Inhibitory Effect of Lactobacillus plantarum Extracts on HT-29 Colon Cancer Cell Apoptosis Induced by Staphylococcus aureus and Its Alpha-Toxin

Abstract

Staphylococcus aureus plays an important role in sepsis, septic shock, pneumonia, and wound infections. Here, we demonstrate that Lactobacillus plantarum extracts inhibited S. aureus-induced cell death of a human epithelial cell line, HT-29. In particular, we have shown that S. aureus-induced cell death was abolished by neutralization of α-toxin, indicating that α-toxin is the major mediator of S. aureus-induced cell death. DNA fragmentation experiment and caspase assay revealed that the S. aureus-induced cell death was apoptosis. L. plantarum extracts inhibited the generation of effector caspase-3 and the initiator caspase-9 in S. aureus- or α-toxin-induced cell death. Moreover, expression of Bcl-2, an anti-apoptotic protein, was activated in L. plantarum extract-treated cells as compared with the S. aureus- or α-toxin-treated only cells. Furthermore, S. aureus-induced apoptosis was efficiently inhibited by lipoteichoic acid and peptidoglycan of L. plantarum. Together, our results suggest that L. plantarum extracts can inhibit the S. aureus-mediated apoptosis, which is associated with S. aureus spreading, in intestinal epithelial cells, and may provide a new therapeutic reagent to treat bacterial infections.

Introduction

Staphylococcus aureus is the most common pathogenic bacterium that is found in hospitalized patients. It induces serious infections such as pneumonia, abscess, endocarditis, and osteomyelitis [3]. S. aureus infections also mediate sepsis and septic shock associated with vascular damage and multiple organ failure. Apoptosis plays a key role during S. aureus-mediated sepsis, and the ability of S. aureus to induce apoptosis in endothelial cells might contribute to metastatic infection. S. aureus-induced apoptosis and caspase activation were mediated by α-toxin [4]. Expression of α-toxin in a non-hemolytic strain recovered apoptosis induction, suggesting a role of α-toxin as a trigger of apoptosis [10]. The α-toxin secreted by S. aureus induces caspase cleavage through the intrinsic caspase pathway, and increases cytochrome c release from intact cells, which is controlled by Bcl-2 [4].

Apoptosis of mammalian cells plays a key role in the regulation of the immune system. Apoptosis is a programmed cell death, which eliminates mutated and damaged cells, mostly without accompanying an inflammatory response [2,26]. Apoptotic cells clearly show morphological and biochemical features such as nucleus shrinkage and chromatin condensation. Caspases, a family of intracellular cysteine proteases, mediate these alterations by cleaving numerous cellular substrates. Inactive proenzymes are proteolytically cleaved to an active complex composed of two heterodimeric subunits having a molecular mass of ~10 and 20 kDa. Caspases are divided into initiator caspases (e.g., caspase-8 and caspase-9) and effector caspases (e.g., caspase-3 and caspase-7) based on the structure and order in cell death pathways [19]. Initiator caspases regulate downstream effector caspases, which finally cleave numerous cellular substrates and induce cell death [5].

Lactic acid bacteria (LAB), including Lactobacillus genus, are considered as probiotics. They are members of the commensal microorganisms of the gastrointestinal tract [8,13]. Intestinal LAB contribute beneficial effects, including modulation of immune responses. They participate in the development and maintenance of homeostasis in the intestinal immune systems [1,27]. L. plantarum promotes the health of the host through regulation of immune responses in the host digestive tract [23]. L. plantarum also has beneficial effects on inflammatory disorders, suggesting that there is some possibility of using L. plantarum as a therapeutic bacterial strain.

Here, we investigated the effect of L. plantarum extracts on the inhibition of apoptosis mediated by S. aureus and α-toxin secreted from S. aureus. We showed that L. plantarum extracts, especially cell wall components, inhibited S. aureus- or α-toxin-induced HT-29 cell death, which was mediated by the inhibition of caspase activities and the induction of Bcl2 expression.

 

Materials and Methods

Cell Culture, Reagent, and Antibodies

Human colon cancer cell line HT-29 (Korean Cell Line Bank, Seoul, Korea) was maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco BRL, USA), 100 U penicillin, and 100 μg/ml streptomycin. The cells were cultured at 37℃ in a humidified chamber containing 5% CO2. α-Toxin (α-hemolysin), and anti-α-toxin and anti-Bcl-2 antibodies were purchased from Sigma (MO, USA). Caspase 3 (Promega, WI, USA) and caspase 9 (R&D Systems, MN, USA) activity assay kits were used in this study.

Bacteria Strains, Culture, and Preparation of Bacterial Extracts

L. plantarum K8 (KCTC10887BP) and S. aureus (KCTC 1621) were used in this study. L. plantarum, Leuconostoc (Leu.) mesenteroides (KCTC3100), and Lactobacillus acidophilus (KCTC3140) were cultured with MRS (de Man Rogosa Sharpe) broth at 37℃ for 18 h and S. aureus was cultured with Brain-heart infusion (BHI) broth at 37℃ for 24 h. Escherichia coli DH5α was amplified with Luria-Bertani broth at 37℃ for 18 h. In general, 1 × 107 CFU/ml live S. aureus or 50 ng/ml α-toxin were treated to HT-29 cells. To prepare bacterial extracts, cells were harvested by centrifugation at 8,000 rpm for 10 min. After washing with phosphate-buffered saline (PBS) three times, 50 g of cells (wet weight) was disrupted by sonication to make crude extracts. After centrifugation at 8,000 rpm for 30 min, supernatants were collected for the cytosol fraction. To prepare the cell wall fraction from the cell lysates, centrifugation (8,000 rpm, 30 min, 4℃) was performed and the pellet was washed with distilled water (DW) three times. The precipitated pellet was incubated with 100 mg/ml RNase and 50 mg/ml DNase for 18 h, and then the pellet was incubated for another 18 h at 37℃ after addition of 200 mg/ml trypsin. The cell wall fraction was sedimented by centrifugation (8,000 rpm, 30 min, 4℃) followed by four washes with DW, and then it was lyophilized. To prepare the peptidoglycan (PGN) fraction, 1 g of cell walls was incubated with 5% trichloroacetic acid (TCA) for 18 h at 22℃, and sedimented by centrifugation at 8,000 rpm for 30 min. The sediment from TCA extraction was washed three times with water and three times with acetone, and dried to make peptidoglycan powder. Lipoteichoic acid (LTA) was prepared from L. plantarum K8 using the methods previously described [16]. Harvested cells were broken up by sonication. The sonicated cells were extracted with n-butanol, and then hydrophobic interaction chromatography using an Octyl-Sepharose CL-4B column was applied to purify LTA. Purified LTA was stored by freeze-drying.

Cell Viability Test

The cytoxicity of HT-29 cells was determined by WST-1 assay (Takara, Japan). The numbers of viable cells are measured by the detection of cleaved tetrazolium salts added into the medium. HT-29 cells stimulated with bacteria or bacterial extracts were incubated with 10 μl of premix WST-1. After incubating for 30 min, absorbance was measured using an ELISA reader. Metabolically active cells form formazen dye that can be quantitated by measuring its absorbance. The measuring wavelength is 420 nm and the reference wavelength is 600 nm.

DNA Fragmentation

Stimulated HT-29 cells were detached from a 6-well plate using Trypsin-EDTA, washed with PBS, and lysed with lysis buffer (0.25% NP-40, 20 mM Tris, 0.5 mM EDTA (pH 8.0), and 50 μg/ml RNase A) at 37℃ for 30 min. Proteinase K (200 μg/ml) was added to the lysis sample and additionally incubated at 37℃ for 30 min. Fragmented DNA was confirmed by agarose gel electrophoresis.

Caspase Assay

Stimulated HT-29 cells were detached from a 6-well plate using Trypsin-EDTA and washed with PBS three times. Cells were incubated with lysis buffer provided by the manufacturer and then underwent five repeated freeze-thawing cycles. After centrifugation, the total protein content from supernatants was examined by the Bradford assay, and then the caspase activity was examined according to the manufacturer’s manual.

Western Blotting

Proteins from stimulated HT-29 cell lysates were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with proper primary antibodies. Peroxidase-conjugated secondary antibodies were applied to detect positive signals. Protein bands were visualized by enhanced chemiluminescence with the Super Signal West Detection Kit (Thermo Chemical Company, IL, USA). A β-actin antibody was used to confirm equal protein loading.

Replication and Internalization Assay

For the S. aureus replication assay, HT-29 cells (1 × 105 cells/well) were seeded onto a 6-well tissue culture plate and cultured for 2 days, until 70-80% confluent. Cells were washed twice with 1 ml of PBS and then incubated with 1 ml of antibiotics-free medium containing S. aureus (1 × 105 CFU/well) for 2 h. Cells were washed four times with PBS and incubated with fresh medium containing 50 μg/ml gentamycin. Different doses of L. plantarum extracts were added to each well, and incubated for 18 h. Cells were washed four times with PBS and lysed with hypotonic lysis buffer (20 mM Tris, pH 7.5, 5 mM MgCl2, 5 mM CaCl2, 1 mM DTT, 1 mM EDTA, and 0.2% Triton X-100). The CFU was determined by plating on BHI plates with serial dilutions of the lysates. For the internalization assay, HT-29 cells were pre-treated with L. plantarum lysates for 18 h. Cells were washed and infected with S. aureus (1 × 105 cells/well) for 1 h. Culture supernatants were aspirated and washed four times with PBS. Cells were incubated with fresh culture media containing 50 μg/ml gentamicin for 24 h. Colony-forming units were determined as described above.

Statistical Analysis

Results were expressed as the mean ± standard deviation, and statistical analyses were performed using a two-tailed unpaired Student t test. A p-value of <0.05 was considered statistically significant.

 

Results and Discussion

S. aureus is a virulent pathogen that causes α-toxin-mediated apoptosis in mammalian cells [4]. To examine the effect of L. plantarum on the S. aureus-mediated apoptosis, live L. plantarum or L. plantarum extracts were treated on S. aureus-infected HT-29 cells. As shown in Fig. 1A, S. aureus (1 × 107 CFU/ml) reduced cell viability by 6-folds as compared with untreated cells. HT-29 cells co-treated with L. plantarum and S. aureus showed about 70% viability. Cell viability was increased by 3-folds in the co-treated cells as compared with the cells treated with S. aureus alone. When S. aureus was co-treated with L. plantarum extracts, the survival rate of HT-29 was over 90%, indicating that L. plantarum or L. plantarum extracts significantly increased HT-29 cell viability. The viability of HT-29 was increased when over 2 × 109 CFU/ml L. plantarum extracts was co-treated with S. aureus. Cell death was not observed by L. plantarum extracts only (Fig. 1B). Apoptosis of mammalian cells significantly contributes to the host defense against the bacteria to ensure homeostasis of the organism. Host responses include cytokine release, defensin secretion, and production of oxygen radicals [12,20]. On the other hand, pathogens have evolved means to subvert host defense systems to create a suitable environment for their replication. S. aureus escapes the phagosome where it replicates via professional and non-professional phagocytes, collapses autophagy, and induces apoptosis and pyronecrosis [9]. Thus, it is most important to remove infected cells before S. aureus is diffused from the cells using apoptosis. In this study, we have shown that L. plantarum, especially its extracts, effectively inhibited S. aureus-mediated cell death, indicating that infected host cells can take their time to recruit immune cells to remove themselves.

Fig. 1.Effects of L. plantarum on HT-29 cell viability treated with S. aureus. (A) HT-29 cells were treated with 1 × 107 CFU/ml live S. aureus alone or together with or without 1 × 109 CFU/ml live L. plantarum or L. plantarum extracts for 12 h. (B) HT-29 cells were treated with S. aureus, or L. plantarum extracts with or without 1 × 107 CFU/ml S. aureus for 12 h. In both experiments, cell viability was examined by WST-1 assay. **p < 0.01; ***p < 0.001 as compared with untreated or S. aureus-treated samples.

Next, we examined the role of α-toxin in S. aureus-mediated cell death. α-Toxin mediates the intrinsic death pathway of apoptosis, independently of death [4]. S. aureus produces a 33 kDa pore-forming protein that can lyse a wide range of human cells and induce apoptosis in T-lymphocytes [6]. Secreted α-toxin induces an inflammatory response, and high concentrations of α-toxin cause necrosis of the target cells, whereas sublytic concentrations predominantly induce apoptosis [4,7,11,21]. In this study, we have shown that α-toxin-induced apoptosis was inhibited by anti-α-toxin neutralization antibody. S. aureus-mediated apoptosis was also significantly inhibited by anti-α-toxin antibody, but it did not inhibit a high dose of S. aureus (1 × 109 CFU/ml)-mediated apoptosis (Fig. 2A). These results suggest that α-toxin is one of the key factors in S. aureus-mediated apoptosis. When HT-29 cells were stimulated with bacterial extracts, such as L. plantarum, Leu. mesenteroides, L. acidophilus, and E. coli, together with S. aureus, only L. plantarum extracts reduced S. aureus-induced cell death (Fig. 2B). We do not know why other bacterial extracts have no inhibitory effects on S. aureus-mediated cell death. Thus, further studies may need to evaluate bacterial extracts in the immune regulatory effects. Besides the anti-apoptotic effect, L. plantarum extracts have interesting roles in skin-related issues. For example, L. plantarum extracts increased the water content in the human face and forearm skin and decreased the horny layer thickness and TEWL value in the experimental group as compared with control group [14]. Similarly, L. plantarum extracts reduced water loss in mice skin, resulting in the alleviation of atopic dermatitis lesion [15].

Fig. 2.Role of α-toxin in S. aureus-induced cell death. (A) HT-29 cells were incubated with the indicated dose of anti-α-toxin neutralization antibody for 30 min, and then incubated with the indicated dose of S. aureus or 50 ng/ml α-toxin for 12 h. (B) HT-29 cells were incubated with 50 ng/ml α-toxin and 1 × 109 CFU/ml bacterial extracts for 12 h. In both experiments, cell viability was examined by WST-1 assay. *p < 0.05; **p < 0.01 as compared with 0%of anti-α-toxin antibody or α-toxin treated only.

To identify the inhibitory role of L. plantarum extracts on S. aureus- or α-toxin-mediated apoptosis, the variation of apoptosis-related molecules was examined. Apoptosis is accompanied by morphological changes of cells and nuclei, including cell shrinkage, condensation, fragmentation of nuclei, and blebbing of the plasma membranes. Thus, chromosomal DNA fragmentation is the first evidence of apoptosis [22]. As shown in Fig. 3A, DNA fragmentation in HT-29 cells co-treated with L. plantarum extracts and α-toxin was reduced as compared with α-toxin-treated only. The activity of caspase-3 increased by S. aureus and α-toxin (Figs. 3B and 3C, respectively) was significantly inhibited by co-treatment with L. plantarum extracts. Similar results were shown in caspase-9 activities in HT-29 cells treated with S. aureus and α-toxin together with L. plantarum extracts (Figs. 3D and 3E, respectively). The activation of caspase-3, caused by the release of cytochrome c from mitochondria and Apaf-1-mediated processing of caspase-9, indicates the activation of the intrinsic pathway [24], which is suggesting that L. plantarum extracts inhibit the mitochondrial-mediated pathway to apoptosis. The Bcl-2 family determines the commitment of cells to apoptosis, consisting of anti-apoptotic and pro-apoptotic members. Bcl-2 and Bcl-XL, the anti-apoptotic members of this family, inhibit apoptosis either by sequestering proforms of death-driving cysteine proteases or by preventing the release of cytochrome c and AIF into the cytoplasm [25]. To examine the effect of L. plantarum extracts on Bcl-2 expression, HT-29 cells were co-treated with L. plantarum extracts and S. aureus (Fig. 3F) or α-toxin (Fig. 3G). In both experiments, Bcl-2 expression was dramatically increased by co-treatment. These results suggest that L. plantarum extracts have an anti-apoptotic effect on S. aureus-induced cell death.

Fig. 3.Inhibition of S. aureus- or α-toxin-induced apoptosis by L. plantarum extracts. (A) Stimulated HT-29 cells were lysed and extracted DNA was separated by agarose gel electrophoresis (1.2%). The activation of caspase-3 by S. aureus and L. plantarum extracts (B) and α-toxin and L. plantarum (C) was examined with a caspase-3 assay kit. Caspase-9 activation by S. aureus and L. plantarum extracts (D) and α-toxin and L. plantarum (E) was also examined. Bcl-2 expression from HT-29 after treating with S. aureus and L. plantarum extracts (F) or α-toxin and L. plantarum extracts (G) was examined by western blotting. *p < 0.05; **p < 0.01.

L. plantarum plays an important role in promoting the health of the host through antioxidative effects, antitumor effects, maintenance of the normal flora of the digestive tract, prevention of enteritis, and regulation of immune responses [23]. In the previous study, we have shown that L. plantarum lysates have a moisture effect in both human and mouse models [14,15]. In the current study, we showed that L. plantarum crude extracts inhibited S. aureus-induced apoptosis through inhibition of the intrinsic pathway. Next, we tried to identify which fraction of L. plantarum contributes the anti-apoptotic effect on S. aureus-infected HT-29 cells. First, HT-29 cells were co-treated with α-toxin and L. plantarum total extracts, culture supernatants, or pellet. L. plantarum total extracts and lysate pellet showed inhibitory effect against α-toxin (Fig. 4A). In the further study, we prepared whole-cell crude extracts, lipoteichoic acid, peptidoglycan, cell wall, and cytosol fractions. As shown in Fig. 4B, α-toxin-mediated apoptosis was inhibited by LTA, PGN, and cell wall fractions, indicating that cell wall components of L. plantarum contributed anti-apoptotic effects. Interestingly, LTA has many useful effects on the immune regulation. For examples, LTA inhibits excessive inflammation, which elevates the survival rate of sepsis in a mice model [16]. It is also suppresses LPS-mediated atherosclerotic plaque inflammation [18] and inhibits melanogenesis in B16F10 mouse melanoma cells [17]. These studies suggest that LTA isolated from L. plantarum would have a strong anti-inflammatory effect and could be a good candidate for the inflammation immune therapy. Through the current study, we identified another effect of L. plantarum LTA; its anti-apoptotic effect in S. aureus-induced cell death.

Fig. 4.Inhibitory effect of cell wall components of L. plantarum on α-toxin-mediated apoptosis. (A) HT-29 cells were stimulated with α-toxin and L. plantarum total extracts (total ext.), culture supernatants (sup.), or L. plantarum lysates pellet (pellet). (B) Cells were stimulated with α-toxin and L. plantarum whole cells (whole cells), lipoteichoic acid (LTA), peptidoglycan (PGN), cell wall, or cytosol fraction (cytosol). In both experiments, the WST-1 assay was used to determine cell viability. *p < 0.05; **p < 0.01 as compared with α-toxin-treated only.

S. aureus infection induces apoptosis, which is associated with intracellular replication of bacteria. Apoptosis mediates the release of S. aureus from the host cells [9]. Next, we examined whether L. plantarum extracts can inhibit S. aureus infection or replication in HT-29 cells. When HT-29 cells were pre-infected with S. aureus followed by incubation with L. plantarum extracts-containing media, the replication of S. aureus within HT-29 cells was decreased by L. plantarum extracts ( Fig. 5A). In the further study, HT- 29 cells were pretreated with L. plantarum extracts for 18 h, and then cells were infected with S. aureus. As shown in the figure, S. aureus infection was significantly inhibited by L. plantarum extracts in a dose-dependent manner (Fig. 5B). These results suggest that L. plantarum extracts can reduce S. aureus infection into HT-29 cells as well as S. aureus replication within the cells, which results in the inhibition of S. aureus spreading to other cells.

Fig. 5.L. plantarum extracts inhibit S. aureus infection into HT-29 cells. (A) HT-29 cells were infected with S. aureus (1 × 105 cell/well) for 2 h, and cells were washed and incubated with L. plantarum extracts-contained media for 18 h. (B) HT-29 cells were pre-treated with the indicated dose of L. plantarum extracts for 18 h and infected with S. aureus (1 × 105 cells/well) for 1 h. Culture media were removed and washed with PBS four times. Cells were incubated with fresh media containing 50 μg/ml gentamicin for 24 h. After incubation, cells were washed with PBS and lysed. The lysates were serially diluted with PBS and plated onto BHI agar plates. The number of bacterial colonies was counted and the total CFU was determined, after overnight culture at 37℃. *p < 0.05; **p < 0.01 as compared with untreated (Unt).

In conclusion, S. aureus has evolved to subvert host defense systems to create a suitable environment for their replication, and apoptosis is one of its strategy. On the other hand, L. plantarum has a beneficial influence by inhibiting S. aureus-mediated cell death. S. aureus-mediated apoptosis was mainly due to its α-toxin, and L. plantarum crude extracts inhibited α-toxin-mediated caspase-3 and caspase-9 activation and increased the Bcl2 expression. In particular, L. plantarum cell wall components such as LTA and PGN contributed these anti-apoptotic effects. Our study suggests that L. plantarum extracts can help to effectively maintain intestinal homeostasis by inhibiting S. aureus spreading.