAD33_SPN1S lysRz did not show a clear zone (Figure 1C). These results suggest that SPN1S lysRz-induced lysis of E. coli allowed active chimeric endolysins to form clear zone on agar plates containing target bacteria and that this system can be used as a method for the successful screening of novel chimeric endolysins.

The random library containing 480 clones were cotransformed into E. coli containing the SPN1S lysRz-harboring vector and applied to an agar plate containing S. aureus for screening. The clones displaying clear lysis zones were sequenced to determine the combination of the EADs (Supplementary Table S2). Most of the selected clones contained a CHAP domain from LysSA12 or LysSA11, which showed high staphylolytic activity (Chang et al., 2017a, b). In particular, LysSA12 CHAP domain-containing clones, which account for 68% of selected clones, showed a large and clear lysis zone. There was also a single clone containing a LysSAP4 CHAP domain. These results suggest that CHAP domain is necessary to degrade S. aureus cell wall peptidoglycan, and this is consistently observed in other chimeric endolysins to control S. aureus (Daniel et al., 2010; Schmelcher et al., 2012c; Yang et al., 2014). Among LysSA12 CHAP domain-containing clones, five promising chimeric endolysins were selected for the further comparative analysis (Figure 2A). All selected proteins were expressed in E. coli in soluble form and evaluated for their lytic activity against S. aureus. As a result, a chimeric endolysin consisting of a LysSA12 CHAP domain in the N-terminal region, a LysSA97 amidase domain at the central and a LysSA97 CBD in the C-terminal region showed the highest lytic activity among the five candidates, and was designated Lys109 (Figure 2B). BLAST analysis revealed that Lys109 has 78% overall amino acid sequence identity with an amidase from S. aureus phage StauST398-1 (Van Der Mee-Marquet et al., 2013) and 80% with endolysins from ΦB166 and ΦB236 S. aureus phage (Botka et al., 2015). Although the three endolysins showed high similarity with Lys109, they have not yet been studied, suggesting that further research on Lys109 will be meaningful. Moreover, LysSA12, a CHAP domain donor of Lys109, has high amino acid similarity with LysH5 of S. aureus phage vB_SauS_phiIPLA88 (98% identity to LysSA12) and S. aureus Φ11 endolysin (96% identity to LysSA12) (Sass and Bierbaum, 2007; García et al., 2010). These endolysins have a conserved catalytic triad (C32, H95, and Q112) in their CHAP domains (Supplementary Figure S1). Considering that the lytic activity of S. aureus endolysin depends mostly on a CHAP domain, we selected Lys109 to investigate the effect of domain replacement on the activity of the endolysin.

Lys109 and its parental endolysins, LysSA12 and LysSA97, were highly expressed as a soluble form in E. coli and were purified via Ni-NTA affinity chromatography. The predicted molecular weights of Lys109, LysSA12 and LysSA97 were approximately 54 kDa and the proteins migrated as expected in SDS-PAGE gel (Figure 2C). The antibacterial activity of Lys109 was compared with those of LysSA12 and LysSA97 at various concentrations (Figure 2D). Lys109 exhibited clear cell lysis against S. aureus CCARM 3090, displaying at least 2-fold higher lytic activity than that of LysSA12 and LysSA97 at all tested concentrations. LysSA12 did not show lytic activity at concentrations below 30 nM and LysSA97 barely exhibited staphylolytic activity at all tested concentrations. These results indicate the superiority of lytic activity of LysSA12 CHAP over LysSA97 CHAP despite their sequence similarity (44% identity). Comparative analysis of the lytic activity of Lys109 with its parental endolysins against other S. aureus strains including clinical isolates and MRSA also showed an evident improvement in the lytic activity of Lys109 (Figure 2E). The MIC of Lys109 was compared with those of its parental endolysins. LysSA97, a donor for the amidase domain and CBD of Lys109, did not show inhibition of cell growth at the maximum concentrations available (5.85 μM) (Figure 2F), leading us to exclude LysSA97 in the following experiments. LysSA12 inhibited the growth of S. aureus CCARM 3090 at concentration of 0.843 μM. The MIC of Lys109 was 0.375 μM, which was at least 2.25-fold lower than that of LysSA12, indicating that the antimicrobial activity of Lys109 was significantly improved compared to its parental endolysins.

The biofilm reduction efficacy of Lys109 against biofilms formed by S. aureus CCARM 3090 and S. aureus RN4220 was evaluated by a crystal violet staining-based assay (Figure 3). Lys109 showed biofilm reduction activity in a dose-dependent manner, and Lys109 appeared to have a higher biofilm reduction efficacy than LysSA12. When 300 nM of endolysins were added to the biofilms, Lys109 exhibited more than 3-fold enhanced efficacy in removing the biofilms compared to LysSA12. These results demonstrate that Lys109 has strong lytic activity against not only planktonic cells but also biofilms, which is an important contributing factor for many treatment failures (Otto, 2013).

The thermostability of endolysins was determined (Supplementary Figure S2). Lys109 retained over 95% of its activity after 1 h of incubation at 4 to 37°C, and the lytic activity of Lys109 started to decrease at 45°C. Approximately 40% decrease in its hydrolytic activity was observed at 50°C, and higher temperatures (55 and 65°C) caused complete inactivation of Lys109. LysSA12 showed similar pattern of thermal stability to that of Lys109, indicating that the structural stabilization by the fusion of LysSA12 CHAP to LysSA97 amidase domain plus CBD might not be the reason of the enzymatic improvement of Lys109. Next, the effects of temperature and pH on the lytic activity of Lys109 were evaluated to determine the optimum working conditions of Lys109 (Figures 4A,B). The maximal activity of Lys109 was exhibited at 25–37°C and pH 6.5–9.0. The wide optimum pH range of Lys109 suggest that Lys109 can be used for a wide variety of applications, including foods associated with a high risk for S. aureus contamination, such as milk products (Marino et al., 2000) and disinfectants for hospital cleaning (Dancer, 2008).

The antimicrobial activity of Lys109 was examined against staphylococcal strains other Gram-positive and Gram-negative bacteria with different amounts of endolysins, and was compared to that of LysSA12 (Table 1). Lys109 displayed effective lytic activity against all tested staphylococcal strains but not the other Gram-positive and Gram-negative bacteria tested. Although Lys109 has the same antibacterial spectrum as LysSA12, Lys109 showed higher lytic activity against most staphylococcal strains tested compared with LysSA12. These results demonstrated that Lys109 retains the specificity of the original endolysin but has stronger antimicrobial properties.

The comparative antibacterial activity of Lys109 and LysSA12 against a MRSA (S. aureus CCARM 3090) strain was examined at various concentrations in milk. Milk was chosen for the test because they have been frequently implicated in staphylococcal foodborne illnesses (Kadariya et al., 2014). In milk artificially contaminated with S. aureus, treatment with LysSA12 did not show any reduction in CFU at all tested concentrations even though LysSA12 exerted lytic activity at 100 nM in reaction buffer (Figure 5A), demonstrating the importance of in vivo experiments to evaluate the potential of a new antimicrobial. Similarly, LysH5, a LysSA12 homolog, could not kill bacteria cells in milk at 0.15 μM, and only 1-log reduction was observed with 0.8 μM LysH5 (Obeso et al., 2008; García et al., 2010). On the other hand, treatment with 300 nM of Lys109 showed an apparent inhibitory effect on S. aureus within 15 min and resulted in a 2-log reduction of bacterial cells after 1h (Figures 5B,C). In addition, the number of S. aureus in milk decreased below the detection limit with 900 nM Lys109 within 45 min (Figure 5B), indicating improved antimicrobial activity of the chimeric endolysin compared to parental endolysins. These findings showed that Lys109 has high staphylolytic activity in a complex biomatrix as well as in buffer and suggest that Lys109 has great potential as an antimicrobial agent to control S. aureus from dairy products.

Staphylococcus aureus is a common bacterium encountered in hospital-acquired and device-associated infections (Gwisai et al., 2017). Stainless steel is commonly used for medical devices, and previous reports revealed that MRSA can be viable and grow on it, leading to human infections (Michels et al., 2015; Craft et al., 2019). In this regard, the staphylolytic efficacy of Lys109 was evaluated and compared with its parental endolysin, LysSA12, on stainless steel coupons artificially contaminated with S. aureus CCARM 3090. At 100 nM, treatment with Lys109 on the surface of a stainless steel coupon caused the number of bacterial cells to decrease below the detection limit, whereas its parental endolysin was less effective in bacterial cell killing, resulting in only a 1-log bacterial reduction after 1 h (Figure 6). These results suggest the possible use of Lys109 as a disinfectant in hospital settings.