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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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Inhibitory activity against G. duodenalis growth and BSH-like activity co-elute in the same fractions after separation of L. johnsonii La1 supernatant by gel filtration chromatography. (A) Chromatography profile. (B) BSH-like activity measured after 24 h of incubation of GDC with the 6 first eluated gel filtration fractions. (C)G. duodenalis growth inhibitory activity after 24 h of incubation with the same gel filtration chromatography fractions as in B. Letters indicate significant differences between treatments (Kruskal-Wallis, p < 0.05).
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Figure 5: Inhibitory activity against G. duodenalis growth and BSH-like activity co-elute in the same fractions after separation of L. johnsonii La1 supernatant by gel filtration chromatography. (A) Chromatography profile. (B) BSH-like activity measured after 24 h of incubation of GDC with the 6 first eluated gel filtration fractions. (C)G. duodenalis growth inhibitory activity after 24 h of incubation with the same gel filtration chromatography fractions as in B. Letters indicate significant differences between treatments (Kruskal-Wallis, p < 0.05).

Mentions: To test the hypothesis of bacterial BSH involvement in the inhibitory activity of L. johnsonii La1 supernatant, the bacterial supernatant was fractionated by gel filtration chromatography on a Sephacryl S300 column, and eluted fractions were assayed for both parasite inhibition and bile salt deconjugating activity. A typical elution profile is shown in Figure 5A. Fractions were collected, tested for G. duodenalis growth inhibition and for BSH-like activity by measuring release of glycine from GDC. In our experimental conditions, 1 unit of the commercial C. perfringens BSH induced the release of 0.76 mM glycine from 2.5 mM GDC within 24 h. Parasite growth inhibitory activity was reproducibly recovered from the first eluted fractions containing proteins with molecular weight between 13.7 and 67 kDa (Figures 5A,C) confirming the estimate of the molecular weight of the inhibitory factor(s) to be >10 kDa (Figure 2C). Those active fractions also exhibited a BSH-like activity (Figure 5B), with the most inhibitory fractions showing significantly higher BSH-like activity (p < 0.05). Our attempts to further purify elements responsible for the inhibitory activity and/or BSH-like activity by combining steps of ion-exchange, hydrophobic interaction and chromatofocusing chromatographies were unsuccessful, with a rapid loss of activities.


Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth
Inhibitory activity against G. duodenalis growth and BSH-like activity co-elute in the same fractions after separation of L. johnsonii La1 supernatant by gel filtration chromatography. (A) Chromatography profile. (B) BSH-like activity measured after 24 h of incubation of GDC with the 6 first eluated gel filtration fractions. (C)G. duodenalis growth inhibitory activity after 24 h of incubation with the same gel filtration chromatography fractions as in B. Letters indicate significant differences between treatments (Kruskal-Wallis, p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037171&req=5

Figure 5: Inhibitory activity against G. duodenalis growth and BSH-like activity co-elute in the same fractions after separation of L. johnsonii La1 supernatant by gel filtration chromatography. (A) Chromatography profile. (B) BSH-like activity measured after 24 h of incubation of GDC with the 6 first eluated gel filtration fractions. (C)G. duodenalis growth inhibitory activity after 24 h of incubation with the same gel filtration chromatography fractions as in B. Letters indicate significant differences between treatments (Kruskal-Wallis, p < 0.05).
Mentions: To test the hypothesis of bacterial BSH involvement in the inhibitory activity of L. johnsonii La1 supernatant, the bacterial supernatant was fractionated by gel filtration chromatography on a Sephacryl S300 column, and eluted fractions were assayed for both parasite inhibition and bile salt deconjugating activity. A typical elution profile is shown in Figure 5A. Fractions were collected, tested for G. duodenalis growth inhibition and for BSH-like activity by measuring release of glycine from GDC. In our experimental conditions, 1 unit of the commercial C. perfringens BSH induced the release of 0.76 mM glycine from 2.5 mM GDC within 24 h. Parasite growth inhibitory activity was reproducibly recovered from the first eluted fractions containing proteins with molecular weight between 13.7 and 67 kDa (Figures 5A,C) confirming the estimate of the molecular weight of the inhibitory factor(s) to be >10 kDa (Figure 2C). Those active fractions also exhibited a BSH-like activity (Figure 5B), with the most inhibitory fractions showing significantly higher BSH-like activity (p < 0.05). Our attempts to further purify elements responsible for the inhibitory activity and/or BSH-like activity by combining steps of ion-exchange, hydrophobic interaction and chromatofocusing chromatographies were unsuccessful, with a rapid loss of activities.

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