Potassium Acetate Blocks Clostridium difficile Toxin A-Induced Microtubule Disassembly by Directly Inhibiting Histone Deacetylase 6, Thereby Ameliorating Inflammatory Responses in the Gut

Abstract

Clostridium difficile toxin A is known to cause deacetylation of tubulin proteins, which blocks microtubule formation and triggers barrier dysfunction in the gut. Based on our previous finding that the Clostridium difficile toxin A-dependent activation of histone deacetylase 6 (HDAC-6) is responsible for tubulin deacetylation and subsequent microtubule disassembly, we herein examined the possible effect of potassium acetate (PA; whose acetyl group prevents the binding of tubulin to HDAC-6) as a competitive/false substrate. Our results revealed that PA inhibited toxin A-induced deacetylation of tubulin and recovered toxin A-induced microtubule disassembly. In addition, PA treatment significantly decreased the production of IL-6 (a marker of inflamed tissue) in the toxin A-induced mouse enteritis model. An in vitro HDAC assay revealed that PA directly inhibited HDAC-6-mediated tubulin deacetylation, indicating that PA acted as a false substrate for HDAC-6. These results collectively indicate that PA treatment inhibits HDAC-6, thereby reducing the cytotoxicity and inflammatory responses caused by C. difficile toxin A.

Introduction

Clostridium difficile (C. difficile) causes pseudomembranous colitis in humans and animals [3,9,21] by releasing two bacterial toxins: toxin A and toxin B [14,22]. These toxins trigger cytoskeletal disorganization and apoptosis of the gut epithelial cells that are responsible for mucosal barrier function [6,22], leading to a severe inflammatory reaction [6,18]. Many antibiotics, including vancomycin and metronidazole, have been used to effectively treat the disease symptoms [16], but there have been problems with drug-resistant C. difficile [23]. Thus, we need to develop new non-antibiotic therapies.

C. difficile toxins have been shown to prevent Rho family proteins (e.g., Rho, Rac, and cdc42) from participating in the formation of actin filaments [8]. This mechanism is believed to be a main cause for the cell rounding that is characteristic of toxin-exposed cells [6,18]. However, we recently reported that the toxin A-induced cytoskeletal disorganization of colonocytes was highly enhanced by the microtubule disassembly resulted from tubulin deacetylation [18]. In the same work, we further showed that toxin A activated histone deacetylase 6 (HDAC-6), which deacetylates tubulin proteins and thereby governs microtubule disassembly.

Microtubules are composed of α-tubulin and β-tubulin, and are regulated by post-translational modifications such as tyrosination and acetylation [19,20]. Acetylation of α-tubulin at Lys-40 (K40) has been reported to enhance microtubule polymerization [28], whereas HDAC-6 has been implicated in the deacetylation of α-tubulin [7,18,28]. Indeed, we previously showed that treatment with trichostatin A (TSA), a chemical inhibitor of HDAC-6, significantly reduced the cytoskeletal disorganization in colonic epithelial cells and inhibited the severe gut inflammation seen in the toxin A-treated mouse model of enteritis [18]. These results suggest that HDAC-6 could be a critical target for inhibiting the toxins produced by C. difficile.

Potassium acetate (PA) has been used to remove proteins during the isolations of DNA and RNA [2,15,24,25], for fixation of animal tissues in biological studies, as a food preservative [13], and to treat diabetic ketoacidosis via its ability to break down into bicarbonate [27]. Moreover, PA can help neutralize the human body from an acidotic state [27]. However, no previous study has examined the possible effect of PA as a therapeutic agent for the treatment of gut inflammation.

Here, we report that PA directly inhibits the enzymatic activity of HDAC-6, rescues the α-tubulin deacetylation seen in toxin A-treated cells, and reduces the signs of cellular toxicity (e.g., cell rounding in vitro and gut inflammation in vivo) caused by C. difficile toxin A. Collectively, these results suggest that PA specifically inhibits HDAC-6 in colonocytes, and could be a candidate drug for the treatment of toxin A-induced gut inflammation.

 

Materials and Methods

Toxin A Preparation and Cell Culture

Toxin A was purified from C. difficile strain 10463 (American Type Culture Collection, Rockville, MD, USA) as previously described [12]. The purity of the native toxin A was assessed by gel electrophoresis, which confirmed a single protein band at the expected molecular mass of 307 kDa [26]. HT29 human colorectal adenocarcinoma cells were cultured in McCoy’s 5A medium (Invitrogen, Carlsbad, CA, USA) in a 37℃ humidified incubator supplemented with 5% CO2.

Antibodies and Reagents

Polyclonal antibodies against α-tubulin, acetylated α-tubulin, acetylated histone H3, HSP90, c-Src, and HDAC-6 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PA, TSA, and the antibody against β-actin were purchased from Sigma Aldrich (St. Louis, MO, USA). The polyclonal antibody against caspase-3 was obtained from Cell Signaling Technology (Beverly, MA, USA).

Immunoblot Analysis

HT29 cells were washed with cold phosphate-buffered saline (PBS) and lysed in a buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, and 1% Nonidet P-40 [17]. Equal amounts of protein were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The appropriate antibodies were applied, and antigen-antibody complexes were detected with the LumiGlo reagent (New England Biolabs, Ipswich, MA, USA).

Enzymatic Activity Assay for HDAC-6

HDAC-6 activity was measured as the absence or presence of PA, which was assessed using immunoprecipitation with an antibody against HDAC-6 and visualized using a fluorescence activity assay kit (Cayman Chemical, Ann Arbor, MI, USA), as previously described [18]. Briefly, each extract (1 μg) or immunoprecipitant was incubated at 37℃ with 100 μM acetylated fluorogenic substrate in HDAC assay buffer. After 30 min, the lysine developer was added, and the mixture was incubated for another 15 min at room temperature. Fluorescence was measured using a Spectra Max M5 fluorescent plate reader (Molecular Devices, Sunnyvale, CA, USA) with excitation at 360 nm and emission at 460 nm.

Animals

This study was approved by the Animal Care and Use Committee of Daejin University (Pocheon, Korea). Male CD1 mice (Daehan Biolink, Daejeon, Korea) weighing 30-35 g were used; they had free access to food and water and were acclimated to these conditions for at least 7 days prior to experiments [10]. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). Ileal loops (2 cm) were prepared and injected with control buffer (PBS), PA (10 μM), toxin A (3 nM), or toxin A plus PA in a volume of 100 μl of PBS. After 4 h, animals were sacrificed and ileal loop tissues were collected.

Cell Viability

HT29 cells treated with the various agents were incubated with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye for 2 h. The solubilization reagent was added, and absorbance was determined at 570 nm by a spectrophotometer (Model 3550; Bio-Rad, Mississauga, Canada) [18].

Measurement of Mouse IL-6

Toxin A was previously reported to stimulate the production of interleukin- (IL-6) from target cells [12]. To evaluate whether this occurred in our mouse model of gut inflammation, ileal loops of the above-described mice were homogenized (40 sec) and centrifuged (11,000 rpm, 10 min at 4℃), and supernatants were collected [18]. Mouse IL-6 was measured by ELISA using a commercially available kit (R&D Systems, Minneapolis, MN, USA).

Confocal Scanning Laser Microscopy

For visualization of microtubule structures, colonocytes were exposed for 5 h to control medium, PA, TSA, toxin A alone (1 nM), or toxin A plus PA, and then fixed in 4% paraformaldehyde dissolved in PBS. After being washed with PBS, the cells were incubated with a polyclonal anti-acetylated α-tubulin antibody (diluted 1:500) in PBS containing 0.05% Tween 20 for 6 h at room temperature. After three washings, the anti-acetylated α-tubulin antibody-treated cells were incubated with FITC-labeled antimouse IgG (1:500) for 2 h at room temperature. The cells were then mounted and analyzed using a Bio-Rad MRC 1024 confocal scanning laser microscope equipped with a krypton/argon mixed gas laser as a light source. Excitation was carried out using the 480 or 594 nm lines from the laser, as previously described [1].

Statistical Analysis

The results are presented as the mean values ± SEM. Data were analyzed using the SIGMA-STAT professional statistics software program (Jandel Scientific Software, San Rafael, CA, USA). Analyses of variance with protected t tests were used for intergroup comparisons.

 

Results and Discussion

Potassium Acetate Increases α-Tubulin Acetylation in Human Colonocytes

We first tested whether PA, which contains an acetyl group, affected the acetylation levels of tubulin in human colonocytes. We treated HT29 cells with and without various concentrations of PA for 5 h, and then measured the tubulin acetylation. As shown in Fig. 1A, PA treatment dose-dependently increased the acetylation level of α-tubulin, but did not alter the amounts of total α-tubulin or the acetylation level of histone H3 (a nuclear protein). This indicates that PA affects the acetylation in the cytosol but not in the nucleus. Here, we found that PA-mediated α-tubulin acetylation increased rapidly beginning at 0.5 h post-treatment, with the maximum rate observed at 12 h post-treatment (Fig. 1B). The rapid effects of PA on cytosolic α-tubulin may reflect that PA easily translocates to the cytosol through the cell plasma membrane owing to its small size and simple structure [5].

Fig. 1.Potassium acetate (PA) increases the acetylation and polymerization of α-tubulin in HT29 human colonocytes. (A) Colonocytes were incubated with 0, 10, 50, and 100 mM PA for 5 h, cell lysates were subjected to 10% polyacrylamide gel electrophoresis, and blots were probed with antibodies against acetylated α-tubulin, α-tubulin, acetylated histone H3, HSP90, c-Src, and β-actin. The presented results are representative of three independent experiments. (B) Cells were incubated with PA (50 mM) for the indicated times. (C) Cells were treated with PA (50 mM) or trichostatin A (TSA, 10 μM) for 5 h. The presented results are representative of three independent experiments. (D) Cells were grown on coverslips, incubated with PA or TSA for 5 h, fixed, and immunostained with an anti-acetylated α-tubulin antibody. Microtubule organization was visualized by confocal microscopy. The results shown are representative of three independent experiments. (E) Cells were incubated with medium (con), PA, or TSA for 24 or 48 h, and cell viability was measured by MTT assay. The results are expressed as a percentage of the value from untreated controls. The bars represent the mean ± SE of three experiments performed in triplicates.

We noted that the PA-mediated increase of α-tubulin acetylation was comparable to that induced by TSA [4], which strongly increases α-tubulin acetylation by inhibiting HDAC-6 (Fig. 1C). We thus speculated that PA may inhibit the enzyme activity of HDAC-6 that is known as a tubulin deacetylase. These results also suggest that although PA has a simple acetyl group, PA is likely able to specifically inhibit HDAC-6 in the cytosol but does not affect other deacetylase activity in the nucleus. Next, to assess whether the PA-dependent increase of tubulin acetylation enhanced microtubule polymerization, we performed immunofluorescence staining with an antibody against acetylated α-tubulin. PA treatment (50 mM) of colonocytes for 5 h substantially increased the level of polymerized microtubules compared with that in control cells (Fig. 1D). Moreover, 50 mM of PA did not induce any cellular toxicity in human colonic epithelial cells (Fig. 1E). However, TSA-treated cells exhibited a marked reduction in cell viability. These results suggest that PA treatment increases α-tubulin acetylation, thereby enhancing microtubule polymerization.

PA Directly Inhibits HDAC-6 Activity as a False Substrate

As our observations suggested that HDAC-6 may largely mediate the PA-induced enhancement of α-tubulin acetylation in colonocytes, we investigated whether PA could directly inhibit HDAC-6 in these cells. Colonocytes were treated with PA and lysed, HDAC-6 was isolated by immunoprecipitation with an HDAC-6 antibody, and the immunoprecipitates were incubated with an acetylated fluorogenic substrate for 30 min. As shown in Fig. 2, the enzymatic activity of HDAC-6 was significantly higher in experiments containing the substrate, compared with substrate-free control experiments. However, this increase was significantly reduced by PA treatment, indicating that the inhibitory effect of PA against HDAC-6 could be due to a competitive inhibition mechanism in which PA (containing an acetyl group) acts as a false substrate. This finding also suggests that the increased acetylation of α-tubulin observed in PA-treated colonocytes is likely to be associated with the inhibition of HDAC-6.

Fig. 2.PA directly inhibits HDAC-6 activity in colonocytes. To assess enzymatic activity in vitro, HDAC-6 proteins were immunoprecipitated from colonocyte extracts, and their enzymatic activity was measured after 30 min incubation in the absence or presence of PA. The results shown are representative of three independent experiments (*, p < 0.005).

PA Inhibits C. difficile Toxin A-Induced Cellular Toxicity in Colonocytes

Toxin A is known to cause a marked deacetylation of tubulin in colonic epithelial cells [6,11], and this deacetylation has been associated with gut inflammation [11]. Moreover, we previously reported that TSA (an HDAC-6 inhibitor) significantly rescued the gut inflammation caused by toxin A [6,11]. Here, we tested whether PA (through its ability to increase α-tubulin acetylation by inhibiting HDAC-6) could prevent toxin A-induced cytotoxicity. HT29 cells were pretreated with 50 mM PA for 1 h and further incubated with toxin A (1 nM) for 4 h. As shown in Fig. 3A, toxin A treatment decreased α-tubulin acetylation, but this was significantly restored by PA pretreatment. PA treatment alone also markedly increased the acetylation of α-tubulin in colonocytes.

Fig. 3.PA inhibits toxin A-induced deacetylation of α-tubulin. (A) HT29 cells were incubated with 50 mM PA for 1 h and then exposed to toxin A for 4 h. Cell lysates were subjected to 10% polyacrylamide gel electrophoresis, and blots were probed with antibodies against acetylated α-tubulin and β-actin. The presented results are representative of three independent experiments. (B) HT29 cells were grown on coverslips, pretreated with PA (50 mM) for 1 h, incubated with medium (con), toxin A (1 nM, Tx), or toxin A plus PA (Tx + PA) for 4 h, and then immunostained with anti-acetylated α-tubulin. (C) Cells were grown on coverslips, pretreated with PA for 1 h, and then incubated with medium (con), toxin A (Tx), or toxin A plus PA (Tx + PA) for 4 h. Changes in cell shape were detected by light microscopy. D. Cells were incubated with 50 mM PA for 1 h and then exposed to toxin A for 48 h. Cell viability was measured by MTT assay and expressed as a percentage of the results from untreated controls. The bars represent the mean ± SE of three experiments performed in triplicates (*, p < 0.05).

Since toxin A is known to cause cell rounding via both tubulin disassembly and actin disaggregation [6], we also tested whether PA prevented the rapid cytoskeletal disorganization caused by toxin A. Colonocytes were pretreated with PA (50 mM) for 1 h and then exposed to toxin A alone (1 nM) or toxin A plus PA (50 mM) for 4h. Microtubules were visualized with anti-acetylated α-tubulin and FITC-labeled anti-mouse IgG. As expected, toxin A treatment caused strong microtubule disassembly, and this was significantly recovered by PA pretreatment (Fig. 3B). Light microscopy also revealed that PA treatment could inhibit the severe cell rounding caused by toxin A (Fig. 3C). We further assessed whether the PA-induced increase of α-tubulin acetylation could recover the marked decrease in cell viability of toxin A-treated cells. However, our results showed that PA treatment only slightly blocked the toxin A-induced loss of cell viability, and treatment with PA alone did not alter the viability of colonocytes (Fig. 3D). This suggests that the PA-induced formation of microtubules inhibits the cytoskeletal disorganization induced by toxin A, but does not affect toxin A-induced cytotoxicity.

PA Treatment Attenuates the Inflammatory Response and Villus Disruption in a Toxin A-Induced Mouse Model of Enteritis

Finally, we investigated whether PA could inhibit toxin A-induced mucosal damage and inflammatory responses in mice. We first determined the minimal dose of PA that showed a therapeutic effect on the mouse gut epithelium, and thus would presumably minimize cellular toxicity. Treatment with 10 mM PA did not cause epithelial damage in the gut (data not shown), so this dosage was used in the following experiments. Closed ileal loops of CD1 mice were treated with PBS (control), PA alone, toxin A alone (3 nM), or PA plus toxin A for 4 h, and inflammatory responses were evaluated. Our results revealed that toxin A-treated mice showed increased mucosal damage, but this was abolished by PA co-treatment (Fig. 4A). Next, we assessed whether PA treatment could inhibit the inflammatory response in the guts of toxin A-exposed mice. As shown in Fig. 4B, toxin A strongly increased the levels of the proinflammatory cytokine IL-6, but this was significantly reduced by PA co-treatment. Interestingly, treatment of mice with PA alone reduced the basal level of IL-6 in the guts of control mice. These results suggest that the PA-dependent enhancement of microtubule assembly in epithelial cells may increase their barrier function, thereby decreasing the villus disruption and inflammatory responses induced by toxin A.

Fig. 4.Therapeutic effect of PA on toxin A-induced inflammatory responses and villus destruction in the gut. (A) Mouse ileal loops were injected with 100 ml of PBS alone (con), or PBS containing PA (10 mM), toxin A (3 nM), or toxin A plus PA. After 4 h, the animals were killed and the ileal loops were removed and processed. Light micrographs of mouse ileum (H&E staining; original magnification, ×200). The results shown are representative of three independent experiments. (B) Concentration of IL-6 in the ileal tissues. Data are expressed as the mean ± SE of three experiments performed in triplicates (*, p < 0.05).

Although actin disaggregation was traditionally considered to be the main cause of toxin A-induced cell rounding [6], we previously demonstrated that toxin A-induced α-tubulin deacetylation is also critical for the cytoskeletal disruption that causes severe rounding of toxin A-treated cells. The results of the current study also strongly suggest that the regulation of α-tubulin is a critical mediator of the cell damage and gut inflammation caused by toxin A.

In summary, we herein show for the first time that potassium acetate increases the acetylation of α-tubulin by directly inhibiting the activity of HDAC-6 in human colonocytes. The PA-dependent increase of α-tubulin acetylation was found to prevent severe cell rounding and the inflammatory response seen in the toxin A-induced mouse model of enteritis. Moreover, the PA-induced inhibition of HDAC-6 was found to be due to its direct interaction with HDAC-6 as a competitive/false substrate. Our findings collectively indicate that PA could prove useful as a candidate drug for preventing the gut inflammation induced by the toxins of C. difficile.