Combination Therapy of LysGH15 and Apigenin as a New Strategy for Treating Pneumonia Caused by Staphylococcus aureus

Abstract

Pneumonia is one of the most prevalent Staphylococcus aureus-mediated diseases, and the treatment of this infection is becoming challenging due to the emergence of multidrug-resistant S. aureus, especially methicillin-resistant S. aureus (MRSA) strains. It has been reported that LysGH15, the lysin derived from phage GH15, displays high efficiency and a broad lytic spectrum against MRSA and that apigenin can markedly diminish the alpha-hemolysin of S. aureus. In this study, the combination therapy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model. No mutual adverse influence was detected between LysGH15 and apigenin in vitro. In animal experiments, the combination therapy showed a more effective treatment effect than LysGH15 or apigenin monotherapy (P < 0.05). The bacterial load in the lungs of mice administered the combination therapy was 1.5 log units within 24 h after challenge, whereas the loads in unprotected mice or mice treated with apigenin or LysGH15 alone were 10.2, 4.7, and 2.6 log units, respectively. The combination therapy group showed the best health status, the lowest ratio of wet tissue to dry tissue of the lungs, the smallest amount of total protein and cells in the lung, the fewest pathological manifestations, and the lowest cytokine level compared with the other groups (P < 0.05). With regard to its better protective efficacy, the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumonia caused by MRSA. This paper reports the combination therapy of lysin and natural products derived from traditional Chinese medicine.

INTRODUCTION

Staphylococcus aureus is a ubiquitous and zoonotic pathogen that causes high morbidity and mortality in a variety of diseases, ranging from skin and soft tissue infections to necrotizing pneumonia and overwhelming sepsis (1, 2). S. aureus pneumonia is one of the most prevalent S. aureus-mediated diseases and accounts for 13.3% of all invasive S. aureus infections (3). Treatment of S. aureus infection has become increasingly difficult, given the prevalence of multidrug-resistant S. aureus strains, especially the widespread existence of methicillin-resistant S. aureus (MRSA) strains (4). MRSA strains are typically resistant to multiple antibiotics, including gentamicin, erythromycin, fluoroquinolones, and ofloxacin, among others (5). There are also reports of vancomycin-resistant S. aureus (VRSA), raising serious concerns within the medical community (6–8). Therefore, there is an urgent need for novel therapeutic strategies that are efficient against this pathogen.

Lysin, which is encoded by the phage (bacterial virus) genome at the end of the phage lytic life cycle to lyse the host cell, can rapidly and specifically lyse Gram-positive bacteria when exogenously applied (9). Because the bacterial cell wall is conserved and necessary for the life cycle, the current lack of bacterial resistance against lysin is not surprising (10). In addition, its species specificity or type specificity ensures that lysin will not affect the normal microflora (11). Thus, lysin may be a promising potential antibacterial agent. The phage lysin LysGH15 is specific for S. aureus and exhibits especially highly efficient lytic activity against MRSA strains in vitro and in vivo (12). In addition, to explore the molecular mechanism of this lytic activity, the structures of three individual domains of LysGH15 were determined (13). However, the disintegration of S. aureus that is caused by LysGH15 could result in the release of toxins, which leads to damage to the body and inflammation (14). Additionally, it has been demonstrated that the rapid release of abundant peptidoglycans, lipoteichoic acid, and exotoxins from S. aureus induces an inflammatory response and cytokine release (15, 16).

Several reports have demonstrated that traditional Chinese medicines target virulence and display therapeutic potential (17–19). In particular, apigenin, a natural flavonoid that is found in a variety of fruits and vegetables (20, 21), inhibits the transcription of the hla and agrA genes that encode alpha-hemolysin (Hla), which ultimately reduces the production of Hla in S. aureus (22) and plays an anti-inflammatory role (23). Hla is the most important S. aureus virulence factor and belongs to a channel-forming cytotoxin that can form a membrane-inserted heptamer to cause cell lysis (24, 25). However, apigenin showed only slight antimicrobial activity against S. aureus (22).

Based on this information, we hypothesized that the combination therapy of LysGH15 and apigenin might rapidly kill S. aureus and, at the same time, decrease the damage caused by Hla. Thus, the combination therapy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model.

MATERIALS AND METHODS

Bacterial strains and animals.

All S. aureus bacteria were routinely grown in brain heart infusion (BHI) broth (Becton, Dickinson and Company, USA) at 37°C with shaking at 200 rpm. For the hemolytic assay and Western blot assay, S. aureus was cultured to an optical density at 600 nm (OD₆₀₀) value of 0.3; then, the culture was cultured for 10 h after the addition of LysGH15 and/or apigenin. For other experiments, S. aureus was cultured to the exponential growth phase (at an OD₆₀₀ value of 0.6) at 37°C with shaking at 200 rpm.

All of the animal studies were conducted according to the National Guidelines for Experimental Animal Welfare (Ministry of Science and Technology of China, 2006) and approved by the Animal Welfare and Research Ethics Committee at Jilin University. The animals were treated humanely, and all possible effort was made to minimize suffering. The animal experiments were performed on 18- to 20-g (6 to 8 weeks of age) female C57BL/6J mice that were purchased from the Experimental Animal Center of Jilin University (Changchun, China). The mice were maintained in a temperature-controlled animal room with a 12-h light/12-h dark cycle. Food and fresh water were available ad libitum.

The preparation of LysGH15 and apigenin.

An Escherichia coli BL21(DE3) strain that expressed full-length LysGH15 protein was constructed by our laboratory, and LysGH15 was expressed and purified according to a previous report (13). Apigenin (see Fig. S1A in the supplemental material) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Apigenin and LysGH15 were dissolved in the same buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.5).

SAXS analysis of full-length LysGH15.

Small-angle X-ray scattering (SAXS) data on full-length LysGH15 were collected at the SIBYLS beamline (Advanced Light Source, Lorenz Berkeley National Lab, USA), as described in a previous report, with some modifications (26). Briefly, each sample was measured with four exposures (0.5, 1, 2, and 4 s) at 10°C at three concentrations (2.5 × 10³, 5.0 × 10³, and 1.0 × 10⁴ μg/ml) in a buffer composed of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% glycerin. The scattering intensity, I(Q), was measured for Q values (Q = 4πsinθ/λ, where 2θ is the scattering angle) that ranged from 0.01/Å to 0.3/Å. The resulting scattering curves for each sample were radially averaged, and buffer was subtracted. The data were integrated and scaled and the buffer was subtracted to obtain standard scattering curves. Multiple curves with different concentrations and different exposure times were scaled and averaged to generate an average scattering curve. The initial radius of gyration (R_g) values were analyzed by Primus (27) from the Guinier plot analysis. The P(r) distribution function was calculated with the program GNOM (28). The molecular weight was estimated directly using the P(r) distribution function from the Web server (http://www.if.sc.usp.br/∼saxs/saxsmow.html) (29). Low-resolution shape reconstructions were modeled by GASBOR (30) from the calculated P(r) distribution curve.

Scanning electron microscopy of S. aureus.

The community-associated MRSA strain USA300-TCH1516 (USA300) was obtained from the American Type Culture Collection (ATCC) and used throughout this study. USA300 was grown to the exponential growth phase (OD₆₀₀ = 0.6) in BHI broth with shaking at 200 rpm at 37°C. The bacteria were collected and washed three times (5,500 × g for 1 min at 4°C) with phosphate buffer solution (PBS). LysGH15 (final concentration, 0.001 μg/ml) was added to S. aureus suspensions. Bacterial lysates were harvested by centrifugation (1,100 × g for 1 min) at 0, 1, or 2 min after LysGH15 treatment. Then, the bacterial lysates were fixed with glutaraldehyde and were dehydrated and freeze dried for scanning electron microscopy (SEM) (Hitachi S-3400N, Hitachi High-Technologies Europe GmbH, Krefeld, Germany).

Measurement of the mutual influence of LysGH15 and apigenin in vitro.

The MRSA strain USA300 was cultured in BHI medium at 37°C with shaking at 200 rpm. Bacteria at the exponential phase (OD₆₀₀ = 0.6) were collected and washed three times with PBS (5,500 × g for 1 min at 4°C). To detect the influence of apigenin on the lytic activity of LysGH15, LysGH15 and apigenin were added to S. aureus suspensions simultaneously at a final concentration of 50 μg/ml and 8 μg/ml, respectively, based on previous studies with modification (12). This mixture was incubated at 37°C, and the absorption at 600 nm was measured for 10 min at 1-min intervals. The OD₆₀₀ of S. aureus suspensions treated with 50 μg/ml LysGH15 alone, 8 μg/ml apigenin alone, or buffer alone was also determined.

Hemolytic activity was measured as described previously using rabbit erythrocytes (31). Briefly, LysGH15 and apigenin were added to S. aureus cultures simultaneously. LysGH15 was added to USA300 cultures at a final concentration of 25, 50, or 100 μg/ml. Apigenin was added to USA300 cultures to obtain a final concentration of 8 μg/ml (22). The absorption of the bacterial culture solutions at 600 nm was measured at 10 h after culturing, and the supernatant was harvested via centrifugation (5,500 × g for 5 min at 4°C). A 0.1-ml supernatant of bacterial culture and 25 μl of defibrinated rabbit erythrocytes were added to PBS to achieve a final volume of 1 ml. The mixture was incubated for 30 min at 37°C; then, unlysed blood cells were removed by centrifugation (5,500 × g for 1 min at 4°C). Following centrifugation, the OD₄₅₀ of the supernatant was determined. The supernatants of pure USA300 culture served as 100% hemolysis control. The percentage of hemolysis was calculated by comparison with the control culture supernatant.

The content of alpha-hemolysin in the USA300 supernatants was analyzed by Western blotting (32). Samples (20 μl) of the supernatant were mixed with the Laemmli SDS sample buffer (5 μl), heat denatured (100°C, 8 min), and loaded onto standard 12% SDS-PAGE gels to separate the proteins (33). The Western blot protocol was performed as previously described (32) and according to the product guide for Millipore Immobilon Western chemiluminescent horseradish peroxidase (HRP) substrate. Antibodies to alpha-hemolysin were purchased from Sigma-Aldrich.

The combination therapy of LysGH15 and apigenin in vivo.

A model of mouse pneumonia caused by S. aureus was established using the USA300 strain, as previously described, with some modifications (34). Groups of five mice per experiment were anesthetized intraperitoneally with ketamine and xylazine. The anesthetized mice were then intranasally administered different inoculations of USA300 (5 × 10⁶, 5 × 10⁷, 5 × 10⁸, 5 × 10⁹, or 5 × 10¹⁰ CFU/mouse) to determine the minimal dose required to produce pneumonia and a 100% mortality rate over a 7-day follow-up period (the minimal lethal dose [MLD]). The number of dead mice was recorded daily.

The protective effects of LysGH15 and apigenin combination therapy or of either agent alone on the mouse pneumonia model were determined using survival studies. Following infection with 2× MLD (1 × 10⁸ CFU) of USA300, the mice were treated intranasally with only LysGH15 (60 μg/mice), subcutaneously with only apigenin (500 μg), or with the combination therapy at 1 h after infection (n = 10 in each group). The control group was treated with an equal amount of buffer under the same conditions. The survival rate was recorded every day for 2 weeks.

The health status of the mice in the pneumonia model was monitored according to the following symptoms to determine the progression of the disease state in the mice at 12 h after treatment with the different therapies: reduced physical activity, depressed spirit, unkempt fur, increased respiratory rate, dry and white apex nasi, white secretions around partially closed eyes, dyspnea, moribundity, and death.

To detect the bacterial load in the lungs and blood, the mice (n = 24 in each group) were treated with LysGH15, apigenin, or both at 1 h following the USA300 challenge (5 × 10⁷ CFU). Buffer-treated mice served as a control. Three mice from each treatment group were euthanized with an injection of Fatal Plus (sodium pentobarbital) at 1, 2, 3, 4, 5, 6, 12, and 24 h following the USA300 challenge. Blood samples were collected by cardiac puncture from euthanized animals and collected in tubes that contained 1 mM EDTA. Lung tissue samples were also collected from the euthanized animals. The lung tissue samples were weighed, suspended in filter-sterilized PBS, and homogenized with sterile mortars and motor-driven Teflon pestles (Kimble, USA). Bacterial loads in homogenates of the blood and lung tissue were measured by serial dilution and plating.

A histopathology analysis was performed on the lungs of USA300-infected (5 × 10⁷ CFU) mice that were treated with LysGH15, apigenin, or both as well as on the lungs of the uninfected mice. Three mice in each group (n = 6) were euthanized at 24 h after the USA300 challenge, and the lungs of these mice were removed and immediately placed in 4% formalin. The formalin-fixed tissues were processed and stained with hematoxylin and eosin (H&E) using a routine staining procedure and then analyzed by microscopy (35). The ratio of wet lung tissue to dry lung tissue (W/D ratio) was also calculated. The lungs of three mice in each group that were euthanized at 24 h after the USA300 challenge were removed and weighed immediately after the removal of the surface moisture. The lungs were then dehydrated at 80°C for 48 h in an oven (36). The weights of the dried lung tissue samples were determined.

The total cell counts, protein analysis, and quantification of cytokines in the bronchoalveolar lavage fluid (BALF) in the different groups (n = 5) were determined at 24 h after the USA300 challenge (5 × 10⁷ CFU). BALF was collected from the upper part of the trachea by lavage two times with 500 μl of PBS (37). The lavage samples from the mice were centrifuged (600 × g for 5 min at 4°C). The precipitated cells were resuspended in 2 ml of PBS and used to determine cell counts in a cytometer. In addition, the supernatant was collected to detect and quantify the amount of protein and cytokines. Protein concentrations were measured using a bicinchoninic acid protein quantitative analysis kit (Thermo Scientific), and the cytokines were quantified using an enzyme-linked immunosorbent assay (ELISA) (eBioscience), according to the manufacturer's instructions (38).

Data analysis.

SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. The survival rates were assessed using a Fisher exact test; other experimental data were analyzed by a one-way analysis of variance. A P value of <0.05 was considered to be significant. Error bars represent the standard deviation.

RESULTS

The structural model and efficient lytic activity of LysGH15.

To characterize the structure of full-length LysGH15 in solution, LysGH15 was purified (see Fig. S2 in the supplemental material), and SAXS analysis was performed. The data indicated that LysGH15 was a monomer in solution. The low-resolution envelope that was generated for the shape of LysGH15 indicated that it assumed a V shape that was composed of three spatially separated domains, as shown in Fig. S1B in the supplemental material.

LysGH15 exhibited efficient lytic activity against the MRSA strain USA300 (Fig. 1). One minute after being treated with LysGH15, the morphology of the USA300 cells changed, and most of the cells appeared cracked compared with their normal morphology. Moreover, only bacterial debris could be observed at 2 min after the LysGH15 treatment.

FIG 1.

Observation of the highly efficient lytic activity of LysGH15: effects on the strain USA300 after treatment with LysGH15 for 0 (A), 1 (B), and 2 (C) min. The pictures were obtained by SEM. The bars indicate 1 μm.

No mutual influence exists between LysGH15 and apigenin.

As shown in Fig. 2A, no significant difference (P > 0.05) in lytic activity was noted between LysGH15 treatment alone (the OD₆₀₀ value of USA300 suspensions decreased from 0.785 to 0.141 within 10 min) and the combination therapy of LysGH15 and apigenin treatment (the OD₆₀₀ value of USA300 suspensions decreased from 0.839 to 0.258 within 10 min). In addition, the other S. aureus strains (12) that express alpha-hemolysin (see Fig. S3 in the supplemental material) that were used in this study were tested by this method, and apigenin did not affect the bactericidal activity of LysGH15 on S. aureus.

FIG 2.

The mutual influence of LysGH15 and apigenin in vitro. (A) The influence of apigenin on LysGH15. Suspensions of S. aureus USA300 with the following treatments: untreated (●), buffer (■), apigenin alone (▲), LysGH15 alone (▼), and cotreatment with both LysGH15 and apigenin (◆). The OD₆₀₀ of each group was detected at the indicated times. Each symbol represents the mean of the results of three independent experiments. (B) Hemolytic assays. The influence of LysGH15 on apigenin was measured by hemolytic assay. Rabbit erythrocytes were treated with supernatants derived from the following different USA300 cultures: untreated USA300 cultures (positive control) (a), erythrocyte suspensions (negative control) (b), buffer that was shared by LysGH15 and apigenin (c), and cultures treated with 25 μg/ml LysGH15 (d), 50 μg/ml LysGH15 (e), 100 μg/ml LysGH15 (f), 8 μg/ml apigenin (g), 8 μg/ml apigenin and 25 μg/ml LysGH15 (h), 8 μg/ml apigenin and 50 μg/ml LysGH15 (i), and 8 μg/ml apigenin and 100 μg/ml LysGH15 (j). (C) Western blot analysis. The influence of LysGH15 on apigenin was measured by Western blot analysis. Lane 1 represents the untreated supernatant; lane 2 represents the supernatant treated with buffer; lanes 3, 4, and 5 represent supernatants treated with 25, 50, and 100 μg/ml LysGH15, respectively; lane 6 represents the supernatant treated with 8 μg/ml apigenin; and lanes 7, 8, and 9 represent supernatants that were cotreated with 8 μg/ml apigenin and 25, 50, and 100 μg/ml LysGH15, respectively.

As shown in Fig. 2B, the USA300 culture supernatant, which contains alpha-hemolysin, induces the hemolysis of rabbit erythrocytes. The hemolytic activity of USA300 culture supernatants in the presence of apigenin and/or LysGH15 was detected. When apigenin was added to the USA300 culture at a final concentration of 8 μg/ml, the hemolytic rate was only 2% that of the control culture supernatant. After treatment with LysGH15 alone at a final concentration of 25, 50, or 100 μg/ml, the hemolytic rates of the USA300 culture supernatants were 86.5%, 67.4%, and 44.78%, respectively. However, no detectable hemolysis was noted when the rabbit erythrocytes were incubated with the combination of apigenin (8 μg/ml) and LysGH15 (50 μg/ml or 100 μg/ml) simultaneously. The other S. aureus strains that expressed alpha-hemolysin were also tested by this method in this study, and similar results were obtained.

The combination of LysGH15 and apigenin shows better protective efficacy than monotherapy in the mouse pneumonia model.

Intranasal injection of 5 × 10⁷, 5 × 10⁸, 5 × 10⁹, or 5 × 10¹⁰ CFU of USA300 per mouse was sufficient to produce a 100% mortality rate within 3 days. The average MLD of USA300 was determined to be 5 × 10⁷ CFU. In addition, the infected mice developed the clinical symptoms of pneumonia (difficulty breathing and slow action). Gross inspection indicated that the lung tissue of the infected mice was crimson and exhibited a swollen texture (Fig. 3). Histopathological observation of the lungs from an infected mouse revealed serious pathological injury. A significant accumulation of inflammatory cells (dark blue or purple) and pink slurry in the alveolar space was noted (Fig. 3). The alveolar wall exhibited telangiectasia and congestion. All of these histopathological alterations are typical features of pneumonia.

FIG 3.

Gross pathological changes and histopathology of lung tissue. C57BL/6J mice infected with 5 × 10⁷ CFU of strain USA300 were treated with LysGH15 and/or apigenin. At 24 h after infection, the lungs were removed from the euthanized mice. The tissues were stained with hematoxylin and eosin.

LysGH15 or apigenin monotherapy and the combination therapy were administered to determine the therapeutic effect at 1 h after the USA300 challenge. As shown in Fig. 4A, the combination therapy of LysGH15 (60 μg) and apigenin (500 μg) was sufficient to protect mice against S. aureus pneumonia. In contrast, the group that was treated with LysGH15 alone (60 μg) exhibited an 80% survival rate. However, all of the mice in the groups that were treated with apigenin alone or buffer were dead at 2 and 3 days, respectively. Additionally, the mice that were treated by the combination therapy were healthier than the mice treated with LysGH15 or apigenin alone at 12 h after treatment (Fig. 4B).

FIG 4.

Combination therapy rescued mice from fatal S. aureus pneumonia. (A) Rescue of mice from lethal MRSA USA300 infection by LysGH15 and/or apigenin. C57BL/6J mice were infected with 1 × 10⁸ CFU of strain USA300 and then intranasally treated (1 h after infection) with buffer (●), 500 μg apigenin (■), 60 μg LysGH15 (▲), or a combination therapy of 500 μg apigenin and 60 μg LysGH15 (▼). (B) Combination therapy improved the health status of mice in the S. aureus pneumonia model. After 1 h of infection with strain USA300 (5 × 10⁷ CFU), LysGH15 and/or apigenin was administered, respectively, intranasally and by subcutaneous injection. The mice were scored for their states of health on a scale of 5 to 0 based on disease progression at 12 h after treatment with a specific therapy. A score of 5 indicated normal health and an unremarkable condition. Slight illness was defined as decreased physical activity and ruffled fur and was scored as 4. Moderate illness was defined as lethargy and a hunched back and was scored as 3. Severe illness was defined as the aforementioned signs plus exudative accumulation around partially closed eyes and was scored as 2. A moribund state was scored as 1; death was scored as 0. Each dot indicates the state of health of a single mouse. **, P < 0.01. (C) Bacterial load in the lungs 24 h after infection. Twenty-four hours after infection with strain USA300 (5 × 10⁷ CFU), the lungs were isolated and homogenized. The bacterial burden of the lung homogenates was determined via serial dilution plating: untreated (●), apigenin treated (▲), LysGH15 treated (■), and cotreatment with both LysGH15 and apigenin (▼). (D) Bacterial load in the blood at 24 h after infection by strain USA300 (5 × 10⁷ CFU). Blood was drawn by cardiac puncture from euthanized animals, and bacteremia was examined: untreated (●), apigenin treated (▲), LysGH15 treated (■), and cotreatment with both LysGH15 and apigenin (▼). Each symbol represents the average of the results of three experiments.

The ability of the different treatments to reduce the bacterial counts in the blood and lungs was investigated (Fig. 4C and D). No significant difference in the bacterial counts in the blood was noted between the groups treated with LysGH15 alone and those treated with the combination therapy (P > 0.05). The bacterial counts in the blood could not be detected at 24 h after treatment with either LysGH15 alone or the combination therapy. The bacterial loads in the lungs of the groups that were treated with LysGH15 alone and those treated with the combination therapy decreased to 3.0 × 10² CFU/mg and 3.5 × 10¹ CFU/mg, respectively, at 24 h after treatment. The bacterial loads in the lungs and blood of the group that was treated with apigenin alone reached 1 × 10⁵ CFU/mg and 3.2 × 10² CFU/ml, respectively, at 24 h after treatment. Finally, the bacterial counts in the untreated mice increased to 3.5 × 10¹⁰ CFU/mg and 5.7 × 10⁵ CFU/ml in the lungs and blood, respectively, at 24 h after treatment, which ultimately caused death within 3 days.

Combination therapy alleviated the injury and inflammation of the lung tissue.

The morphology of the lung tissues is presented in Fig. 3. The lung tissues of USA300-infected mice without any treatment were crimson in color and hyperemic and exhibited a firm texture. In contrast, the lung tissues of the infected mice following treatment with LysGH15 alone or combination therapy were pink and supple, which was similar to the exterior of the lungs in healthy mice (Fig. 3). Based on the histopathological observations, the lungs in the mice treated with a combination of LysGH15 and apigenin showed no severe inflammation or other pathological changes. Apigenin alone only slightly relieved the pathological changes.

As shown in Fig. 5A, the W/D ratio of the group that was treated with the combination of LysGH15 and apigenin (3.96) was the closest to that of the healthy group (3.76), followed by that of the LysGH15-treated group (4.93). The W/D ratio of the apigenin-treated group (6.90) was similar to that of the buffer-treated group (7.37; P > 0.05). To determine the level of inflammation in the mouse pneumonia model after treatment with LysGH15 and/or apigenin, the total number of cells and the protein in the BALF were measured. As shown in Fig. 5B and C, the total number of cells and the protein in the group treated with the combination therapy (0.22 × 10⁶ cells and 1.1 mg/ml, respectively) were significantly reduced compared with those in the nontreated group (10.8 × 10⁶ cells and 2.6 mg/ml, respectively), and the values were lower than those in the groups treated with LysGH15 alone (0.6 × 10⁶ cells and 1.5 mg/ml, respectively; P < 0.05) and with apigenin alone (8.3 × 10⁶ cells and 2.0 mg/ml, respectively; P < 0.01). Furthermore, the tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 levels in the group that was treated with the combination therapy were the most similar to those of healthy mice, followed by those of the LysGH15-treated, apigenin-treated, and untreated groups, as shown in Fig. 6.

FIG 5.

Determination of the W/D ratio, total protein levels in the BALF, and total cell numbers in the BALF. C57BL/6J mice infected with 5 × 10⁷ CFU of strain USA300 were treated with LysGH15 and/or apigenin. At 24 h after infection, the lung W/D ratio (A), total protein levels in the BALF (B), and total cell numbers in the BALF (C) were determined. Compared with the control group: ###, P < 0.001; ##, P < 0.01. Compared with S. aureus alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6.

Cytokine levels in the bronchoalveolar lavage fluid. C57BL/6J mice infected with 5 × 10⁷ CFU of strain USA300 were treated with LysGH15 and/or apigenin. At 24 h after infection, the levels of these cytokines in the BALF were determined: TNF-α (A); IL-1β (B); IL-6 (C). Compared with the control group: ###, P < 0.001; ##, P < 0.01. Compared with the untreated group: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

DISCUSSION

In this study, the biochemical characterization and lytic activity of LysGH15 were determined. In addition, the combination therapy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model. The data indicated that the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumonia caused by MRSA.

The structures of three individual domains of LysGH15 have been determined (13). To further understand LysGH15, the structure of the integral LysGH15 in solution was detected by SAXS. The results indicated that LysGH15 formed a V-shaped surface structure in solution and that the three domains of LysGH15 were relatively independent. This finding is consistent with the results of a previous study that demonstrated that single CHAP and SH3b domains displayed lytic and binding activity, respectively (13). SEM analysis further indicated that LysGH15 could lyse USA300 strains completely in only a few seconds. Together with a previous study (12), these data demonstrated that LysGH15 possesses highly efficient bactericidal activity in vitro and in vivo.

The mutual influence of LysGH15 and apigenin was first determined in vitro. The results indicated that apigenin had no effect on the lytic activity of LysGH15. In addition, LysGH15 and apigenin exhibited a slightly synergistic effect in a hemolysis inhibition test. The secretion of toxins by S. aureus triggers hemolytic activity in erythrocytes, and alpha-hemolysin is the most important of these toxins (39). Although LysGH15 does not inhibit the secretion and assembly of alpha-hemolysin, it does reduce the amount of S. aureus in a lytic manner, which ultimately decreases the production of alpha-hemolysin.

A previous study demonstrated that a single intraperitoneal injection of LysGH15 (50 μg) was sufficient to protect mice against MRSA bacteremia (12). In this study, LysGH15 alone (60 μg) also protected mice (80%) from MRSA pneumonia. Thus, the phage lysin LysGH15 exhibits therapeutic potential for treating pneumonia caused by MRSA.

Although apigenin reduces the production of alpha-hemolysin by S. aureus, it showed only slight antimicrobial activity against S. aureus (22). Because of this characteristic, apigenin is thought to reduce the selective pressure against the growth of this bacterial species (40). It has been reported that the curative dose of apigenin against S. aureus pneumonia is 50 mg/kg of body weight in mice (22). We found that a decreased dose of apigenin (500 μg) did not provide any protective effect against S. aureus pneumonia in mice. The load of S. aureus in the lungs and blood was only slightly reduced compared with that in untreated mice. Nevertheless, we found that this subtherapeutic dose of apigenin significantly improved the capacity of LysGH15 treatment against S. aureus pneumonia. In contrast, the combination of apigenin (500 μg) and LysGH15 (60 μg) was sufficient to protect mice (100%). More importantly, both the health score and the lung pathology exhibited substantial improvement when the combination therapy was administered compared with treatment with LysGH15 (60 μg) or apigenin (500 μg) alone. In addition, the colony count of bacteria in the lung and blood was reduced significantly when the combination therapy was administered. This result can be mainly attributed to the lytic activity of LysGH15 on S. aureus.

Acute pneumonia is accompanied by the activation of the inflammatory system, which is characterized by the presence of inflammatory cells and proteinaceous fluid in air spaces (41). Acute injury to the alveolocapillary barrier can result in increased permeability and can also cause protein-rich exudative edema (42). Additionally, TNF-α, IL-1β, and IL-6 are multifunctional cytokines that control systems that are involved in cell proliferation, inflammation, and immunity at both the local and systemic level (43). These proinflammatory cytokines cause severe cell injury and tissue damage (44). Compared with monotherapy, the combination therapy of LysGH15 and apigenin not only demonstrated an exceedingly curative effect in terms of the total cell numbers and total protein levels in the BALF but also brought down the proinflammatory cytokines. This result can be mainly attributed to the anti-inflammatory and antitoxin effects of apigenin (22, 45).

Due to the prevalence of multidrug-resistant bacteria, especially the emergence of superbugs (46, 47), there is an urgent need for novel therapeutic agents that are directed against such pathogens. Phage lysin (48) and the natural products derived from traditional Chinese medicine (49) are considered novel therapeutic agents because their mechanism of action is different from that of antibiotics. Additionally, several studies have reported on the efficacy of the combination therapy of lysin and antibiotics (50). In addition, other studies have investigated the synergy of natural products with antibiotics (51). This paper reports the systematic in vitro and in vivo investigation and evaluation of the combination therapy of lysin and natural products derived from traditional Chinese medicine.

Overall, the data presented in this study showed that the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumonia that is caused by MRSA. This research provides evidence for the viability of the combination of lysin and natural products derived from traditional Chinese medicine.