LysGH15, a novel bacteriophage lysin, protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection

Abstract

Phage-coded lysin is an enzyme that destroys the cell walls of bacteria. Phage lysin could be an alternative to conventional antibiotic therapy against pathogens that are resistant to multiple antibiotics. In this study, a novel staphylococcal phage, GH15, was isolated, and the endogenous lytic enzyme (LysGH15) was expressed and purified. The lysin LysGH15 displayed a broad lytic spectrum; in vitro treatment killed a number of Staphylococcus aureus strains rapidly and completely, including methicillin-resistant S. aureus (MRSA). In animal experiments, a single intraperitoneal injection of LysGH15 (50 μg) administered 1 h after MRSA injections at double the minimum lethal dose was sufficient to protect mice (P < 0.01). Bacteremia in unprotected mice reached colony counts of about 10(7) CFU/ml within 3.5 h after challenge, whereas the mean colony count in lysin-protected mice was less than 10(4) CFU/ml (and ultimately became undetectable). These results indicate that LysGH15 can kill S. aureus in vitro and can protect mice efficiently from bacteremia in vivo. The phage lysin LysGH15 might be an alternative treatment strategy for infections caused by MRSA.

FIG. 1.

One-step growth curve of phage GH15 in MRSA strain W3275. GH15 was added at an MOI of 0.1 and allowed to adsorb to W3275 cells for 15 min at 4°C. The GH15-exposed bacterial cells were thoroughly washed, transferred into 10 ml fresh BHI medium, and cultured at 37°C with shaking at 200 rpm. The culture samples were harvested at regular intervals, and the quantity of phage particles was measured during the incubation.

FIG. 2.

The morphology of phage GH15 as revealed by transmission electron micrographs of GH15 negatively stained with 2% phosphotungstic acid. The phage has an isometrically hexagonal head 65 ± 3 nm in diameter, a contractile tail of approximately 125 ± 2 nm, and a double-layer baseplate about 60 nm in diameter. The head is always separated from the tail sheath by a collar. These morphological characteristics confirmed that the phage is a member of the family Myoviridae. The bar represents 200 nm.

FIG. 3.

Protein profiles of N-terminal His-6-tagged LysGH15 on a 12% Coomassie brilliant blue (CBB)-stained gel. The lanes were loaded as follows: lane 1, uninduced E. coli BL21 (Coden Plus) cells containing a pET15b-LysGH15 construct; lane 2, pET15b-LysGH15-containing E. coli BL21 (Coden Plus) cells induced with 1 mM IPTG at 25°C; lane 3, supernatant of the induced E. coli BL21 Coden Plus cells containing a pET15b-LysGH15 construct after being crushed; lane 4, the purified LysGH15 fraction eluted from Ni-NTA His-Bind slurry; lane M, molecular mass marker. The arrow indicates the purified LysGH15 protein of about 55 kDa.

FIG. 4.

Characteristics of LysGH15. Shown is the percent reduction in the turbidity of a MRSA W3275 suspension after treatment with 40 μg/ml lysin for 30 min. (A) pH profile of LysGH15 activity. (B) Temperature profile of lysin activity. The values represent means and standard deviations (SD) (n = 3).

FIG. 5.

Lytic activities of the lysin LysGH15 on various strains of S. aureus. LysGH15 caused specific killing of S. aureus. Log-phase cultures of different bacteria were exposed to LysGH15 (final concentration, 40 μg/ml) for 30 min. The results are displayed as the number of viable bacteria after buffer treatment divided by the number of viable bacteria after exposure to LysGH15. (A) MRSA strains. (B) MSSA strains. Averages of triplicate samples for each assay were used for the calculations. The error bars indicate SD.

FIG. 6.

Rescue of mice from lethal MRSA YB57 infection by LysGH15. The mice were inoculated i.p. with 2× MLD YB57. (A) One hour later, 50 μg (black diamonds), 10 μg (black triangles), or 5 μg (black squares) of lysin was injected into the mouse peritoneal cavity. Control mice (white squares) were treated with elution buffer under the same conditions. (B) LysGH15 (50 μg) or buffer was administered into the peritoneal cavities of mice at the indicated time intervals after challenge with 2× MLD YB57. Lysin was given at 1 h (black circles), 2 h (black squares), 3 h (black triangles), or 4 h (black diamond) after infection. As controls, infected mice were treated with buffer (white squares) under the same conditions. Each symbol represents the average of three experiments.

FIG. 7.

Colony counts of blood samples at regular intervals. At the indicated times, bacterial counts (CFU/ml) in five mice treated with either 50 μg of LysGH15 (black symbols) or buffer (white symbols) were determined from peripheral blood samples (10 μl) taken from the caudal vein. Every line with black or white symbols represents the bacterial counts of a single mouse treated with lysin or buffer. The arrow indicates the moment at which the lysin or elution buffer was injected (1 h after challenge).