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Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

View Article: PubMed Central - PubMed

ABSTRACT

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

No MeSH data available.


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G. duodenalis growth inhibition by L. johnsonii La1 supernatant is affected by supernatant incubation with proteases, heat-treatment and pH. (A) Supernatant of L. johnsonii La1 was incubated for 6 h with 1 mg of immobilized proteases (L. johnsonii La1 supernatant: no protease treatment; L. johnsonii La1 supernatant + PK: treatment with proteinase K; L. johnsonii La1 supernatant + Pronase: treatment with pronase; L. johnsonii La1 supernatant + Catalase: treatment with catalase) or heated at 90°C for 10 min before G. duodenalis growth inhibition assay. Growth inhibition (%) was normalized according to matched control: lactic acid-adjusted MTYI medium incubated with protease-coupled beads or treated for 10 min at 90°C. (B) Supernatant from L. johnsonii La1 in MTYI medium was adjusted to pH 6.2, 6.7, 6.9, or 7.2 before Giardia growth inhibition assays. Growth inhibition (%) was normalized according to control, i.e., lactic acid-adjusted MTYI subsequently raised to pH 6.2, 6.7, 6.9, or 7.2. Values are the mean ± SD of three independent experiments. (C) Molecular weight determination of inhibitory compounds from L. johnsonii La1 supernatant. Supernatant from a culture of L. johnsonii La1 in KM-FCS was filtrated through 10, 30, or 50 kDa MW cut-off membranes. Acidified KM-FCS alone was processed similarly. Fractions above and under respective thresholds were assayed for Giardia growth inhibition in the presence of bile (0.5 g/L). Inhibition values (%) were normalized according to KM-FCS controls. Data are the mean ± SD of three independent experiments performed in triplicate. Letters indicate significant differences between treatments (Kruskal-Wallis, p < 0.05).
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Figure 2: G. duodenalis growth inhibition by L. johnsonii La1 supernatant is affected by supernatant incubation with proteases, heat-treatment and pH. (A) Supernatant of L. johnsonii La1 was incubated for 6 h with 1 mg of immobilized proteases (L. johnsonii La1 supernatant: no protease treatment; L. johnsonii La1 supernatant + PK: treatment with proteinase K; L. johnsonii La1 supernatant + Pronase: treatment with pronase; L. johnsonii La1 supernatant + Catalase: treatment with catalase) or heated at 90°C for 10 min before G. duodenalis growth inhibition assay. Growth inhibition (%) was normalized according to matched control: lactic acid-adjusted MTYI medium incubated with protease-coupled beads or treated for 10 min at 90°C. (B) Supernatant from L. johnsonii La1 in MTYI medium was adjusted to pH 6.2, 6.7, 6.9, or 7.2 before Giardia growth inhibition assays. Growth inhibition (%) was normalized according to control, i.e., lactic acid-adjusted MTYI subsequently raised to pH 6.2, 6.7, 6.9, or 7.2. Values are the mean ± SD of three independent experiments. (C) Molecular weight determination of inhibitory compounds from L. johnsonii La1 supernatant. Supernatant from a culture of L. johnsonii La1 in KM-FCS was filtrated through 10, 30, or 50 kDa MW cut-off membranes. Acidified KM-FCS alone was processed similarly. Fractions above and under respective thresholds were assayed for Giardia growth inhibition in the presence of bile (0.5 g/L). Inhibition values (%) were normalized according to KM-FCS controls. Data are the mean ± SD of three independent experiments performed in triplicate. Letters indicate significant differences between treatments (Kruskal-Wallis, p < 0.05).

Mentions: To biochemically characterize the inhibitory activity present in L. johnsonii La1 supernatant, the supernatant was treated with immobilized enzymes prior to contact with parasites. Trophozoite growth inhibition was totally abolished by proteinase K and pronase treatments, suggesting involvement of inhibitory factor(s) of peptidic nature (Figure 2A). Heat-treatment also led to inactivation of L. johnsonii La1 supernatant inhibitory properties (Figure 2A). Additionally, in a pH range similar to the ones experienced by G. duodenalis in vivo (Biagini et al., 2001), a strong pH influence on the inhibitory activity was noticed, with the highest inhibition occurring at pH 6.2 (Figure 2B).


Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth
G. duodenalis growth inhibition by L. johnsonii La1 supernatant is affected by supernatant incubation with proteases, heat-treatment and pH. (A) Supernatant of L. johnsonii La1 was incubated for 6 h with 1 mg of immobilized proteases (L. johnsonii La1 supernatant: no protease treatment; L. johnsonii La1 supernatant + PK: treatment with proteinase K; L. johnsonii La1 supernatant + Pronase: treatment with pronase; L. johnsonii La1 supernatant + Catalase: treatment with catalase) or heated at 90°C for 10 min before G. duodenalis growth inhibition assay. Growth inhibition (%) was normalized according to matched control: lactic acid-adjusted MTYI medium incubated with protease-coupled beads or treated for 10 min at 90°C. (B) Supernatant from L. johnsonii La1 in MTYI medium was adjusted to pH 6.2, 6.7, 6.9, or 7.2 before Giardia growth inhibition assays. Growth inhibition (%) was normalized according to control, i.e., lactic acid-adjusted MTYI subsequently raised to pH 6.2, 6.7, 6.9, or 7.2. Values are the mean ± SD of three independent experiments. (C) Molecular weight determination of inhibitory compounds from L. johnsonii La1 supernatant. Supernatant from a culture of L. johnsonii La1 in KM-FCS was filtrated through 10, 30, or 50 kDa MW cut-off membranes. Acidified KM-FCS alone was processed similarly. Fractions above and under respective thresholds were assayed for Giardia growth inhibition in the presence of bile (0.5 g/L). Inhibition values (%) were normalized according to KM-FCS controls. Data are the mean ± SD of three independent experiments performed in triplicate. Letters indicate significant differences between treatments (Kruskal-Wallis, p < 0.05).
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Figure 2: G. duodenalis growth inhibition by L. johnsonii La1 supernatant is affected by supernatant incubation with proteases, heat-treatment and pH. (A) Supernatant of L. johnsonii La1 was incubated for 6 h with 1 mg of immobilized proteases (L. johnsonii La1 supernatant: no protease treatment; L. johnsonii La1 supernatant + PK: treatment with proteinase K; L. johnsonii La1 supernatant + Pronase: treatment with pronase; L. johnsonii La1 supernatant + Catalase: treatment with catalase) or heated at 90°C for 10 min before G. duodenalis growth inhibition assay. Growth inhibition (%) was normalized according to matched control: lactic acid-adjusted MTYI medium incubated with protease-coupled beads or treated for 10 min at 90°C. (B) Supernatant from L. johnsonii La1 in MTYI medium was adjusted to pH 6.2, 6.7, 6.9, or 7.2 before Giardia growth inhibition assays. Growth inhibition (%) was normalized according to control, i.e., lactic acid-adjusted MTYI subsequently raised to pH 6.2, 6.7, 6.9, or 7.2. Values are the mean ± SD of three independent experiments. (C) Molecular weight determination of inhibitory compounds from L. johnsonii La1 supernatant. Supernatant from a culture of L. johnsonii La1 in KM-FCS was filtrated through 10, 30, or 50 kDa MW cut-off membranes. Acidified KM-FCS alone was processed similarly. Fractions above and under respective thresholds were assayed for Giardia growth inhibition in the presence of bile (0.5 g/L). Inhibition values (%) were normalized according to KM-FCS controls. Data are the mean ± SD of three independent experiments performed in triplicate. Letters indicate significant differences between treatments (Kruskal-Wallis, p < 0.05).
Mentions: To biochemically characterize the inhibitory activity present in L. johnsonii La1 supernatant, the supernatant was treated with immobilized enzymes prior to contact with parasites. Trophozoite growth inhibition was totally abolished by proteinase K and pronase treatments, suggesting involvement of inhibitory factor(s) of peptidic nature (Figure 2A). Heat-treatment also led to inactivation of L. johnsonii La1 supernatant inhibitory properties (Figure 2A). Additionally, in a pH range similar to the ones experienced by G. duodenalis in vivo (Biagini et al., 2001), a strong pH influence on the inhibitory activity was noticed, with the highest inhibition occurring at pH 6.2 (Figure 2B).

View Article: PubMed Central - PubMed

ABSTRACT

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

No MeSH data available.


Related in: MedlinePlus