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

Feifei Xia,a Xin Li,a Bin Wang,a Pengjuan Gong,a Feng Xiao,a Mei Yang,a Lei Zhang,a Jun Song,a Liyuan Hu,a Mengjun Cheng,a

Changjiang Sun,a Xin Feng,a Liancheng Lei,a Songying Ouyang,b Zhi-Jie Liu,b Xinwei Li,a Jingmin Gu,a Wenyu Hana,c

Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, People’s Republic of Chinaa; National Laboratory ofBiomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of Chinab; Jiangsu Coinnovation Center for the Prevention andControl of Important Animal Infectious Disease and Zoonoses, Yangzhou, People’s Republic of Chinac

Pneumonia is one of the most prevalent Staphylococcus aureus-mediated diseases, and the treatment of this infection is becom-ing 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 spectrumagainst MRSA and that apigenin can markedly diminish the alpha-hemolysin of S. aureus. In this study, the combination ther-apy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model. No mutual adverse influencewas detected between LysGH15 and apigenin in vitro. In animal experiments, the combination therapy showed a more effectivetreatment effect than LysGH15 or apigenin monotherapy (P < 0.05). The bacterial load in the lungs of mice administered thecombination therapy was 1.5 log units within 24 h after challenge, whereas the loads in unprotected mice or mice treated withapigenin or LysGH15 alone were 10.2, 4.7, and 2.6 log units, respectively. The combination therapy group showed the best healthstatus, the lowest ratio of wet tissue to dry tissue of the lungs, the smallest amount of total protein and cells in the lung, the few-est pathological manifestations, and the lowest cytokine level compared with the other groups (P < 0.05). With regard to its bet-ter protective efficacy, the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumoniacaused by MRSA. This paper reports the combination therapy of lysin and natural products derived from traditional Chinesemedicine.

Staphylococcus aureus is a ubiquitous and zoonotic pathogenthat causes high morbidity and mortality in a variety of dis-

eases, ranging from skin and soft tissue infections to necrotizingpneumonia and overwhelming sepsis (1, 2). S. aureus pneumoniais one of the most prevalent S. aureus-mediated diseases and ac-counts for 13.3% of all invasive S. aureus infections (3). Treatmentof S. aureus infection has become increasingly difficult, given theprevalence of multidrug-resistant S. aureus strains, especially thewidespread existence of methicillin-resistant S. aureus (MRSA)strains (4). MRSA strains are typically resistant to multiple anti-biotics, including gentamicin, erythromycin, fluoroquinolones,and ofloxacin, among others (5). There are also reports of vanco-mycin-resistant S. aureus (VRSA), raising serious concerns withinthe medical community (6–8). Therefore, there is an urgent needfor novel therapeutic strategies that are efficient against thispathogen.

Lysin, which is encoded by the phage (bacterial virus) genomeat the end of the phage lytic life cycle to lyse the host cell, canrapidly and specifically lyse Gram-positive bacteria when exoge-nously applied (9). Because the bacterial cell wall is conserved andnecessary for the life cycle, the current lack of bacterial resistanceagainst lysin is not surprising (10). In addition, its species speci-ficity or type specificity ensures that lysin will not affect the normalmicroflora (11). Thus, lysin may be a promising potential antibac-terial agent. The phage lysin LysGH15 is specific for S. aureus andexhibits especially highly efficient lytic activity against MRSAstrains in vitro and in vivo (12). In addition, to explore the molec-ular mechanism of this lytic activity, the structures of three indi-vidual domains of LysGH15 were determined (13). However, thedisintegration of S. aureus that is caused by LysGH15 could resultin the release of toxins, which leads to damage to the body and

inflammation (14). Additionally, it has been demonstrated thatthe rapid release of abundant peptidoglycans, lipoteichoic acid,and exotoxins from S. aureus induces an inflammatory responseand cytokine release (15, 16).

Several reports have demonstrated that traditional Chinesemedicines target virulence and display therapeutic potential (17–19). In particular, apigenin, a natural flavonoid that is found in avariety of fruits and vegetables (20, 21), inhibits the transcriptionof 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 impor-tant S. aureus virulence factor and belongs to a channel-formingcytotoxin that can form a membrane-inserted heptamer to causecell lysis (24, 25). However, apigenin showed only slight antimi-crobial activity against S. aureus (22).

Based on this information, we hypothesized that the combina-

Received 9 August 2015 Accepted 10 October 2015

Accepted manuscript posted online 16 October 2015

Citation Xia F, Li X, Wang B, Gong P, Xiao F, Yang M, Zhang L, Song J, Hu L, ChengM, Sun C, Feng X, Lei L, Ouyang S, Liu Z-J, Li X, Gu J, Han W. 2016. Combinationtherapy of LysGH15 and apigenin as a new strategy for treating pneumoniacaused by Staphylococcus aureus. Appl Environ Microbiol 82:87–94.doi:10.1128/AEM.02581-15.

Editor: H. L. Drake

Address correspondence to Jingmin Gu, jingmin0629@163.com, or Wenyu Han,hanwy@jlu.edu.cn.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02581-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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tion therapy of LysGH15 and apigenin might rapidly kill S. aureusand, at the same time, decrease the damage caused by Hla. Thus,the combination therapy of LysGH15 and apigenin was evaluatedin vitro and in a mouse S. aureus pneumonia model.

MATERIALS AND METHODSBacterial strains and animals. All S. aureus bacteria were routinely grownin brain heart infusion (BHI) broth (Becton, Dickinson and Company,USA) at 37°C with shaking at 200 rpm. For the hemolytic assay and West-ern 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 theaddition of LysGH15 and/or apigenin. For other experiments, S. aureuswas cultured to the exponential growth phase (at an OD600 value of 0.6) at37°C with shaking at 200 rpm.

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

The preparation of LysGH15 and apigenin. An Escherichia coliBL21(DE3) strain that expressed full-length LysGH15 protein was con-structed by our laboratory, and LysGH15 was expressed and purified ac-cording to a previous report (13). Apigenin (see Fig. S1A in the supple-mental material) was purchased from the National Institutes for Food andDrug Control (Beijing, China). Apigenin and LysGH15 were dissolved inthe 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 beam-line (Advanced Light Source, Lorenz Berkeley National Lab, USA), asdescribed in a previous report, with some modifications (26). Briefly, eachsample was measured with four exposures (0.5, 1, 2, and 4 s) at 10°C atthree concentrations (2.5 � 103, 5.0 � 103, and 1.0 � 104 �g/ml) in abuffer 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 �4�sin�/�, where 2� is the scattering angle) that ranged from 0.01/Å to0.3/Å. The resulting scattering curves for each sample were radially aver-aged, and buffer was subtracted. The data were integrated and scaled andthe buffer was subtracted to obtain standard scattering curves. Multiplecurves with different concentrations and different exposure times werescaled and averaged to generate an average scattering curve. The initialradius of gyration (Rg) values were analyzed by Primus (27) from theGuinier plot analysis. The P(r) distribution function was calculated withthe program GNOM (28). The molecular weight was estimated directlyusing the P(r) distribution function from the Web server (http://www.if.sc.usp.br/�saxs/saxsmow.html) (29). Low-resolution shape reconstruc-tions were modeled by GASBOR (30) from the calculated P(r) distribu-tion curve.

Scanning electron microscopy of S. aureus. The community-asso-ciated MRSA strain USA300-TCH1516 (USA300) was obtained fromthe American Type Culture Collection (ATCC) and used throughoutthis study. USA300 was grown to the exponential growth phase(OD600 � 0.6) in BHI broth with shaking at 200 rpm at 37°C. Thebacteria were collected and washed three times (5,500 � g for 1 min at4°C) with phosphate buffer solution (PBS). LysGH15 (final concen-tration, 0.001 �g/ml) was added to S. aureus suspensions. Bacterial ly-sates were harvested by centrifugation (1,100 � g for 1 min) at 0, 1, or 2min after LysGH15 treatment. Then, the bacterial lysates were fixed withglutaraldehyde and were dehydrated and freeze dried for scanning elec-tron microscopy (SEM) (Hitachi S-3400N, Hitachi High-TechnologiesEurope GmbH, Krefeld, Germany).

Measurement of the mutual influence of LysGH15 and apigenin invitro. The MRSA strain USA300 was cultured in BHI medium at 37°Cwith 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 at4°C). To detect the influence of apigenin on the lytic activity of LysGH15,LysGH15 and apigenin were added to S. aureus suspensions simultane-ously at a final concentration of 50 �g/ml and 8 �g/ml, respectively, basedon previous studies with modification (12). This mixture was incubated at37°C, and the absorption at 600 nm was measured for 10 min at 1-minintervals. The OD600 of S. aureus suspensions treated with 50 �g/mlLysGH15 alone, 8 �g/ml apigenin alone, or buffer alone was also deter-mined.

Hemolytic activity was measured as described previously using rabbiterythrocytes (31). Briefly, LysGH15 and apigenin were added to S. aureuscultures simultaneously. LysGH15 was added to USA300 cultures at afinal concentration of 25, 50, or 100 �g/ml. Apigenin was added toUSA300 cultures to obtain a final concentration of 8 �g/ml (22). Theabsorption of the bacterial culture solutions at 600 nm was measured at 10h 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 and25 �l of defibrinated rabbit erythrocytes were added to PBS to achieve afinal 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 minat 4°C). Following centrifugation, the OD450 of the supernatant was de-termined. The supernatants of pure USA300 culture served as 100% he-molysis control. The percentage of hemolysis was calculated by compari-son with the control culture supernatant.

The content of alpha-hemolysin in the USA300 supernatants was an-alyzed by Western blotting (32). Samples (20 �l) of the supernatant weremixed with the Laemmli SDS sample buffer (5 �l), heat denatured (100°C,8 min), and loaded onto standard 12% SDS-PAGE gels to separate theproteins (33). The Western blot protocol was performed as previouslydescribed (32) and according to the product guide for Millipore Immo-bilon Western chemiluminescent horseradish peroxidase (HRP) sub-strate. Antibodies to alpha-hemolysin were purchased from Sigma-Al-drich.

The combination therapy of LysGH15 and apigenin in vivo. A modelof mouse pneumonia caused by S. aureus was established using theUSA300 strain, as previously described, with some modifications (34).Groups of five mice per experiment were anesthetized intraperitoneallywith ketamine and xylazine. The anesthetized mice were then intranasallyadministered different inoculations of USA300 (5 � 106, 5 � 107, 5 � 108,5 � 109, or 5 � 1010 CFU/mouse) to determine the minimal dose requiredto produce pneumonia and a 100% mortality rate over a 7-day follow-upperiod (the minimal lethal dose [MLD]). The number of dead mice wasrecorded daily.

The protective effects of LysGH15 and apigenin combination therapyor of either agent alone on the mouse pneumonia model were determinedusing survival studies. Following infection with 2� MLD (1 � 108 CFU)of USA300, the mice were treated intranasally with only LysGH15 (60�g/mice), subcutaneously with only apigenin (500 �g), or with the com-bination therapy at 1 h after infection (n � 10 in each group). The controlgroup was treated with an equal amount of buffer under the same condi-tions. The survival rate was recorded every day for 2 weeks.

The health status of the mice in the pneumonia model was monitoredaccording to the following symptoms to determine the progression of thedisease state in the mice at 12 h after treatment with the different thera-pies: reduced physical activity, depressed spirit, unkempt fur, increasedrespiratory rate, dry and white apex nasi, white secretions around partiallyclosed eyes, dyspnea, moribundity, and death.

To detect the bacterial load in the lungs and blood, the mice (n � 24 ineach group) were treated with LysGH15, apigenin, or both at 1 h followingthe USA300 challenge (5 � 107 CFU). Buffer-treated mice served as acontrol. Three mice from each treatment group were euthanized with aninjection of Fatal Plus (sodium pentobarbital) at 1, 2, 3, 4, 5, 6, 12, and 24

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h following the USA300 challenge. Blood samples were collected by car-diac puncture from euthanized animals and collected in tubes that con-tained 1 mM EDTA. Lung tissue samples were also collected from theeuthanized animals. The lung tissue samples were weighed, suspended infilter-sterilized PBS, and homogenized with sterile mortars and motor-driven Teflon pestles (Kimble, USA). Bacterial loads in homogenates ofthe 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, orboth as well as on the lungs of the uninfected mice. Three mice in eachgroup (n � 6) were euthanized at 24 h after the USA300 challenge, and thelungs of these mice were removed and immediately placed in 4% forma-lin. The formalin-fixed tissues were processed and stained with hematox-ylin and eosin (H&E) using a routine staining procedure and then ana-lyzed 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 groupthat were euthanized at 24 h after the USA300 challenge were removedand weighed immediately after the removal of the surface moisture. Thelungs were then dehydrated at 80°C for 48 h in an oven (36). The weightsof the dried lung tissue samples were determined.

The total cell counts, protein analysis, and quantification of cytokinesin the bronchoalveolar lavage fluid (BALF) in the different groups (n � 5)were determined at 24 h after the USA300 challenge (5 � 107 CFU). BALFwas collected from the upper part of the trachea by lavage two times with500 �l 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, thesupernatant was collected to detect and quantify the amount of proteinand cytokines. Protein concentrations were measured using a bicin-choninic acid protein quantitative analysis kit (Thermo Scientific), andthe cytokines were quantified using an enzyme-linked immunosorbentassay (ELISA) (eBioscience), according to the manufacturer’s instructions(38).

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

RESULTSThe structural model and efficient lytic activity of LysGH15. Tocharacterize 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 thatLysGH15 was a monomer in solution. The low-resolution enve-lope that was generated for the shape of LysGH15 indicated that itassumed a V shape that was composed of three spatially separateddomains, as shown in Fig. S1B in the supplemental material.

LysGH15 exhibited efficient lytic activity against the MRSAstrain USA300 (Fig. 1). One minute after being treated withLysGH15, the morphology of the USA300 cells changed, and most

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 followingtreatments: untreated (�), buffer (�), apigenin alone (Œ), LysGH15 alone (�), and cotreatment with both LysGH15 and apigenin (}). The OD600 of each groupwas detected at the indicated times. Each symbol represents the mean of the results of three independent experiments. (B) Hemolytic assays. The influence ofLysGH15 on apigenin was measured by hemolytic assay. Rabbit erythrocytes were treated with supernatants derived from the following different USA300cultures: 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 �g/ml LysGH15 (d), 50 �g/ml LysGH15 (e), 100 �g/ml LysGH15 (f), 8 �g/ml apigenin (g), 8 �g/ml apigenin and 25 �g/ml LysGH15(h), 8 �g/ml apigenin and 50 �g/ml LysGH15 (i), and 8 �g/ml apigenin and 100 �g/ml LysGH15 (j). (C) Western blot analysis. The influence of LysGH15 onapigenin was measured by Western blot analysis. Lane 1 represents the untreated supernatant; lane 2 represents the supernatant treated with buffer; lanes 3, 4, and5 represent supernatants treated with 25, 50, and 100 �g/ml LysGH15, respectively; lane 6 represents the supernatant treated with 8 �g/ml apigenin; and lanes7, 8, and 9 represent supernatants that were cotreated with 8 �g/ml apigenin and 25, 50, and 100 �g/ml LysGH15, respectively.

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 �m.

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of the cells appeared cracked compared with their normal mor-phology. Moreover, only bacterial debris could be observed at 2min after the LysGH15 treatment.

No mutual influence exists between LysGH15 and apigenin.As shown in Fig. 2A, no significant difference (P 0.05) in lyticactivity was noted between LysGH15 treatment alone (the OD600

value of USA300 suspensions decreased from 0.785 to 0.141within 10 min) and the combination therapy of LysGH15 andapigenin treatment (the OD600 value of USA300 suspensions de-creased from 0.839 to 0.258 within 10 min). In addition, the otherS. aureus strains (12) that express alpha-hemolysin (see Fig. S3 inthe supplemental material) that were used in this study were testedby this method, and apigenin did not affect the bactericidal activ-ity of LysGH15 on S. aureus.

As shown in Fig. 2B, the USA300 culture supernatant, whichcontains alpha-hemolysin, induces the hemolysis of rabbit eryth-rocytes. The hemolytic activity of USA300 culture supernatants inthe presence of apigenin and/or LysGH15 was detected. Whenapigenin was added to the USA300 culture at a final concentrationof 8 �g/ml, the hemolytic rate was only 2% that of the controlculture supernatant. After treatment with LysGH15 alone at a fi-nal concentration of 25, 50, or 100 �g/ml, the hemolytic ratesof the USA300 culture supernatants were 86.5%, 67.4%, and44.78%, respectively. However, no detectable hemolysis wasnoted when the rabbit erythrocytes were incubated with the com-bination of apigenin (8 �g/ml) and LysGH15 (50 �g/ml or 100�g/ml) simultaneously. The other S. aureus strains that expressedalpha-hemolysin were also tested by this method in this study, andsimilar results were obtained.

The combination of LysGH15 and apigenin shows betterprotective efficacy than monotherapy in the mouse pneumoniamodel. 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 wasdetermined to be 5 � 107 CFU. In addition, the infected micedeveloped the clinical symptoms of pneumonia (difficulty breath-ing and slow action). Gross inspection indicated that the lungtissue of the infected mice was crimson and exhibited a swollentexture (Fig. 3). Histopathological observation of the lungs froman infected mouse revealed serious pathological injury. A signifi-cant accumulation of inflammatory cells (dark blue or purple)and pink slurry in the alveolar space was noted (Fig. 3). The alve-olar wall exhibited telangiectasia and congestion. All of these his-topathological alterations are typical features of pneumonia.

LysGH15 or apigenin monotherapy and the combination ther-apy were administered to determine the therapeutic effect at 1 hafter the USA300 challenge. As shown in Fig. 4A, the combinationtherapy of LysGH15 (60 �g) and apigenin (500 �g) was sufficientto protect mice against S. aureus pneumonia. In contrast, thegroup that was treated with LysGH15 alone (60 �g) exhibited an80% survival rate. However, all of the mice in the groups that weretreated with apigenin alone or buffer were dead at 2 and 3 days,respectively. Additionally, the mice that were treated by the com-bination therapy were healthier than the mice treated withLysGH15 or apigenin alone at 12 h after treatment (Fig. 4B).

The ability of the different treatments to reduce the bacterialcounts in the blood and lungs was investigated (Fig. 4C and D). Nosignificant difference in the bacterial counts in the blood wasnoted between the groups treated with LysGH15 alone and thosetreated with the combination therapy (P 0.05). The bacterial

counts in the blood could not be detected at 24 h after treatmentwith either LysGH15 alone or the combination therapy. The bac-terial loads in the lungs of the groups that were treated withLysGH15 alone and those treated with the combination therapydecreased to 3.0 � 102 CFU/mg and 3.5 � 101 CFU/mg, respec-tively, at 24 h after treatment. The bacterial loads in the lungs andblood of the group that was treated with apigenin alone reached1 � 105 CFU/mg and 3.2 � 102 CFU/ml, respectively, at 24 h aftertreatment. Finally, the bacterial counts in the untreated mice in-creased to 3.5 � 1010 CFU/mg and 5.7 � 105 CFU/ml in the lungsand blood, respectively, at 24 h after treatment, which ultimatelycaused death within 3 days.

Combination therapy alleviated the injury and inflamma-tion of the lung tissue. The morphology of the lung tissues ispresented in Fig. 3. The lung tissues of USA300-infected micewithout any treatment were crimson in color and hyperemic andexhibited a firm texture. In contrast, the lung tissues of the in-

FIG 3 Gross pathological changes and histopathology of lung tissue.C57BL/6J mice infected with 5 � 107 CFU of strain USA300 were treated withLysGH15 and/or apigenin. At 24 h after infection, the lungs were removedfrom the euthanized mice. The tissues were stained with hematoxylin andeosin.

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fected mice following treatment with LysGH15 alone or combina-tion therapy were pink and supple, which was similar to theexterior of the lungs in healthy mice (Fig. 3). Based on the histo-pathological observations, the lungs in the mice treated with acombination of LysGH15 and apigenin showed no severe inflam-mation or other pathological changes. Apigenin alone onlyslightly relieved the pathological changes.

As shown in Fig. 5A, the W/D ratio of the group that wastreated with the combination of LysGH15 and apigenin (3.96) wasthe closest to that of the healthy group (3.76), followed by that ofthe LysGH15-treated group (4.93). The W/D ratio of the apige-nin-treated group (6.90) was similar to that of the buffer-treatedgroup (7.37; P 0.05). To determine the level of inflammation inthe mouse pneumonia model after treatment with LysGH15and/or apigenin, the total number of cells and the protein in theBALF were measured. As shown in Fig. 5B and C, the total numberof cells and the protein in the group treated with the combinationtherapy (0.22 � 106 cells and 1.1 mg/ml, respectively) were signif-icantly reduced compared with those in the nontreated group

(10.8 � 106 cells and 2.6 mg/ml, respectively), and the values werelower than those in the groups treated with LysGH15 alone (0.6 �106 cells and 1.5 mg/ml, respectively; P 0.05) and with apigeninalone (8.3 � 106 cells and 2.0 mg/ml, respectively; P 0.01).Furthermore, the tumor necrosis factor alpha (TNF-�), interleu-kin-1� (IL-1�), and IL-6 levels in the group that was treated withthe combination therapy were the most similar to those of healthymice, followed by those of the LysGH15-treated, apigenin-treated,and untreated groups, as shown in Fig. 6.

DISCUSSION

In this study, the biochemical characterization and lytic activity ofLysGH15 were determined. In addition, the combination therapyof LysGH15 and apigenin was evaluated in vitro and in a mouse S.aureus pneumonia model. The data indicated that the combina-tion therapy of LysGH15 and apigenin exhibits therapeutic poten-tial for treating pneumonia caused by MRSA.

The structures of three individual domains of LysGH15 havebeen determined (13). To further understand LysGH15, the struc-

FIG 4 Combination therapy rescued mice from fatal S. aureus pneumonia. (A) Rescue of mice from lethal MRSA USA300 infection by LysGH15 and/orapigenin. C57BL/6J mice were infected with 1 � 108 CFU of strain USA300 and then intranasally treated (1 h after infection) with buffer (�), 500 �g apigenin(�), 60 �g LysGH15 (Œ), or a combination therapy of 500 �g apigenin and 60 �g LysGH15 (�). (B) Combination therapy improved the health status of micein 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 treatmentwith a specific therapy. A score of 5 indicated normal health and an unremarkable condition. Slight illness was defined as decreased physical activity and ruffledfur 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 signsplus 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 stateof 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 (�), apigenintreated (Œ), 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 (Œ), LysGH15treated (�), and cotreatment with both LysGH15 and apigenin (�). Each symbol represents the average of the results of three experiments.

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ture of the integral LysGH15 in solution was detected by SAXS.The results indicated that LysGH15 formed a V-shaped surfacestructure in solution and that the three domains of LysGH15 wererelatively independent. This finding is consistent with the resultsof a previous study that demonstrated that single CHAP and SH3bdomains displayed lytic and binding activity, respectively (13).SEM analysis further indicated that LysGH15 could lyse USA300strains completely in only a few seconds. Together with a previousstudy (12), these data demonstrated that LysGH15 possesseshighly efficient bactericidal activity in vitro and in vivo.

The mutual influence of LysGH15 and apigenin was first de-termined in vitro. The results indicated that apigenin had no effecton the lytic activity of LysGH15. In addition, LysGH15 and apige-nin exhibited a slightly synergistic effect in a hemolysis inhibitiontest. The secretion of toxins by S. aureus triggers hemolytic activityin erythrocytes, and alpha-hemolysin is the most important ofthese toxins (39). Although LysGH15 does not inhibit the secre-tion and assembly of alpha-hemolysin, it does reduce the amountof S. aureus in a lytic manner, which ultimately decreases the pro-duction of alpha-hemolysin.

A previous study demonstrated that a single intraperitonealinjection of LysGH15 (50 �g) was sufficient to protect miceagainst MRSA bacteremia (12). In this study, LysGH15 alone (60�g) also protected mice (80%) from MRSA pneumonia. Thus, the

phage lysin LysGH15 exhibits therapeutic potential for treatingpneumonia caused by MRSA.

Although apigenin reduces the production of alpha-hemolysinby S. aureus, it showed only slight antimicrobial activity against S.aureus (22). Because of this characteristic, apigenin is thought toreduce the selective pressure against the growth of this bacterialspecies (40). It has been reported that the curative dose of apigeninagainst S. aureus pneumonia is 50 mg/kg of body weight in mice(22). We found that a decreased dose of apigenin (500 �g) did notprovide any protective effect against S. aureus pneumonia in mice.The load of S. aureus in the lungs and blood was only slightlyreduced compared with that in untreated mice. Nevertheless, wefound that this subtherapeutic dose of apigenin significantly im-proved the capacity of LysGH15 treatment against S. aureus pneu-monia. In contrast, the combination of apigenin (500 �g) andLysGH15 (60 �g) was sufficient to protect mice (100%). Moreimportantly, both the health score and the lung pathology exhib-ited substantial improvement when the combination therapy wasadministered compared with treatment with LysGH15 (60 �g) orapigenin (500 �g) alone. In addition, the colony count of bacteriain the lung and blood was reduced significantly when the combi-nation therapy was administered. This result can be mainly attrib-uted to the lytic activity of LysGH15 on S. aureus.

Acute pneumonia is accompanied by the activation of the in-

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 strainUSA300 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 numbersin 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/orapigenin. At 24 h after infection, the levels of these cytokines in the BALF were determined: TNF-� (A); IL-1� (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.

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flammatory system, which is characterized by the presence of in-flammatory cells and proteinaceous fluid in air spaces (41). Acuteinjury to the alveolocapillary barrier can result in increased per-meability and can also cause protein-rich exudative edema (42).Additionally, TNF-�, IL-1�, and IL-6 are multifunctional cyto-kines 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 injuryand tissue damage (44). Compared with monotherapy, thecombination therapy of LysGH15 and apigenin not only dem-onstrated an exceedingly curative effect in terms of the total cellnumbers and total protein levels in the BALF but also broughtdown the proinflammatory cytokines. This result can bemainly attributed to the anti-inflammatory and antitoxin ef-fects of apigenin (22, 45).

Due to the prevalence of multidrug-resistant bacteria, espe-cially the emergence of superbugs (46, 47), there is an urgent needfor novel therapeutic agents that are directed against such patho-gens. Phage lysin (48) and the natural products derived from tra-ditional Chinese medicine (49) are considered novel therapeuticagents because their mechanism of action is different from that ofantibiotics. Additionally, several studies have reported on the ef-ficacy of the combination therapy of lysin and antibiotics (50). Inaddition, other studies have investigated the synergy of naturalproducts with antibiotics (51). This paper reports the systematicin vitro and in vivo investigation and evaluation of the combina-tion therapy of lysin and natural products derived from traditionalChinese medicine.

Overall, the data presented in this study showed that the com-bination therapy of LysGH15 and apigenin exhibits therapeuticpotential for treating pneumonia that is caused by MRSA. Thisresearch provides evidence for the viability of the combination oflysin and natural products derived from traditional Chinese med-icine.

FUNDING INFORMATIONNational Natural Science Foundation of China (NSFC) provided fundingto Jingmin Gu and Wenyu Han under grant numbers 31502103,31572553, and 31130072.

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