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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.

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(A) The inhibitory effect of L. johnsonii La1 supernatant on G. duodenalis growth is observed after 24 h in the presence of bovine bile. G. duodenalis trophozoites were grown in KM-FCS with bovine bile (0.75 g/L, final concentration) in the presence (■) or in the absence (♦) of bacterial supernatant, or without bovine bile in the presence (□) or in the absence (◊) of bacterial supernatant. The parasite concentration was estimated by counting live cells with a Malassez cell chamber. Values are the mean ± SD of two independent experiments performed in triplicate. Letters indicate significant differences between treatments (Kruskall-Wallis, p < 0.05). (B)G. duodenalis growth inhibition by L. johnsonii La1 supernatant depends on the presence of bile, more specifically of bile salts. G. duodenalis trophozoites in KM-FCS were incubated for 24 h with L. johnsonii La1 supernatant and various concentrations of mixed bile salts (0.016, 0.032, 0.048 g/L, final concentration) or complete bovine bile (0, 0.5, 0.6, 0.75 g/L, final concentration). Growth inhibition values (%) were normalized according to controls in lactic acid-acidified KM-FCS supplemented with similar concentrations of bovine bile or mixed bile salts. Values are the mean ± SD of three independent experiments. Letters indicate significant differences between treatments (Kruskall-Wallis, p < 0.05).
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Figure 1: (A) The inhibitory effect of L. johnsonii La1 supernatant on G. duodenalis growth is observed after 24 h in the presence of bovine bile. G. duodenalis trophozoites were grown in KM-FCS with bovine bile (0.75 g/L, final concentration) in the presence (■) or in the absence (♦) of bacterial supernatant, or without bovine bile in the presence (□) or in the absence (◊) of bacterial supernatant. The parasite concentration was estimated by counting live cells with a Malassez cell chamber. Values are the mean ± SD of two independent experiments performed in triplicate. Letters indicate significant differences between treatments (Kruskall-Wallis, p < 0.05). (B)G. duodenalis growth inhibition by L. johnsonii La1 supernatant depends on the presence of bile, more specifically of bile salts. G. duodenalis trophozoites in KM-FCS were incubated for 24 h with L. johnsonii La1 supernatant and various concentrations of mixed bile salts (0.016, 0.032, 0.048 g/L, final concentration) or complete bovine bile (0, 0.5, 0.6, 0.75 g/L, final concentration). Growth inhibition values (%) were normalized according to controls in lactic acid-acidified KM-FCS supplemented with similar concentrations of bovine bile or mixed bile salts. Values are the mean ± SD of three independent experiments. Letters indicate significant differences between treatments (Kruskall-Wallis, p < 0.05).

Mentions: Most media previously described to support G. duodenalis growth in vitro commonly contains bile as a supply for parasite cholesterol and fatty acid requirements (Farthing et al., 1985; Gillin et al., 1986; Halliday et al., 1995). In our hands, G. duodenalis trophozoites growth was observed in the absence of bovine bile. Bile, at the recommended concentration (0.75 g/L) (Pérez et al., 2001), appeared to reduce trophozoite proliferation when added to KM-FCS (Figure 1A). We next confirmed the in vitro inhibitory effect of L. johnsonii La1 supernatant upon G. duodenalis growth. Interestingly, the inhibitory effect was only observed in the presence of bovine bile. No inhibitory effect was observed in the absence of bovine bile, even after 24 h of culture (Figure 1A). After 10 h in the presence of 0.75 g/L of bovine bile, G. duodenalis trophozoite survival was slightly impacted by L. johnsonii La1 supernatant, but survival was largely affected after 24 h of contact (9.104 and 1.104 trophozoite/ml, respectively, Figure 1A, Video S2, Figure S1). Thus, a 24 h incubation time-period was retained for all subsequent inhibitory growth assays.


Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth
(A) The inhibitory effect of L. johnsonii La1 supernatant on G. duodenalis growth is observed after 24 h in the presence of bovine bile. G. duodenalis trophozoites were grown in KM-FCS with bovine bile (0.75 g/L, final concentration) in the presence (■) or in the absence (♦) of bacterial supernatant, or without bovine bile in the presence (□) or in the absence (◊) of bacterial supernatant. The parasite concentration was estimated by counting live cells with a Malassez cell chamber. Values are the mean ± SD of two independent experiments performed in triplicate. Letters indicate significant differences between treatments (Kruskall-Wallis, p < 0.05). (B)G. duodenalis growth inhibition by L. johnsonii La1 supernatant depends on the presence of bile, more specifically of bile salts. G. duodenalis trophozoites in KM-FCS were incubated for 24 h with L. johnsonii La1 supernatant and various concentrations of mixed bile salts (0.016, 0.032, 0.048 g/L, final concentration) or complete bovine bile (0, 0.5, 0.6, 0.75 g/L, final concentration). Growth inhibition values (%) were normalized according to controls in lactic acid-acidified KM-FCS supplemented with similar concentrations of bovine bile or mixed bile salts. Values are the mean ± SD of three independent experiments. Letters indicate significant differences between treatments (Kruskall-Wallis, p < 0.05).
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Figure 1: (A) The inhibitory effect of L. johnsonii La1 supernatant on G. duodenalis growth is observed after 24 h in the presence of bovine bile. G. duodenalis trophozoites were grown in KM-FCS with bovine bile (0.75 g/L, final concentration) in the presence (■) or in the absence (♦) of bacterial supernatant, or without bovine bile in the presence (□) or in the absence (◊) of bacterial supernatant. The parasite concentration was estimated by counting live cells with a Malassez cell chamber. Values are the mean ± SD of two independent experiments performed in triplicate. Letters indicate significant differences between treatments (Kruskall-Wallis, p < 0.05). (B)G. duodenalis growth inhibition by L. johnsonii La1 supernatant depends on the presence of bile, more specifically of bile salts. G. duodenalis trophozoites in KM-FCS were incubated for 24 h with L. johnsonii La1 supernatant and various concentrations of mixed bile salts (0.016, 0.032, 0.048 g/L, final concentration) or complete bovine bile (0, 0.5, 0.6, 0.75 g/L, final concentration). Growth inhibition values (%) were normalized according to controls in lactic acid-acidified KM-FCS supplemented with similar concentrations of bovine bile or mixed bile salts. Values are the mean ± SD of three independent experiments. Letters indicate significant differences between treatments (Kruskall-Wallis, p < 0.05).
Mentions: Most media previously described to support G. duodenalis growth in vitro commonly contains bile as a supply for parasite cholesterol and fatty acid requirements (Farthing et al., 1985; Gillin et al., 1986; Halliday et al., 1995). In our hands, G. duodenalis trophozoites growth was observed in the absence of bovine bile. Bile, at the recommended concentration (0.75 g/L) (Pérez et al., 2001), appeared to reduce trophozoite proliferation when added to KM-FCS (Figure 1A). We next confirmed the in vitro inhibitory effect of L. johnsonii La1 supernatant upon G. duodenalis growth. Interestingly, the inhibitory effect was only observed in the presence of bovine bile. No inhibitory effect was observed in the absence of bovine bile, even after 24 h of culture (Figure 1A). After 10 h in the presence of 0.75 g/L of bovine bile, G. duodenalis trophozoite survival was slightly impacted by L. johnsonii La1 supernatant, but survival was largely affected after 24 h of contact (9.104 and 1.104 trophozoite/ml, respectively, Figure 1A, Video S2, Figure S1). Thus, a 24 h incubation time-period was retained for all subsequent inhibitory growth assays.

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