Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea. The symptoms of CDI are caused by two exotoxins, TcdA and TcdB, which are structurally and functionally highly homologous. Both toxins bind to specific receptors on mammalian cells, are internalized through endocytosis, translocate to the cytoplasm, and inactivate Rho-type GTPases via covalent glucosylation. This leads to downstream events that include morphological changes and disruption of epithelial tight junctions, release of pro-inflammatory mediators, and cell death. Assays used to assess the effects of toxins on cells have historically relied on evaluation of cell rounding or quantitation of ATP levels to estimate cell death—assays which can be qualitative and variable. In this chapter, several assays are described that robustly and quantitatively measure early and late toxin-dependent events in cells, including (i) toxin binding, (ii) Rac1 glucosylation, (iii) changes in cellular morphology (measured as dynamic mass redistribution), (iv) loss of epithelial integrity (measured as transepithelial electrical resistance), and (v) cell death (measured as total cellular protein using a colorimetric assay). The assays were validated using the highly specific monoclonal antitoxin antibodies, actoxumab and bezlotoxumab, which neutralize TcdA and TcdB, respectively.
- C. difficile
- cell-based assays
- epithelial cells
Current treatment for
The renewed interest in toxin-directed therapies underscores the importance of having robust quantitative assays in place to assess the activity of the
2. Materials and methods
2.1. TcdA- and TcdB-binding assay (Western blot)
TcdA (1 μg/ml) or TcdB (0.1 μg/ml) (The Native Antigen Company, Upper Heyford, the UK and tgcBIOMICS, Bingen, Germany) was incubated with or without 200 µg/ml actoxumab or bezlotoxumab in Vero cell culture medium (Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin) for 30 min at 37°C; these mixtures were then chilled on ice and added to plates of pre-chilled Vero cells (ATCC, Rockville, MD). Plates were incubated for 30 min on ice to allow binding of toxins. Following incubation, plates were washed three times with cold phosphate buffered saline (PBS) and cells were harvested by scraping. Cell membranes were isolated at 4°C with the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific, Grand Island, NY), according to the manufacturer’s instructions, and solubilized in a total volume of 100 µL solubilization buffer with HALT protease/phosphatase inhibitors (Thermo Scientific). Following addition of Laemmli sample buffer, samples were incubated for 5 min at 95°C and resolved by SDS PAGE in 4–12% polyacrylamide gels and transferred to a nitrocellulose membrane. The nitrocellulose membrane containing transferred protein was blocked in Odyssey blocking buffer (Li-Cor) followed by incubation with actoxumab, bezlotoxumab, or an anti-cadherin antibody (Cell Signaling Technology, Beverly, MA) as the primary antibody for 1 h at room temperature (RT). After washing, the nitrocellulose membrane was incubated with a goat anti-human IgG antibody coupled to IRDye® 800CW (Li-Cor) for 30 min at RT. After additional washing, bands were visualized using the Odyssey imaging system (Li-Cor).
2.2. TcdA-binding assay (flow cytometry)
TcdA, from ribotype 087 (The Native Antigen Company, Upper Heyford, the UK), was fluorescently labeled using the Lightning Link Atto488 Antibody Labeling kit (Novus Biosciences, Littleton, CO) as directed by the manufacturer. About 50 μg of lyophilized TcdA was reconstituted for a minimum of 30 min in sterile ddH2O at RT before adding the LL-modifier buffer. The toxin/LL-modifier buffer solution was transferred to a vial containing the lyophilized Lightning Link mix. The mixture was pipetted up and down and incubated at RT in the dark. After 5 h, LL-quencher buffer was added and incubated at RT in the dark for 30 min and then stored at 4°C until use the following day. Several concentrations of TcdA-Atto488 were incubated with or without 200 µg/ml actoxumab at RT for 60 min, protected from light. Samples were then chilled on ice. Adherent HT29 cells (ATCC, Rockville, MD) were resuspended in the cell medium (McCoy’s 5A Modified medium supplemented with 10% FBS, 2 mM glutamine, 0.75% sodium bicarbonate, 100 U/ml penicillin, and 100 U/ml streptomycin), following treatment with Accutase (Innovative Cell Technologies), washed once with cold Dulbecco’s phosphate-buffered saline with calcium and magnesium (DPBS++) containing 1% bovine serum albumin (BSA), and then chilled on ice. 100 µL of each toxin/antibody sample was added to separate vials containing 3 × 105 cells. After mixing, samples were incubated on ice in the dark. After 30 min, 1 ml of ice cold DPBS++/1% BSA was added to each sample. To remove unbound toxin, cell suspensions were washed twice with ice cold DPBS++/1% BSA by centrifuging for 5 min at 4°C at 200 × g and removing the supernatant. Washed cells were resuspended in 500 µl cold DPBS++/1% BSA and analyzed by flow cytometry using an LSRII instrument (BD Biosciences) with excitation and emission wavelengths of 488 and 530 nm, respectively. 10,000 events were measured for each sample.
2.3. Rac1 glucosylation assay
Vero cells were seeded at a cell density of 5 × 103 cells/well in a 384-well collagen-coated plate and grown overnight at 37°C in 5% CO2. TcdA and TcdB (The Native Antigen Company, Upper Heyford, the UK and tgcBIOMICS, Bingen, Germany) were serially diluted in Vero cell culture medium, and 50 μl was added to each well. For assays determining neutralization effects of actoxumab and bezlotoxumab, TcdA and TcdB were pre-incubated at 90% effective concentrations (EC90) with actoxumab and bezlotoxumab, respectively (various concentrations), for 1 h at RT in Vero cell culture medium, prior to addition of cells as above. Following incubation at 37°C in 5% CO2 for 3 h, medium containing toxin alone or toxin+antibody was removed by aspiration. Cells were immediately fixed with 50 μl/well fixing solution (4% paraformaldehyde in modified Dulbecco’s phosphate-buffered saline (DPBS/modified)) for 1 h at RT. Following fixation, cells were washed four times for 5 min with 50 μl/well permeabilization solution (0.1% Triton-X-100 in DPBS/modified) at RT with gentle shaking. Cells were then blocked with 50 μl/well Odyssey blocking buffer (Li-Cor) overnight at 4°C. After removing blocking buffer, cells were incubated with 25 μl/well mouse anti-Rac1 (BD Biosciences #610651, recognizing non-glucosylated Rac1), or anti-Rac1 clone 23A8 (Millipore #05-389, recognizing total Rac1), diluted at 1:75 and 1:200, respectively, in Odyssey blocking buffer and incubated for 2 h at RT with gentle shaking. Cells were washed four times for 5 min with 50 μl/well wash solution (0.1% tween 20 in DPBS/modified) at RT with gentle shaking. Cells were then incubated with 25 μl/well secondary antibodies (IRDye 800 CW goat anti-mouse and CellTag 700 stain, diluted at 1:800 and 1:1000, respectively, in Odyssey blocking buffer) at RT for 1 h with gentle shaking protected from light. Cells were again washed four times for 5 min with 50 μl/well wash solution at room temperature with gentle shaking. After the final wash, any remaining solution was removed from the wells, and the plates were scanned on the Li-Cor Odyssey classic (Li-Cor) with detection in both 700 and 800 nm channels (A700 and A800). Cell number normalization/well was calculated using the ratio of A800/A700, and remaining percent of non-glucosylated Rac1 was determined using the ratio of normalized A800 of treated cells/normalized A800 of untreated cells multiplied by 100. Analysis was performed with GraphPad Prism (version 6.04) using the 4-parameter nonlinear regression formula.
2.4. Dynamic mass redistribution (Epic) assay
Vero cells were seeded at a cell density of 5 × 103 cells/well in a 384-well fibronectin-coated Epic plate (Corning #5042) and grown overnight at 37°C in 5% CO2. On the day of assay, medium was aspirated and replaced with 40 μl/well assay buffer (HBSS in 20 mM HEPES) and equilibrated at RT for 1 h. TcdA and TcdB (The Native Antigen Company, Upper Heyford, UK and tgcBIOMICS, Bingen, Germany) were serially diluted in assay buffer and equilibrated at RT for approximately 10 min. For assays determining neutralization effects of actoxumab and bezlotoxumab, TcdA and TcdB were pre-incubated at EC90 concentrations for 1 h at RT with actoxumab and bezlotoxumab, respectively (various concentrations). Following pre-incubations, 10 μl/well of the toxins alone or toxin/antibody solutions were added to Vero cells using a Matrix Platemate (Thermo Scientific) and gently mixed. The plate was read every 12 s for 200 min using the Epic BT-157900 (Corning). As a baseline, wells containing assay buffer alone were used. The dynamic mass redistribution (DMR) values were recorded at 180 min at which point the signal had plateaued (not shown). The recorded DMR values (corrected for assay buffer alone) were collected with EpicAnalyzer software and analyzed with GraphPad Prism (version 6.04) using the four-parameter nonlinear regression formula.
2.5. Transepithelial electrical resistance (TER) assay
To initiate the 2-dimensional culture system, 0.5–1 × 105 Caco-2 cells (ATCC, Rockville, MD) were seeded into each well of the 24-well insert plates (Falcon #351181 HTS Multiwell Insert System—1.0 um pore size/PET membrane), with 250 μl Caco-2 cell culture medium (EMEM supplemented with 10% FBS, 1× non-essential amino acid, 0.075% sodium bicarbonate, 100 U/ml penicillin, and 100 U/ml streptomycin) in the apical chamber and 800 μl in the basolateral chamber. Caco-2 cells were cultured for at least 14 days at 37°C with 5% CO2 to ensure full differentiation and confluency, which were confirmed by plateauing of the TER reading at ≥ 600 Ω cm2. TER was measured using the Epithelial Volt-Ohm Meter Millicell ERS-2 (EMD Millipore, Billerica, MA, USA). To assess the effect of toxins on the cell monolayer, TcdA and TcdB (The Native Antigen Company, Upper Heyford, UK and tgcBIOMICS, Bingen, Germany) were added to the apical chamber. To evaluate the ability of the antibodies to neutralize toxin effects, actoxumab or bezlotoxumab was added to the apical chamber immediately before addition of TcdA or TcdB to the apical chamber. For neutralization studies, 10 ng/ml TcdA was combined with various concentrations (from 0 to 50 μg/ml) of actoxumab, and 100 ng/ml TcdB was combined with various concentrations (from 0 to 100 μg/ml) of bezlotoxumab. TER measurements were obtained immediately before and, at 6, 24, and 48 h, after addition of toxins/antibodies to the apical chamber. TER values were normalized to values obtained in the absence of toxin at each time point to account for minor time-dependent variability.
2.6. Sulforhodamine B assay
To study the effects of
To assess the cytotoxicity of
3.1. Overview of mammalian cell intoxication by TcdA and TcdB
TcdA and TcdB are large, monomeric proteins (300 and 270 kDa, respectively) with similar structures and functions (Figure 1) [17, 18]. The functional domains of the toxins are arranged according to the ABCD model : the N-terminal A domain contains the glucosyltransferase enzymatic activity, the B domain is a putative receptor-binding domain composed of a series of long and short repeats known as combined repetitive oligopeptides (CROPs), the cysteine protease (C) domain is responsible for autocatalytic processing, and the D domain is involved in pore formation and toxin translocation. Both toxins bind to receptors on the surface of the epithelial cells that line the wall of the lower intestine (and possibly other cell types). Once bound, they are internalized via receptor-mediated endocytosis . Acidification of the endosome promotes a conformational change that enables translocation of the N-terminal glucosyltransferase domain of the toxin into the cytoplasm. Cellular inositol hexakisphosphate (InsP6) allows cleavage of the toxin by the cysteine protease domain, releasing the glucosyltransferase domain into the cytoplasm where it inactivates Rho-type GTPases through covalent glucosylation (from UDP-glucose) . This in turn causes changes in epithelial cell morphology due to actin depolymerization, loss of tight junction integrity, and eventually, cell death (Figure 1) . The assays described in this chapter measure many of the various steps, described above, involved in the intoxication cascade (steps 1–5, as denoted in Figure 1).
3.2. Cell surface binding of TcdA and TcdB (step 1 in Figure 1)
Binding of toxins to the cell surface of target cells is the first step in TcdA and TcdB cell entry, leading to the downstream effects of the toxins. We assessed cell surface binding of TcdA and TcdB by Western blotting of cell membranes isolated from Vero cells incubated with TcdA or TcdB at 4°C. As shown in Figure 2, membrane fractions isolated from cells incubated with TcdA (see Figure 2A, top panel) or TcdB (Figure 2B, top panel) contain toxins, indicating cell surface binding of the toxins. Actoxumab and bezlotoxumab bind to and neutralize purified TcdA and TcdB, respectively, from a variety of
Binding of TcdA to cells and the prevention, thereof, by actoxumab were also assessed by flow cytometry (Figure 3). Incubation of HT29 cells with increasing levels of fluorescently labeled TcdA (TcdA-Atto488) led to an elevated mean fluorescence intensity (MFI), indicating binding of TcdA to the cell surface in a concentration-dependent manner. In the presence of actoxumab, however, the MFI for each toxin concentration was reduced to background levels showing that actoxumab blocked binding of TcdA to the cell surface. No significant changes in MFI were measured in the presence of bezlotoxumab, indicating that the effect of actoxumab is specific (data not shown).
3.3. Glucosylation of Rac1 by TcdA and TcdB (step 2 in Figure 1)
Inactivation of Rho-type GTPases is a key step in the intoxication of host cells, leading to the downstream cytopathic and cytotoxic effects of the
Actoxumab and bezlotoxumab neutralized the effects of TcdA and TcdB (at EC90 concentrations), respectively (Figure 4C and D). Notably, the potency of actoxumab and bezlotoxumab on their respective toxins was lower for toxins of ribotype 027 and 078 compared to ribotype 087. This is consistent with the lower affinities of the antibodies against toxins of these ribotypes, as previously described by Hernandez et al. .
3.4. Changes in cell morphology induced by TcdA and TcdB (step 3 in Figure 1)
The cytopathic effects of TcdA and TcdB on gut epithelium are visualized as profound morphological changes, typically cell rounding, due to the glucosylation and inactivation of Rho-type GTPases and subsequent disruption of actin polymerization. Historically, these cytopathic effects have been assessed qualitatively through visual determination of cell rounding . Improved phenotypic assays used to investigate changes in cell morphology involve the quantification of length-to-width ratios of fluorescently labeled cells [23, 24]. This latter technique is quantitative and has an improved throughput, although it requires consistent staining and substantial data analysis. To better understand and quantify toxin-induced morphological changes in unlabeled cells, an assay was developed to examine dynamic mass distribution (DMR) in Vero cells using the Epic instrument. In this assay, plates containing optical sensors are used to capture translocation of cellular mass of unlabeled cells in response to ligand binding, allowing changes in cell shape to be quantified. The concentration-dependent effects of TcdA and TcdB on mass redistribution were determined at 180 min (at which time the effects have plateaued, not shown) (Figure 5A). As with the Rac1 glucosylation assay, Vero cells are much more sensitive to TcdB than TcdA in the DMR assay. The neutralizing effects of actoxumab and bezlotoxumab on toxin-induced morphological changes were assessed at EC90 concentrations of TcdA and TcdB, respectively. Actoxumab and bezlotoxumab fully neutralized the effects of TcdA and TcdB, respectively, on DMR (Figure 5B).
3.5. Toxin-induced disruption of epithelial tight junctions (step 4 in Figure 1)
To gain an understanding of the effect of
3.6. Toxin-induced cytotoxicity (step 5 in Figure 1)
The traditional way of assessing the cytoxic effects of
The robust nature of the SRB assay also allows for the study of the cytotoxic effects of unpurified
In this chapter, we have described novel cell-based assays for analyzing multiple distinct steps in the intoxication cascade associated with TcdA and TcdB. Unlike historical assays that measure toxin effects qualitatively, such as the visual assessment of cell rounding, or are variable and often unreliable, such as quantitation of ATP levels to estimate cell death, the assays presented here can quantitatively and robustly assess the effects of toxins in mammalian cells. We show how the initial event of toxin binding to host cells can be assessed using cell surface binding assays with labeled or unlabeled toxins in flow cytometry and Western blot formats, respectively. The more proximal events that follow internalization of the toxins, namely Rac1 glucosylation and cell rounding, can be studied with novel quantitative assays by in-cell Western and dynamic mass redistribution assays, respectively. Finally, we show how the TER and SRB assays can be utilized to assess the final stages of intoxication, tight junction disruption, and cell death, respectively. We also show how the SRB assay can be used to accurately measure the activities of TcdA and TcdB from unpurified toxins in culture supernatants of
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