Phellinus baumii extract influences pathogenesis of Brucella abortus in phagocyte by disrupting the phagocytic and intracellular trafficking pathway

Authors


Correspondence

Suk Kim, Gyeongsang National University, 900, Gazwa, Jinju, 660-701, Korea. E-mail: kimsuk@gnu.ac.kr

Abstract

Aims

To clarify the effects of Phellinus baumii ethanol extract (PBE) on Brucella abortus pathogenesis in phagocytes focusing on the phagocytic and intracellular trafficking pathway.

Methods and Results

The effects of PBE on Br. abortus infection in macrophages were evaluated through an adherence and infection assays and an analysis of LAMP-1 staining. The phosphorylation of ERK1/2 and the F-actin polymerization associated with PBE during Br. abortus uptake were detected by immunoblotting and FACS, respectively. The survival of Br. abortus in pure culture was remarkably reduced by PBE in a dose-dependent manner. PBE-treated cells showed significantly decreased uptake, intracellular replication and adherence of Br. abortus. The declines of ERK1/2 phosphorylation and F-actin polymerization following Br. abortus entry were apparent in PBE-treated cells compared with the control. Moreover, the co-localization of Br. abortus-containing phagosomes with LAMP-1 was elevated in PBE-treated cells compared with the control during intracellular trafficking.

Conclusion

Phellinus baumii ethanol extract may possess the modulatory effect on pathogenesis of Br. abortus through disrupting the phagocytic and intracellular trafficking pathway in phagocyte.

Significance and Impact of the Study

The potential modulation of PBE to Br. abortus pathogenesis could provide an alternative approach to control of brucellosis, contributing to attenuate Br. abortus manifestation in hosts.

Introduction

Brucellosis is an emerging chronic zoonotic disease of mammals that is responsible for economic loss in livestock industries and represents a considerable public health burden worldwide (Corbel 1997). Brucella abortus is a facultative intracellular pathogen that invades and replicates within a variety of phagocytes, such as macrophages, epithelial cells and placental trophoblasts, displaying a diverse interaction with host cells (Liautard et al. 1996; Guzman-Verri et al. 2001). The pathogenic strategy of Br. abortus that allows it to invade, withstand intracellular killing and attain its preferred niche in phagocytes was established by the factual virulence constituents (Pizarro-Cerda et al. 1998; Celli et al. 2003). Meanwhile, the intracellular replication properties of Brucella and the resulting chronic infection have led to a difficulty in the development of medical therapy for brucellosis. The emerged problems of multiple antibiotic regimens for this disease include financial deliberations of developing countries, therapeutic failures in the form of relapses and the increasing emergence of antibiotic resistance (Solera 2010; Al Dahouk and Nockler 2011). Furthermore, a critical concern of therapeutic regimens against chronic and recurrent diseases is the health and safety of patients. In respect to these important health considerations, the development of natural plant products that possess safe and efficient properties might be one of the tasks needed to be advanced. On the other hand, the modulatory mechanism of Br. abortus pathogenesis and the control of brucellosis using traditional medicine have not been investigated in detail.

Mushrooms have currently received distinctive attention as physiologically functional foods and as commendable sources of natural medicines (Dai et al. 2010). Phellinus linteus, Phellinus baumii and Phellinus gilvus are medicinal mushrooms belonging to the Hymenochaetaceae basidiomycetes family (Hwang et al. 2004), which is a source of many antitumor or immunostimulating polysaccharides and has been utilized in folk medicines for diverse human diseases in several Asian countries (including Korea) for a long time. Research regarding P. linteus demonstrates that it possesses antitumor (Kim et al. 2004a), immunomodulating (Kim et al. 2004c), hepatoprotective (Kim et al. 2004b), antibacterial (Hur et al. 2004), antiangiogenic and antioxidant activity (Song et al. 2003). Furthermore, recent studies have shown that many other genera of Phellinus (e.g. P. baumii, P. gilvus and P. igniarius) also have potent pharmacological activities (Jang et al. 2004; Hwang et al. 2005; Lee et al. 2010).

Therefore, in this study, we investigated the inhibitory effects of P. baumii extract (PBE) on Br. abortus infection in phagocytic cells, placing emphasis on verifying the modulating mechanism that destructs pathogenic strategies of Br. abortus. These antibacterial effects suggest that this natural product could be applicable as a therapeutic agent and a natural alternative for the prevention and control of Brucella infection in hosts.

Materials and methods

Preparation of PBE

Preparation of a polyphenol fraction from the fruiting bodies of P. baumii was previously described (Noh et al. 2011). In brief, fruiting bodies (10 kg) of P. baumii were ground and extracted in ethanol for 24 h. The ethanol extract was filtered through cheese cloth, and the filtrate was concentrated under reduced pressure. The concentrate was partitioned using hexane and water, and the hexane-soluble fraction was discarded. The water-soluble fraction was extracted with ethyl acetate. The ethyl acetate-soluble fraction contained yellowish polyphenols of the styrylpyrone class and various oils, which were eliminated by washing with chloroform. The remaining yellowish polyphenol cluster was dried into powder, and then, the dissolved powder in phosphate-buffered saline (PBS) or RPMI was sterilized by filtering through a 0·22-μm filter (Sartorius Minisart®, Goettingen, Germany) before using for further experiments. The major chemical components of the yellowish polyphenol cluster were identified as interfungin A, davallialactone and hypholomine B by NMR and mass spectral analysis (Noh et al. 2011).

Cells and culture conditions

RAW 264·7 cells, a murine macrophage cell line, were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) grown at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute RPMI 1640 medium, containing 10% (v/v) heat-inactivated foetal bovine serum (FBS), 2 mmol l−1 l-glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (all provided by Gibco, Invitrogen, CA, USA). Cells were regularly assessed to prevent mycoplasma contamination (MycoAlert® Mycoplasma Detection kit, Lonza, Anaheim, CA, USA) and used between passages 5–10 in all experiments. Cells were seeded (1 × 105 cells ml−1) in cell culture plates and incubated for 24 h before infection for all assays. Prior to the bacterial infection, cells were washed three times with a sterile phosphate-buffered saline solution (PBS, pH 7·4) to eliminate antibiotics, and fresh culture medium was changed by RPMI 1640 medium containing 10% (v/v) FBS and 2 mmol l−1 L-glutamine without antibiotics.

Bacterial strains and culture conditions

Brucella abortus strain was derived from 544 (ATCC 23448), a smooth, virulent Br. abortus biovar 1 strains (Kim et al. 2003). The Br. abortus organisms were stored as frozen aliquots in 80% (v/v) glycerol at −70°C and maintained by weekly subculture on Brucella agar (Difco, Becton Dickinson, Bergenfield, NJ, USA) without antibiotics for 3 days at 37°C. Bacteria were grown at 37°C with vigorous shaking until they reached the stationary phase, and bacteria were suspended in PBS, and then, the viable counting was measured by plating serial dilutions on Brucella agar.

Bactericidal analysis

Bacteria grown to stationary phase were diluted with PBS to a concentration of 2 × 104 cells ml−1 as live bacteria and added to PBS containing different concentrations of PBE (0, 25, 50, 100, 200 and 400 μg ml−1) and incubated at 37°C for 0, 1, 4, 8, 16 and 24 h. After incubation and dilution, each diluent was plated onto Brucella agar and cultured for 3 days at 37°C to detect bacterial colony forming units (CFUs). The bacterial survival rates were expressed as a percentage of the survival rate of the treated sample relative to an untreated control (0 μg ml−1), which was set to 100%.

Cytotoxicity assay

The different concentrations of PBE (0, 25, 50, 100, 200 and 400 μg ml−1) in RPMI were chosen to evaluate the cytotoxicity of PBE, and RAW 264·7 cells (1 × 105 cells ml−1) were cultured in RPMI with or without PBE in a 96-well cell culture plate for 48 h. The cell viability was measured using a 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) cleavage assay (Mosmann 1983). In brief, RAW 264·7 cells were inoculated in MTT solution (5 mg ml−1 in PBS) and incubated at 37°C in 5% CO2 for 4 h. After incubation, plates were centrifuged at 450 g, and supernatants were removed. Acid/isopropanol (one portion of 4N HCl: 100 portion of isopropanol) was added to the wells and mixed to completely dissolve crystalline material. Product generation is measured at an optical density (OD) of 570 nm.

Bacterial uptake and intracellular replication assay

RAW 264·7 cells were infected with Br. abortus. For analysis of the bacterial internalization efficiency, the pretreatment of macrophage cells (overnight cultured in 96-well plates at 1 × 105 cells ml−1) with different concentrations of PBE (0, 25, 50 and 100 μg ml−1, which are noncytotoxic concentrations) was carried out 2 h before infection. Following pretreatment, bacteria were deposited onto cells, which were washed three times and replaced with new media without PBE, at multiplicities of infection (MOIs) of 10, centrifuged at 150 g for 10 min at room temperature and incubated at 37°C in 5% CO2 for 0, 15 or 30 min. The infected cells were washed once with media and then incubated with RPMI 1640 media containing 10% (v/v) FBS and gentamicin (30 μg ml−1) for 30 min to kill any remaining extracellular bacteria (Watarai et al. 2002). At different periods of time, the infected cells were thoroughly washed three times with PBS and then lysed with distilled water. The number of viable internalized bacteria was determined by counting the CFUs from serial dilutions of cell lysates that were spread onto Brucella agar plates in triplicate. For the intracellular-replication-efficiency measurements, the infected cells were incubated at 37°C for 1 h and the medium was replaced with RPMI 1640 containing 10% (v/v) FBS and gentamicin (30 μg ml−1) in the presence or absence of different concentrations of PBE (0, 25, 50 and 100 μg ml−1) and then incubated for 2, 8, 24 or 48 h. The cell washing, lysis and plating procedures were the same as for the detection of bacterial uptake efficiency. All of the assays were conducted in triplicate and were repeated at least three times on different days.

Bacterial adherence assay

RAW 264·7 cells were cultured in 12-well plates with 18-mm-diameter glass coverslips (Fisher Scientific, Pittsburgh, PA, USA) at 105 cells well−1 1 day before the infection. Cells were pretreated with PBE (100 μg ml−1) for 2 h prior to the infection. During the last 40 min of pretreatment, cytochalasin D (500 μg ml−1) was added to the cells to block the internalization of bacteria, and subsequently, the cells were infected by Br. abortus for 30 min. The infected cells were fixed in 4% (w/v) periodate–lysine–paraformaldehyde–sucrose for 1 h at 37°C (Watarai et al. 2002). After incubation for 30 min with a blocking buffer (2% (v/v) goat serum in PBS), the preparations were incubated with anti-Br. abortus polyclonal rabbit serum in blocking buffer for 1 h at 37°C to stain for adherent bacteria within 30 min of the infection and stained in a subsequent step with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G (IgG) (Sigma-Aldrich, St Louis, MO, USA) in blocking buffer for 1 h at 37°C. Finally, the preparations were washed and mounted with a fluorescent mounting solution (DakoCytomation, Glostrup, Denmark). Immunofluorescence images were collected with a microscope (DMIRE2; Leica) equipped with a camera (DC350F; Leica, Wetzlar, Germany) and IM50 imaging software (Leica). One hundred macrophages were selected randomly, and the bacteria that adhered to these cells were counted.

F-actin and LAMP-1 staining by immunofluorescence microscopy

The immunofluorescence microscopy assay with F-actin and LAMP-1 staining was performed as previously described with slight modification (Watarai et al. 2002). RAW 264·7 cells were cultured and pretreated with PBE as described above. The cells were infected with unconjugated or FITC-conjugated Br. abortus. In the observation of F-actin reorganization, the bacterial infection was monitored for 10 min. To detect the co-localization of Br. abortus-containing phagosomes (BCPs) with LAMP-1, the infection procedure for 2, 4, 8, 24 or 48 h was performed. The infected macrophages were fixed and then were blocked and permeabilized in a solution containing PBS with 2% (v/v) goat serum, supplemented with 0·1% Triton X-100 for 30 min at 22°C. For staining of F-actin, the cells were incubated with 0·1 μmol ml−1 rhodamine–phalloidin (Cytoskeleton, Denver, CO, USA) for 30 min at 22°C. For staining of LAMP-1, the samples were incubated with an anti-LAMP-1 rat monoclonal antibody (Santa Cruz, CA, USA); then, after being washed, the samples were stained with Texas red goat anti-rat IgG (Santa Cruz, CA). Finally, the preparations were washed and mounted with fluorescent mounting medium (DakoCytomation). Fluorescence images were collected using an Olympus FV1000 (Olympus, Tokyo, Japan) laser scanning confocal microscope. The images were processed with Adobe Photoshop and NIH ImageJ software. For LAMP-1 staining, 100 bacteria within macrophages were randomly selected, and the extent of LAMP-1 acquisition by the bacteria was determined.

FACS assay for F-actin

To evaluate the relative content of F-actin in Br. abortus-infected or -uninfected cells in the presence or absence of PBE (100 μg ml−1) for the indicated times, we performed a Fluorescence activated cell sorting (FACS) assay for F-actin as previously described (Madrid et al. 2001). In brief, cells (1·5 × 106 cells ml−1) were harvested and fixed with 4% (w/v) paraformaldehyde at room temperature for 30 min. And then, they were permeabilized and stained with 20 μg ml−1 lysophosphatidylcholine (Sigma-Aldrich) containing 1 μmol ml−1 TRITC-phalloidin (Sigma-Aldrich). After centrifugation at 300 g, 4°C for 5 min, cells were washed with PBS, and their F-actin content was quantified by FACS analysis using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). Data were collected as log-scaled fluorescence histograms from 10 000 cells, and the average F-actin content of a population was expressed as the mean of the fluorescence intensity. The experiments were performed in duplicates and repeated at least three times.

Immunoblot analysis

The immunoblot analysis methods were performed as previously described (MacPhee 2010). The cultured cells in six-well plates were infected with Br. abortus for indicated times. Cells were then washed twice with ice-cold PBS and lysed in ice-cold lysis buffer for 30 min at 4°C. Protein concentration was assessed by Bradford protein assay (Bio-Rad, Richmond, CA, USA). Samples were separated by SDS-PAGE and electrically transferred to a Polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Blots were blocked for 1 h with 5% (w/v) bovine serum albumin in TBS-T (20 mmol l−1 Tris–HCl, 150 mmol l−1 NaCl, 0·1% (v/v) Tween, pH 7·4) and proved by phospho-specific antibodies against ERK1/2 (Thr183/Tyr185) and p38α (Thr180/Tyr182). Pan antibodies and β-actin antibody were applied on stripped blots to verify that equivalent amounts of proteins were loaded per lane. All of the antibodies were obtained by Cell Signalling (Denver, MA, USA). The binding of primary antibody was visualized using HRP-conjugated anti-rabbit IgG and anti-mouse IgG secondary antibodies (Sigma-Aldrich) and then was detected by enhanced enhanced chemiluminescence (ECL) (Anigen, Genetics Inc, Suwon, Korea). The immunoblot ECL signals were quantified using NIH ImageJ software.

Statistical analysis

The data are expressed as the mean ± standard deviation (SD) for the replicate experiments. Statistical analysis was carried on using Graph Pad Prism software, ver. 4·00 (Graph Pad Software, Inc., San Diego, CA, USA). Student's t-test or one-way anova followed by the Newman–Keuls test was used to make a statistical comparison between the groups. Results with < 0·05 were considered statistically significant.

Results

Effect of PBE on bacterial survival rates of Brucella abortus and cell viability of murine macrophage

The bacterial survival rates were reduced considerably in a dose-dependent manner in the presence of PBE (< 0·01). The PBE concentrations of 200 and 400 μg ml−1 exhibited dramatic reductions in the viability of bacteria with increased incubation time, while the viability in the lower concentrations showed a relatively slower decrease over time. The bacterial survival rates are shown in Fig. 1.

Figure 1.

Bactericidal effect of phosphate-buffered saline on Brucella abortus. Bacterial viability was evaluated by counting colony forming units on culture plates, and the rate of bacterial viability was compared with the zero time point in the untreated phosphate-buffered saline (Control). Data represent the mean ± SD of triplicate samples from three identical experiments. (image_n/jam12072-gra-0001.png) 0 μg ml−1; (image_n/jam12072-gra-0002.png) 200 μg ml−1; (image_n/jam12072-gra-0003.png) 400 μg ml−1.

To evaluate the cytotoxicity of PBE, RAW 264·7 cells were incubated in the presence of various concentrations of PBE (0, 25, 50, 100, 200 and 400 μg ml−1). As a result, the OD values of the cell cultures treated with 200 and 400 μg ml−1 PBE radically decreased. However, the OD values at a concentration of 100 μg ml−1 PBE did not decrease, compared with the OD values of the untreated control cells. These results indicate that PBE did not have any cytotoxicity at concentrations at or below 100 μg ml−1. Thus, PBE was applied at concentrations less than 100 μg ml−1 for the subsequent experiments performed in this study.

Effect of PBE on invasion and intracellular bacterial growth of Brucella abortus by macrophages

To verify the effects of PBE on the invasion and intracellular growth of Br. abortus by RAW 264·7 cells, macrophage cells were pretreated with different concentrations of PBE (0, 25, 50 and 100 μg ml−1, which are noncytotoxic concentrations) and were infected with Br. abortus for the indicated times (0, 15 or 30 min); the PBE concentrations were maintained throughout the experiments. The results indicate that the invasion of Br. abortus into PBE-treated cells was significantly diminished in a dose-dependent manner compared with the untreated cells (< 0·001) (Fig. 2a). Moreover, PBE-treated cells displayed significantly reduced intracellular growth of Br. abortus, especially from 24 to 48 h postinfection treatment, although a similar pattern of replication was observed at times between 2 and 24 h compared with that of the untreated cells (< 0·001) (Fig. 2b).

Figure 2.

Effect of phosphate-buffered saline on the invasion and intracellular growth of Brucella abortus. (a) Bacterial internalization efficiency. (b) Bacterial intracellular growth efficiency. Data represent the mean ± SD of triplicate samples from three identical experiments. Statistically significant differences relative to the untreated control are indicated by asterisks (***< 0·001). (image_n/jam12072-gra-0004.png) 0 μg ml−1; (image_n/jam12072-gra-0005.png) 25 μg ml−1; (□) 50 μg ml−1; (image_n/jam12072-gra-0006.png) 100 μg ml−1; (image_n/jam12072-gra-0007.png) 0 μg ml−1; (image_n/jam12072-gra-0008.png) 25 μg ml−1; (image_n/jam12072-gra-0003.png) 50 μg ml−1; (image_n/jam12072-gra-0007.png) 100 μg ml−1.

Interference effect of PBE on Brucella abortus phagocytosis through modulating adherence and F-actin polymerization

The adherence of Brucella on the host cell membrane has been implicated as a critical step associated with the entry of Brucella into host cells (Campbell et al. 1994; Guzman-Verri et al. 2001). To examine this premise, we tested whether PBE altered the adherence of Br. abortus to the macrophage surface membrane. The results indicated that the number of bacteria that adhered to cells pretreated with PBE (34·41 ± 3·76) was significantly reduced compared with untreated control cells (45·82 ± 1·88), showing a reduction rate of 24·89 ± 1·32% (< 0·05).

The observations using the phalloidin-associated F-actin fluorescence microscopy revealed that F-actin polymerization for Br. abortus invasion was attenuated in the PBE-treated cells compared with the untreated control cells and displayed reduction in filopodia and lamellipodia scattering at the cell periphery (Fig. 3a). Additionally, we assessed the F-actin content in the PBE-treated cells upon Br. abortus invasion, utilizing FACS analysis to quantitatively prove the effect of PBE on the F-actin polymerization required for the phagocytosis of Br. abortus. PBE treatment led to a remarkable decrease (up to 1·64-, 1·75- and 1·47-fold at 0, 15 and 30 min p.i., respectively) in F-actin fluorescence intensity (< 0·01) compared with the Br. abortus-infected control cells; no difference in F-actin fluorescence was found between the uninfected control cells and the PBE-treated uninfected cells (Fig. 3b).

Figure 3.

Effect of phosphate-buffered saline on Brucella abortus phagocytosis by the modulation of F-actin polymerization. (a) F-actin polymerization and bacterial co-localization. The results are representative of three separate experiments (Scale bars = 5 μm). (b) FACS analysis for F-actin content. Statistically significant differences relative to the untreated control are indicated by asterisks (*< 0·05; **< 0·01). (□) Uninfected control; (image_n/jam12072-gra-0008.png) PBE; (■) Br. abortus; (image_n/jam12072-gra-0009.png) PBE + Br. abortus.

Down-regulation of PBE on phagocytic signals for the invasion of Brucella abortus into macrophages

As a result, the phosphorylation levels of ERK1/2, p38α and JNK in the PBE-treated cells at 15 min postinfection were reduced by 30·66%, 35·36% and 20·65%, respectively, compared with those in the infected control cells (Fig. 4). These findings indicate that the suppressive effects of PBE on the activation of MAPKs could negatively affect the invasion of Br. abortus into macrophages.

Figure 4.

Effect of phosphate-buffered saline (PBE) on the activation of intracellular signalling for Brucella abortus phagocytosis. Immunoblot analysis of total cell lysates was assessed with phospho-specific and pan antibodies against ERK1/2, p38α and JNK in RAW264·7 cells pretreated with PBE (100 μg ml−1) at the indicated times. Images shown are representative of three independent experiments.

Disturbance effect of PBE on the intracellular trafficking of Brucella abortus

In untreated cells, the colocalization of LAMP-1 with BCPs increased up to 4 h postinfection and steadily decreased thereafter. Meanwhile, the number of LAMP-1-positive BCPs in PBE-treated cells increased gradually, showing significant increase up to 1·5- and 1·7-fold at 24 and 48 h postinfection, respectively (< 0·05) (Fig. 5).

Figure 5.

Effects of phosphate-buffered saline on intracellular trafficking of Brucella abortus. One hundred fluorescein isothiocyanate-fluorescing BCPs within macrophages were randomly selected, and the fraction of LAMP-1 co-localization of Br. abortus-containing phagosomes was determined. Data represent the mean ± SD of triplicate samples from three identical experiments. Statistically significant differences relative to the untreated control are indicated by asterisks (*< 0·05). (image_n/jam12072-gra-0001.png) Control; (image_n/jam12072-gra-0006.png) PBE.

Discussion

Brucellosis has been competently controlled through vaccination, surveillance and confinement programmes. However, there is currently no safe or effective vaccine that can control brucellosis in humans. The use of antibiotics for the treatment of this disease is also a controversial issue, requiring extended doses and multidrug regimens based on a combination of several antibiotics, including doxycycline, rifampin and gentamicin (Skalsky et al. 2008). Furthermore, Brucella seem to be adapted to survive the harsh conditions they encounter until they reach their intracellular niche inside the host (Gorvel and Moreno 2002). Due to these characteristics, a conventional antibiotic regimen might be not appropriate for brucellosis treatment, and, combined with the emergence of antibiotic resistance, these problems could lead to serious main effects. Despite these drug problems, there has been little investigation in the application of traditional medicine and natural products for the control of brucellosis. Therefore, the establishment of precise mechanisms with respect to how a natural product modulates Brucella infection as well as the identification of alternative and advanced treatments for brucellosis, which could effectively attenuate Brucella infections, is required.

Many extracts obtained from mushrooms, such as P. baumii or P. gilvus, have a variety of medicinal effects (Jang et al. 2004; Hwang et al. 2005). However, the majority of the potential therapeutic effects of P. baumii on bacterial infections have yet to be clinically examined.

This study elucidated the modulating mechanism of PBE to inhibit Br. abortus infection in vitro and ultimately provide the potential advantage for controlling and preventing the diseases caused by Br. abortus.

Among several well-known medicinal mushrooms from Asian countries, Phellinus linteus has been taken orally since ancient times as a health-promoting dietary supplement and an adjuvant to combat viral and bacterial infections (Hur et al. 2004). We first hypothesized that PBE can directly inhibit Br. abortus growth. Consistent with our expectations, our results revealed that treatment with PBE leads to a dose-dependent decline in the in vitro growth of Br. abortus, although the viability of bacteria did not be inhibited in concentrations less than 100 μg ml−1.

The invasion of intracellular bacteria into host cells is likely the most crucial event in bacterial infections and depends on specific cell-to-bacteria interactions, which are directly mediated by specific cell surface adhesion. A recent study investigated the direct effects of Phellinus linteus on melanoma cells by blocking cell adhesion and invasion (Han et al. 2006). In our study, we performed an adherence assay to examine the effect of PBE on bacterial adhesion to their target cells. The results showed that PBE interferes with the in vitro adherence of Br. abortus to macrophage cell membranes, indicating that PBE might negatively affect the in vivo adherence of Br. abortus to macrophage surface membranes during infection by these bacteria. Corresponding to the interfering effect of PBE on the adherence of Br. abortus to macrophages, the uptake of Br. abortus in PBE-treated cells was significantly diminished compared with the control, suggesting that PBE has an inhibitory effect on phagocytosis of Br. abortus. The F-actin polymerization and dynamic rearrangement of the actin cytoskeleton are essential for phagocytic uptake of microbial pathogen (Gruenheid and Finlay 2003). Previous studies demonstrated that M cells, macrophages and neutrophils ingest Brucella by zipper-like phagocytosis (Ackermann et al. 1988) or conventional zipper-type mechanisms (Rittig et al. 2001). Furthermore, F-actin polymerization is a concern in the phagocytosis of Brucella in both epithelial cells and macrophages (Kusumawati et al. 2000; Guzman-Verri et al. 2001). Invasion of epithelial cells by Br. abortus requires small GTPases of the Rho subfamily, which are involved in the induction of actin polymerization. Because we found that PBE inhibited Br. abortus phagocytosis in the infection assay, we monitored whether PBE interrupted the F-actin reorganization during Br. abortus phagocytosis by macrophages. Predictably, we found that PBE interfered with Br. abortus phagocytosis by hindering F-actin polymerization.

A recent study found that P. baumii methanol extract (PBME) inhibited the phosphorylation levels of ERK and JNK, suggesting the therapeutic potential for the treatment of cardiovascular diseases (Kamruzzaman et al. 2011). It has been demonstrated that mitogen-activated protein kinase (MAPK) plays an important role in the phagocytosis of bacteria and remodelling of the actin cytoskeleton (Schorey and Cooper 2003; Doyle et al. 2004). In the present study, we proposed that the inhibitory effects of PBE on the Br. abortus invasion and F-actin polymerization were due to the restraint of MAPKs (ERK1/2, p38α and JNK) activation. To prove this postulation, we tested the phosphorylation of MAPKs in macrophage cells in the presence or absence of PBE during Br. abortus invasion. Consistent with this study and the evidence regarding the involvement of bacterial phagocytosis in MAPKs activation (Schorey and Cooper 2003; Doyle et al. 2004), our study revealed that PBE attenuated the activation of MAPKs (ERK1/2, p38α and JNK) in Br. abortus-infected cells at 15 min postinfection of invasion. These findings suggest that the suppressive effects of PBE on the MAPKs-linked phagocytic signalling pathway could negatively affect the invasion of Br. abortus into macrophages.

In addition to the noticeable relationship between PBE and the invasion mechanism of Br. abortus, our study demonstrates the inhibitory effect of PBE on intracellular survival of Br. abortus in the macrophage. Based on the evidence that PBE hampered the intracellular growth of Br. abortus in macrophages, we assumed that PBE might affect the intracellular trafficking of Br. abortus in macrophages. Currently, one of the most important strategies of Brucella for intracellular survival within their host cells is the maturation of Brucella-containing phagosomes (BCPs) into replicative organelles, accomplished by gradually circumventing phagosome–lysosome fusion in macrophages (Gorvel and Moreno 2002). To verify whether PBE interrupts the intracellular survival strategy of Br. abortus within macrophages, we investigated the effect of PBE treatment on the interaction of Brucella-containing phagosomes (BCPs) with LAMP-1. The observations of LAMP-1 staining indicated that the phagosome–lysosome fusion of Br. abortus inside macrophages was enhanced by PBE treatment and, consequently, suggests that PBE interrupts the intracellular survival strategy of Br. abortus (which was implicated in the evasion of phagosome–lysosome fusion within macrophages).

Several investigations regarding the immunomodulatory activities of Phellinus extracts suggested that these natural products may be helpful in the treatment of diverse inflammatory and immune diseases (Kim et al. 2004c; Chang et al. 2008). In particular, polysaccharides isolated from a variety of mushrooms are immune potentiators, stimulating the production of nitric oxide (NO) and cellular cytokines, including IFN-γ, IL-4 and TNF-α. We postulate that Br. abortus infection is influenced by the regulation of different cytokines by PBE treatment. Thus, we intend to further study the immunomodulatory activities of PBE on Br. abortus pathogenesis, focusing on cytokine analysis in vitro and in vivo.

In conclusion, this study suggested that PBE appears to possess the modulatory effect on pathogenesis of Br. abortus through disrupting the phagocytic and intracellular trafficking pathway in phagocyte. Accordingly, we proposed that the mechanistic findings revealed in this study could provide new insights into an alternative approach of this natural product, which is applicable as a therapeutic agent that is helpful for the prevention and control of Brucella infection in hosts. Furthermore, the implicated mechanism of Br. abortus pathogenesis and the advanced application of alternative therapies using natural material extracts should be required to discover safe and economical candidates that abolish diverse complications resulting from brucellosis. Ultimately, based on this study, an authenticatable practicality of PBE needs to be established to prevent or treat Br. abortus infection. After that, PBE can be useful as a natural alternative available for the feed additives in animals and in the food products or medicine for human.

Acknowledgements

The present work was supported by a grant from the Ministry of Education, Basic Science Research Program, National Research Foundation, Korea (2010-0009080).

Authors' contributions

S. Kim conceived the study and carried out macrophage–pathogen co-cultures, molecular studies and immunoassay. J.J. Lee, D.H. Kim, D.G. Kim, W.G. Min and H.J. Lee carried out macrophage–pathogen co-cultures, molecular studies and immunoassay. M.H. Rhee and B.S. Yun carried out PB extraction, FACS, MTT assay and statistical analysis.

Competing interests

The authors declare that they have no competing interests.

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