Novel Chimeric Lysin with High-Level Antimicrobial Activity against Methicillin-Resistant Staphylococcus aureus In Vitro and In Vivo

Bacterial strains.Bacterial strains (see Table S2 in the supplemental material) used in this work were routinely grown at 37°C. All of the staphylococcal strains were grown in Trypticase soy broth (TSB) medium. Clinical isolates of S. aureus with different genetic backgrounds were collected from various sources in China in order to cover all SCCmec types. MRSA strains were determined by PCR against mecA and femB, as described previously (25), with primers MecA-F (5′-GTAGAAATGACTGAACGTCCGATAA-3′) and MecA-R (5′-CCAATTCCACATTGTTTCGGTCTAA-3′) and FemB-F (5′-TTACAGAGTTAACTGTTACC-3′) and FemB-R (5′-ATACAAATCCAGCACGCTCT-3′), respectively. Once confirmed, their SCCmec types were further determined by multiplex PCR, as described previously (26). The Panton-Valentine leucocidin gene (lukF/lukS-PV) was determined by PCR according to the method described previously (27).

Because some lysins (28) were reported to be active against both S. aureus and streptococcal strains, Streptococcus thermophilus, Streptococcus sobrinus, Streptococcus pyogenes, and Streptococcus suis were tested to evaluate the specificity of ClyH. Other strains used include Lactobacillus acidophilus, Bifidobacterium dentium, Enterococcus faecalis, Enterococcus faecium, Enterobacter sakazakii, Salmonella enterica, Listeria monocytogenes, Pseudomonas aeruginosa, and Xanthomonas oryzae. All of these strains were cultured in brain heart infusion (BHI) medium. Bacillus cereus was tested as well but cultivated in Luria-Bertani (LB) medium. Escherichia coli BL21(DE3) was used for the cloning and expression of recombinant proteins.

Construction of gene-expressing plasmids.The chimeric lysin ClyH was constructed by fusing the N-terminal 157 amino acids of Ply187 (Pc) with the C-terminal 97 amino acids of phiNM3 lysin. To do this, the DNA fragment encoding the chimeric lysin was chemically synthesized by Songon Biotech (Shanghai, China). The resulting gene, corresponding to clyH, was cloned into pBAD24 vector with forward (-F) and reverse (-R) primers ClyH-F (5′-AAAAGAATTCATGGCACTGCCTAAAACGGGTAAAC-3′) and ClyH-R (5′-AAACTCGAGTTAAAACACTTCTTTCACAATC-3′) for expression of untagged ClyH and into pET28a(+) vector with primers pH-F (5′-TTAACCATGGGCATGGCACTGCCTAAAACG-3′) and pH-R (5′-TTAACTCGAGAAACACTTCTTTCACAATCAATC-3′) for expression of His-tagged ClyH (ClyH-His), respectively. To express the His-tagged parental CD (Pc-His), the gene fragment corresponding to Pc was cloned into pET28a(+) vector with primers PC-F (5′-AATTCCATGGGCATGGCACTGCCTAAAACG-3′) and PC-R (5′-TTAACTCGAGTGGTGGTGTAGGTTTCGGTTC-3′). After confirmation by sequencing, the correct plasmids were transformed into E. coli BL21(DE3) for expression.

Purification of recombinant proteins.The recombinant proteins were expressed by the E. coli BL21(DE3) strain in standard LB medium and purified following procedures described previously (24, 29), with minor modifications. ClyH was induced overnight in BL21(DE3) cells with l-arabinose in a final concentration of 0.2% at 20°C. Briefly, cells were harvested by centrifugation and resuspended in 20 mM phosphate buffer (pH 7.4). After sonication, the supernatant was collected by centrifugation at 10,000 × g for 30 min at 4°C. The supernatant was applied to a HiTrap Q Sepharose FF column (GE Healthcare) and then bound to a HiTrap SP Sepharose FF column (GE Healthcare) and eluted in a linear gradient from 0.02 M to 1 M NaCl solution. For the purification of ClyH-His as well as Pc-His, protein was expressed by inducing the bacteria with 1 mM isopropyl β-d-thiogalactoside (IPTG) when an optical density (OD) of 0.6 to 0.8 was reached. After induction, the bacteria were incubated overnight at 16°C to allow expression. Purification was achieved through a His₆ tag by using a nickel nitrilotriacetic acid column, by washing and elution with 60 and 265 mM imidazole solutions, respectively. Active fractions were pooled and dialyzed against 1× phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄·H₂O, 1.4 mM KH₂PO₄ [pH 7.4]). After quantitation by the Bradford assay, the purified proteins were stored at −80°C until use.

Quantification of ClyH activity.Lytic activity was measured as previously described (8), with some modifications. Briefly, S. aureus strain CCTCC AB91118 (also called AB918 for short) was grown to an optical density at 600 nm (OD₆₀₀) of 0.2 to 0.3, centrifuged, and resuspended in PBS (pH 7.4) to a final OD₆₀₀ of 1.0. Next, 100 μl of the purified ClyH in 2-fold serial dilutions was mixed with 100 μl of the bacterial suspension in 96-well plates (Perkin-Elmer), respectively. The drop in OD₆₀₀ was monitored by a microplate reader (Synergy H1; BioTek) for 60 min at 37°C. A unit of ClyH activity was defined as the highest dilution that decreased the absorbance by 50% within 15 min (8). The lytic activities of ClyH at different pH values were also measured using a universal buffer described before (30). The buffer was prepared by mixing equal parts of 20 mM boric acid and 20 mM phosphoric acid, followed by titration with sodium hydroxide from pH 2 to 12.

After 1 U of ClyH was mixed with the suspension of S. aureus strain AB918, the decrease in viable cells corresponding to the loss of turbidity was also tested by plating the aliquots from the lytic assay at various time points (5, 15, 30, and 60 min) to TSB agar for counting of CFU. The action of ClyH on the cell wall was monitored by thin-section transmission electron microscopy (Tecnai G2 20 TWIN transmission electron microscope; FEI, Hillsboro, OR). The bacterial suspensions were incubated with 1 U of ClyH at 37°C for 3, 5, and 10 min, respectively, and then the reaction was terminated by addition of 2.5% glutaraldehyde before the transmission electron microscopy (TEM) analysis.

To compare the activity of ClyH with those of lysostaphin and Pc-His, mid-log-phase cultures of randomly selected S. aureus strains were pelleted and resuspended in PBS to a final OD₆₀₀ of 1.0, respectively. One hundred microliters of ClyH or lysostaphin at the same concentration (0.16 μM) was added to the bacterial suspension (100 μl), respectively. The decrease in OD₆₀₀ was monitored by the spectrophotometer. To minimize the effect of His tag on the enzymatic activity, ClyH-His (1.2 μM) was used for comparison with Pc-His at the same concentration.

To determine the specificity of ClyH, the lytic activities of ClyH to various bacterial strains were measured as the drop in milli-OD₆₀₀ per minute (−mOD₆₀₀/min) in the first 15 min, as described elsewhere (31).

All of the experiments described above were performed in triplicate, and bacterial cells treated with PBS were used as the blank controls.

MIC determinations.MICs of antibiotics (penicillin, gentamicin, vancomycin, and oxacillin) and ClyH were determined by microtiter broth dilution as described by the Clinical and Laboratory Standards Institute (CLSI) (32). The MIC was defined as the lowest concentration of antibiotic producing inhibition of visible growth.

Immunological neutralization test.The neutralization effect of ClyH-specific antibodies to the activity of ClyH was tested by using a standard immunological protocol, as described previously (10). ClyH (200-U) solutions were injected into the peritoneal cavities of mice 3 times, with a 10-day interval between injections. Mouse sera were sampled 15 days after the last injection, and the serum titers were checked by enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-conjugated goat anti-mouse IgG. The detailed procedure of the ELISA was performed following the instructions of the manufacturer of a commercial ELISA kit (QF-Bio, Shanghai, China). Before the neutralization test, ClyH (about 0.5 U) was reacted with 80 μl of the ClyH-immunized mouse serum at 37°C for 15 min, using nonimmunized mouse serum and PBS as the controls. The assay to determine the neutralization effect then was performed immediately by testing the lytic activity of each mixture for S. aureus strain AM025 by the same procedure as the lytic activity assay described above.

Mouse protection experiments.All mouse experiments were conducted with the approval of the Animal Experiments Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (WIVA17201203). Female BALB/c mice (6 to 8 weeks old) were injected intraperitoneally with different concentrations of MRSA strain AM025 to determine the minimal lethal dose (MLD) that caused 100% mortality within 2 days. In the mouse protection assay, mice were inoculated intraperitoneally with 2× the MLD of AM025 cells and then divided into 3 groups randomly. Three hours after the challenge, two groups (6 each) received 180 U and 360 U (900 μg) of ClyH intraperitoneally, respectively, and the other group (n = 8) was injected with PBS buffer. Another group (n = 6) without MRSA infection received 540 U of ClyH only. The survival rates of all the groups were observed for 10 days after the infection. To check the toxicity of ClyH, 5 mice without injection of the bacteria were given the ClyH solution for 7 days (200 U/injection, one injection/day, for a total dose of 1,400 U), and the survival rate, their body weights, and activities were observed for 10 days after the last injection.