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. 1999 Jan;67(1):302-7.

Saccharomyces Boulardii Protease Inhibits the Effects of Clostridium Difficile Toxins A and B in Human Colonic Mucosa

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Free PMC article

Saccharomyces Boulardii Protease Inhibits the Effects of Clostridium Difficile Toxins A and B in Human Colonic Mucosa

I Castagliuolo et al. Infect Immun. .
Free PMC article

Abstract

Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibits C. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225-5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardii on toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of S. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii on C. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease.

Figures

FIG. 1
FIG. 1
Effect of anti-S. boulardii protease IgG on S. boulardii protease activity against [14C]methemoglobin. S. boulardii conditioned medium (CM) (50 μg) or medium alone was incubated (1 h at 22°C) with different dilutions of rabbit anti-S. boulardii protease polyclonal IgG or with control rabbit IgG. To determine the proteolytic activity in the S. boulardii CM, the mixtures were incubated at 37°C with [methyl-14C]methemoglobin. After 20 min the reactions were terminated by adding TCA, and samples were centrifuged. To determine the proteolytic activity, the radioactivity released in the supernatant was measured by scintillation counting. Results are the means ± standard errors of the means of results from three to six separate experiments, each with duplicate determinations. ∗∗, P < 0.01, and ∗, P < 0.05 versus values for respective controls; ++, P < 0.01 versus values for S. boulardii CM alone. Ab, antibody; prot., protease; Sb, S. boulardii.
FIG. 2
FIG. 2
Anti-S. boulardii protease IgG reverses the protective effects of S. boulardii in toxin A-mediated enteritis. Rat ileal loops were injected with 5 μg of toxin A or toxin A preincubated (1 h at 37°C) with S. boulardii conditioned medium (3.0 mg in 200 μl). In other experiments S. boulardii conditioned medium was incubated (22°C for 1 h) with anti-S. boulardii protease IgG or control rabbit IgG (dilution, 1:1,000 for both) before ileal toxin A administration as described above. After 4 h animals were sacrificed and secretion of fluid was measured as the ratio of loop weight (micrograms) to length (centimeters) (A). 3[H]mannitol permeability was expressed as disintegration of [3H]mannitol per minute per centimeter of loop (B). Results are expressed as means ± standard errors of the means of results for each experimental condition (n = 6 to 10). ∗∗, P < 0.01 versus values for the control; +, P < 0.05, and ++, P < 0.01 versus values for toxin A alone. CM, S. boulardii conditioned medium; Tx A, toxin A; Ab, antibody; Sb, S. boulardii.
FIG. 3
FIG. 3
S. boulardii protease prevents toxin A- and B-mediated inhibition of protein synthesis in colonic epithelial (HT-29) cells. Purified toxin A or B (1 μg/ml) was incubated (1 h at 37°C) with either purified S. boulardii conditioned medium (100 μg/ml) or buffer alone before addition to cultured HT-29 cells (106 cells per well). After 4 h [3H]leucine (3 μCi/ml) was added to the culture media and the mixtures were incubated for an additional 16 h. At the end of the incubation period, culture media and cells were collected separately and proteins were precipitated by addition of TCA. Radioactivity contents in the precipitated proteins were measured as an indicator of protein synthesis and expressed as disintegrations per minute per well. Results are expressed as means ± standard errors of the means of results for each group; four to six wells were tested for each experimental condition, and duplicate determinations were made for each. ∗∗, P < 0.01 versus values for the control; ++, P < 0.01 versus values for toxin A or B alone. Tx, toxin; Sb, S. boulardii.
FIG. 4
FIG. 4
S. boulardii protease prevents toxin A- and B-mediated reduction of colonic resistance. Human colonic mucosal sheets were placed in Ussing chambers and incubated (3.5 h at 37°C) with either buffer alone or buffer containing 32 nM toxin A, 3 nM toxin B, or purified S. boulardii protease (1 μg/ml). Where indicated, toxin A or toxin B was preincubated (1 h at 37°C) with S. boulardii protease prior to placement in the Ussing chambers. Potential differences and short-circuit currents were recorded every 30 min to calculate changes in resistance (ohms per square centimeter). Results are expressed as means ± standard errors of the means of results for each group. Resistance baseline values 10 min before application of toxins (light gray bars) and 3.5 h after exposure to toxins (dark gray bars) are shown. Six to eight chambers were tested for each experimental condition. ∗, P < 0.01 versus the 10-min values. Sb, S. boulardii; Tx, toxin.

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