# Bile-Salt-Hydrolases from the Probiotic Strain Lactobacillus johnsonii La1 Mediate Anti-giardial Activity in Vitro and in Vivo

Thibault Allain, Soraya Chaouch, Myriam Thomas, Isabelle Vallée, André Buret, Philippe Langella, Philippe Grellier, Bruno Polack, Luis Bermúdez-Humarán, Isabelle Florent

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Thibault Allain, Soraya Chaouch, Myriam Thomas, Isabelle Vallée, André Buret, et al.. Bile-Salt-Hydrolases from the Probiotic Strain Lactobacillus johnsonii La1 Mediate Anti-giardial Activity in Vitro and in Vivo. Frontiers in Microbiology, 2018, 8, pp.1-15. 10.3389/fmicb.2017.02707. hal-02126890

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# Bile-Salt-Hydrolases from the Probiotic Strain *Lactobacillus johnsonii* La1 Mediate Anti-giardial Activity *in Vitro* and *in Vivo*

Thibault Allain¹,², Soraya Chaouch², Myriam Thomas³, Isabelle Vallée³, André G. Buret⁴, Philippe Langella¹, Philippe Grellier², Bruno Polack⁵, Luis G. Bermúdez-Humarán¹*† and Isabelle Florent²*†

¹ Commensal and Probiotics-Host Interactions Laboratory, Micalis Institute, Institut National de la Recherche Agronomique, AgroParisTech, Jouy-en-Josas, France, ² UMR7245, Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, Sorbonne-Universités, Paris, France, ³ JRU BIPAR, ANSES, Ecole Nationale Vétérinaire d'Alfort, INRA, Université Paris-Est, Animal Health Laboratory, Maisons-Alfort, France, ⁴ Department of Biological Sciences, University of Calgary, Calgary, AB, Canada, ⁵ JRU BIPAR, Ecole Nationale Vétérinaire d'Alfort, ANSES, INRA, Université Paris-Est, Maisons-Alfort, France

*Giardia duodenalis* (syn. *G. lamblia*, *G. intestinalis*) is the protozoan parasite responsible for giardiasis, the most common and widely spread intestinal parasitic disease worldwide, affecting both humans and animals. After cysts ingestion (through either contaminated food or water), *Giardia* excysts in the upper intestinal tract to release replicating trophozoites that are responsible for the production of symptoms. In the gut, *Giardia* cohabits with the host's microbiota, and several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against *G. duodenalis* infection through mechanisms that remain incompletely understood. Recent findings suggest that Bile-Salt-Hydrolase (BSH)-like activities from the probiotic strain of *Lactobacillus johnsonii* La1 may contribute to the anti-giardial activity displayed by this strain. Here, we cloned and expressed each of the three *bsh* genes present in the *L. johnsonii* La1 genome to study their enzymatic and biological properties. While BSH47 and BSH56 were expressed as recombinant active enzymes, no significant enzymatic activity was detected with BSH12. *In vitro* assays allowed determining the substrate specificities of both BSH47 and BSH56, which were different. Modeling of these BSHs indicated a strong conservation of their 3-D structures despite low conservation of their primary structures. Both recombinant enzymes were able to mediate anti-giardial biological activity against *Giardia* trophozoites *in vitro*. Moreover, BSH47 exerted significant anti-giardial effects when tested in a murine model of giardiasis. These results shed new light on the mechanism, whereby active BSH derived from the probiotic strain *Lactobacillus johnsonii* La1 may yield anti-giardial effects *in vitro* and *in vivo*. These findings pave the way toward novel approaches for the treatment of this widely spread but neglected infectious disease, both in human and in veterinary medicine.

**Keywords:** *Giardia duodenalis*, *lactobacilli*, *Lactobacillus johnsonii*, *bile salt hydrolases*, *BSH*, conjugated bile salts, anti-giardial activity

## OPEN ACCESS

**Edited by:**
Andrea Gomez-Zavaglia,
Centro de Investigación y Desarrollo en Criotecnología de Alimentos
(CIDCA), Argentina

**Reviewed by:**
Graciela L. Lorca,
University of Florida, United States
Giuseppe Spano,
University of Foggia, Italy

*Correspondence:
Luis G. Bermúdez-Humarán
luis.bermudez@inra.fr
Isabelle Florent
isabelle.florent@mnhn.fr

† These authors have contributed equally to this work.

**Specialty section:**
This article was submitted to *Food Microbiology*, a section of the journal *Frontiers in Microbiology*

**Received:** 27 September 2017
**Accepted:** 29 December 2017
**Published:** 31 January 2018

**Citation:**
Allain T, Chaouch S, Thomas M, Vallée I, Buret AG, Langella P, Grellier P, Polack B, Bermúdez-Humarán LG and Florent I (2018) Bile-Salt-Hydrolases from the Probiotic Strain *Lactobacillus johnsonii* La1 Mediate Anti-giardial Activity in *Vitro* and *in Vivo*. *Front. Microbiol.* 8:2707. doi: 10.3389/fmicb.2017.02707
# INTRODUCTION

*Giardia duodenalis* (syn. *Giardia lamblia* and *Giardia intestinalis*) is a flagellated protozoan parasite responsible for giardiasis, an intestinal zoonotic disease infection that may cause acute or chronic diarrhea, weight loss, malabsorption, abdominal pain, and nausea (Ankarklev et al., 2010; Cotton et al., 2011). It is one of the most common intestinal parasites and one of the most frequent causes of diarrhea, with over 280 million human symptomatic cases worldwide (Lane and Lloyd, 2002; Platts-Mills et al., 2015). Infections occur mainly by the ingestion of cysts present in contaminated food and water. After ingestion, infectious cysts differentiate into trophozoite stages, which in turn colonize the upper small intestine. Included in the “Neglected Disease Initiative” of the World Health Organization (WHO) in 2004, giardiasis has a significant public health impact in both developed and developing countries (Savioli et al., 2006; Platts-Mills et al., 2015). Metronidazole is the most frequently used drug for treating *G. duodenalis* infections, whereas albendazole, tinidazole, and nitazoxanide may also be used with efficacy (Gardner and Hill, 2001; Petri, 2005). Although these drugs have different modes of action, there is an increasing incidence of parasite resistance, and treatment failure is relatively common (Ansell et al., 2015). Moreover, these standard treatments are commonly associated with undesirable side effects in both medical and veterinary usages (Barr et al., 1994; Gardner and Hill, 2001). A successful vaccine has proven elusive, and *Giardia* is able to escape host immunity by switching its variant-specific surface proteins (Singer et al., 2001). Together, these observations underscore the need for new therapeutic alternatives for the treatment of giardiasis.

In the last decade, some probiotics (i.e., live microorganisms which, when administered in adequate amounts, confer a health benefit on their hosts, WHO 2001), in particular several species belonging to the genus *Lactobacillus*, have shown anti-giardial efficacy in various murine models (see Travers et al., 2011 for review). The mechanisms remain incompletely understood but may involve host immunomodulation and/or extracellular compounds released by the bacteria (Perez et al., 2001; Humen et al., 2005; Shukla et al., 2008; Shukla and Sidhu, 2011; Goyal et al., 2013). In this context, we have recently shown that unconjugated bile salts, generated by secreted or released enzymes by the probiotic strain of *Lactobacillus johnsonii* La1 (also known as *L. johnsonii* NCC533), may contribute to the inhibition of *Giardia* trophozoite growth *in vitro* (Perez et al., 2001; Travers et al., 2016). BSH (also called cholylglycine hydrolase, EC 3.5.1.2) are enzymes that hydrolyze the amide bond of conjugated bile salts, liberating the amino acid moiety from the steroid core and generating deconjugated bile salts (i.e., cholic acid, deoxycholic acid and chenodeoxycholic acid) (Begley et al., 2006). Conjugated-bile salts are synthesized in the liver where conjugation to either glycine or taurine occurs, and these conjugated-bile salts play an important role in the solubility and absorption of lipids and cholesterol in the intestinal tract (Eyssen, 1973; Kim et al., 2005; Begley et al., 2006). Moreover, glyco- and tauro-conjugated bile salts exert detergent and antimicrobial properties (Ruiz et al., 2013). BSH activities lead

to bile salt detoxification and confer a competitive advantage to the microbial communities that express them, such as lactobacilli in the upper part of the small intestine (Ridlon et al., 2006; Ruiz et al., 2013).

In this study, we cloned and expressed each one of the three-*bsh* genes (i.e., *bsh12*, *bsh47*, and *bsh56*) from *L. johnsonii* La1 (Pridmore et al., 2004) in *Escherichia coli* in order to evaluate their substrate specificities and to assess their anti-*Giardia* activities, both *in vitro* and *in vivo*. A comparative structural analysis of the three BSHs was also performed using *in silico* approaches to explore whether structural differences could explain possible differences in substrate specificities. The three recombinant BSHs, rBSH12, rBSH47, and rBSH56, were tested against two different strains of the human assemblage A of *G. duodenalis* (WB6 and NF) *in vitro*. Then, rBSH47 was selected to be tested *in vivo* on OF1 suckling mice infected with the *G. duodenalis* strain WB6.

# MATERIALS AND METHODS

## In Silico Analysis of BSHs

Bile salt hydrolases amino acid sequences from different bacterial species were retrieved from databases using BLASTP program from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and analyzed in silico. Multiple sequence alignments of BSH amino acid sequences were performed using CLUSTALO 1.2.1 (http://www.ebi.ac.uk/Tools/msa/clustalo/) to identify the conserved motifs between the different enzymes. Phylogenetic relationships and phylogenetic clustering of BSHs from different species were established by neighbor-joining methods using MEGA5 software (http://www.megasoftware.net/; Tamura et al., 2011). Three-dimensional modeling of *L. johnsonii* La1-BSHs was performed using I-TASSER from University of Michigan (http://zhanglab.ccmb.med.umich.edu/I-TASSER/; Roy et al., 2010). According to C-score results, the BSH from *Bifidobacterium longum* (Kumar et al., 2006) was chosen as template for modeling BSH56, whereas the BSH from *Clostridium perfringens* was used as template for modeling both BSH12 and BSH47 (Rossocha et al., 2005). Models for structure predictions were selected according to the highest values of their C-score (measured for evaluating global and local similarity between query and template protein). Protein structure analysis was performed using Pymol (PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).

## Bacteria and Growth Conditions

*Lactobacillus johnsonii* La1 strain (Pridmore et al., 2004) was cultured in Man Rogosa Sharpe broth (MRS, Difco) and grown at 37°C in an anaerobic jar using BBL GasPak Anaerobic System (BD), incubated overnight (ON). *E. coli* TOP10 chemically competent cells (Invitrogen) were used for the subcloning of PCR fragments. *E. coli* CYS21 and SE1 chemically competent strains (DelphiGenetic, Belgium) were used, respectively, for the cloning and expression of BSHs. *E. coli* strains were grown in Luria-Bertani (LB) medium at 37°C ON with vigorous shaking at 180 rpm. All bacterial strains were stored at -80°C with 15% (v/v) glycerol for cryoprotection.
## Cloning of bsh Genes from L. johnsonii La1

Genomic DNA of L. johnsonii La1 was extracted from 2 mL of an ON culture using Wizard Genomic DNA Purification Kit Protocol (Promega) and used as template to amplify the 3 bsh genes: bsh12 (Gene ID: 2743525), bsh47 (Gene ID: 2743183), and bsh56 (Gene ID: 2743142) (Pridmore et al., 2004). The coding sequences of bsh12, bsh47, and bsh56 genes (excluding the putative signal sequences) were amplified by PCR (Phusion Taq, Thermo Fisher Scientific) using primers described in Table 1. These primers were designed to incorporate two restriction sites: NheI (forward primer) and XhoI (reverse primer). The amplified PCR fragments were purified using SV Gel and PCR Clean-Up System (Wizard) and were subcloned into the vector pCR®2.1-TOPO® (Invitrogen). The resulting constructions (pLB487, pLB488, and pLB489) were validated by sequencing (MWG-Genomic Company, Germany) before recovering the bsh genes with NheI and XhoI restriction enzymes and cloning them into pStaby 1.2 vector (DelphiGenetics) previously digested with the same enzymes. The pStaby 1.2 plasmid was used for intermediate cloning to introduce a C-terminal six-Histidine tag (His-tag), allowing subsequent purification of rBSHs using affinity chromatography. The resulting plasmids were transferred into E. coli CYS21 strains and transformants were grown at 37°C ON in 10 mL of LB containing ampicillin (Amp, 100 µg/ml) with shaking at 180 rpm. Plasmid DNA were extracted from positive clones, sequenced to confirm identity, and subsequently transformed into E. coli SE1 expression strain. Bacterial strains, plasmids, and primer sequences used in this study are described in Table 1. Immunoblotting experiments were performed on E. coli SE1 (pLB490), E. coli SE1 (pLB491), and E. coli SE1 (pLB492) strains lysates (see below) using mouse monoclonal 6x-His Epitope Tag Antibody (Thermo Fisher Scientific) to detect recombinant BSHs.

## Expression and Purification of Recombinant BSH12, BSH47, and BSH56 in E. coli

E. coli SE1 strains harboring pLB490 (bsh12), pLB491 (bsh47), and pLB492 (bsh56) were grown at 37°C ON in 10 mL of LB supplemented with ampicillin (100 µg/mL) with vigorous shaking at 180 rpm and subsequently grown in 1.5 L of LB/ampicillin (100 µg/mL) at 37°C. When an optical density (OD600nm) = 0.6-0.8 was reached, gene expression was induced by the addition of 1 mM of Isopropyl β-D-1-Thiogalactopyranoside (IPTG), and cultures were incubated at 21°C ON with shaking at 180 rpm. Bacteria were harvested by centrifugation and cell pellets were washed with PBS and resuspended in 15 mL of Tris-KCl buffer (Tris 50 mM, KCl 100 mM, MgCl2 10 mM, pH 7.5) supplemented with Triton-X-100 (Sigma-Aldrich) to a final concentration of 1% and protease inhibitors 1X (Roche). Cells were subsequently sonicated for 4 min with alternated pulses on ice (on: 5 s, off: 30 s). The lysed cells were then placed in ultracentrifuge tubes and spun at 220,000 × g at 4°C for 45 min to separate soluble supernatants from pellets.

The soluble fractions containing the recombinant BSH (rBSH) were then collected and rBSHs were purified using affinity

<table><caption>TABLE 1 | Bacterial strains, plasmids, primers used in this study.</caption><thead><tr><th>Material</th><th>Relevant properties</th><th>Source/reference</th></tr></thead><tbody><tr><td colspan="3"><strong>STRAINS</strong></td></tr><tr><td colspan="3"><strong>L. johnsonii</strong></td></tr><tr><td>La1<br>(NCC533)</td><td>Wild type strain</td><td>Nestlé collection<br>center NCC533</td></tr><tr><td colspan="3"><strong>E. coli</strong></td></tr><tr><td>TOP10</td><td>Cloning strain</td><td>Invitrogen</td></tr><tr><td>CYS21</td><td>Cloning strain</td><td>DelphiGenetics</td></tr><tr><td>SE1</td><td>Expression strain</td><td>DelphiGenetics</td></tr><tr><td colspan="3"><strong>PLASMIDS</strong></td></tr><tr><td>pCR2.1-TOPO</td><td>*Amp<sup>r</sup>, subcloning vector</td><td>Invitrogen</td></tr><tr><td>pLB487</td><td>*Amp<sup>r</sup>, TOPO harboring <i>bsh12</i> gene</td><td>This study</td></tr><tr><td>pLB488</td><td>*Amp<sup>r</sup>, TOPO harboring <i>bsh47</i> gene</td><td>This study</td></tr><tr><td>pLB489</td><td>*Amp<sup>r</sup>, TOPO harboring <i>bsh56</i> gene</td><td>This study</td></tr><tr><td>pStaby express</td><td>Amp<sup>r</sup>, subcloning vector</td><td>DelphiGenetics</td></tr><tr><td>pLB490</td><td>*Amp<sup>r</sup>, pStaby harboring <i>bsh12</i> gene</td><td>This study</td></tr><tr><td>pLB491</td><td>*Amp<sup>r</sup>, pStaby harboring <i>bsh47</i> gene</td><td>This study</td></tr><tr><td>pLB492</td><td>*Amp<sup>r</sup>, pStaby harboring <i>bsh56</i> gene</td><td>This study</td></tr><tr><td colspan="3"><strong>Primers</strong></td></tr><tr><td>bsh12Fw</td><td>5'CCGCTAGCTGTACCTCAATTGTTTATAGTTC 3'</td><td>This study</td></tr><tr><td>bsh12Rev</td><td>3'GGCTCGAGATTTTGATAATTAATTGTTTGC 5'</td><td>This study</td></tr><tr><td>bsh47Fw</td><td>5'CCGCTAGCTGTACTGGTTTAAGATTCAC 3'</td><td>This study</td></tr><tr><td>bsh47Rev</td><td>3'GGCTCGAGGTAAAGTCACAAGACTGGTTG 5'</td><td>This study</td></tr><tr><td>bsh56Fw</td><td>5'CCGCTAGCTGTACATCAATTTTATATAGTCC 3'</td><td>This study</td></tr><tr><td>bsh56Rev</td><td>3'GGCTCGAGATTTTCAAATTTTAATGGCTTG 5'</td><td>This study</td></tr></tbody></table>

chromatography. Briefly, columns kept in nickel-nitrilotriacetic acid (Ni-NTA; Qiagen) agarose were first washed with milliQ water and equilibrated with 50 mM Tris-KCl buffer pH 7.5 according to the supplier's protocol. Soluble lysates were passed through Ni-NTA columns and washed with 50 mM Tris-KCl buffer pH 7.5 to remove unbound proteins. Finally, rBSHs were eluted by increasing imidazole concentrations (25, 75, and 500 mM) as recommended by the supplier. The eluted proteins were desalted using Sephadex G-25 columns (Amersham Biosciences). All fractions were analyzed on Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue.

## Bile Salt Hydrolase Activity Assays

The substrate specificity of each rBSHs was assessed on plates using an agar test. E. coli strains harboring pLB490, pLB491, and pLB492 were cultured in LB broth in presence of ampicillin (100 µg/ml). Overnight cultures were then spotted on LB agar plates supplemented with either 0.5% taurodeoxycholic acid (TDCA, Sigma-Aldrich) or 0.5% glycodeoxycholic acid (GDCA, Merck Millipore) and incubated at 37°C for 48 h.

The BSH hydrolyzing activities were also monitored using purified recombinant enzymes in presence of conjugated bile salts, in solution, by measuring the liberation of amino acids
(glycine or taurine) as previously described (Grill et al., 2000). A volume of 100 µl of rBSH (20 µg) was mixed with 100 µl of 2.4 g/L of each conjugated bile salts (GDCA, TDCA, glycocholic acid, or taurocholic acid) and incubated for 30 min at 37°C. BSH from C. *perfringens* (Sigma-Aldrich, reference C4018) was used as a positive control. A solution without bile salts was used as a negative control. The hydrolysis of bile salts was stopped by adding 200 µl of 15% trichloroacetic acid (TCA) (v/v%) and the mixture was spun at 10,000 g for 15 min to remove precipitated proteins. The supernatant (80 µl) was subsequently collected and added to 680 µl of 0.3 M borate buffer, 1% SDS (pH 9.5), and 80 µl of 0.3% picrylsulfonic acid solution (Sigma-Aldrich). Mixtures were incubated for 30 min in the dark at room temperature and 800 µL of 0.6 mM HCl was added to stop the colorimetric reaction. The amount of glycine or taurine released was measured at 416 nm using a spectrophotometer and standard curves were established with free glycine and taurine.

## Giardia duodenalis Cultures

Two different isolates of assemblage A were used in this study: G. duodenalis strains WB6 clone 6 (WB6, ATCC50803), isolated from a patient with chronic giardiasis, and G. duodenalis NF (kindly provided by Dr. André Buret, University of Calgary), obtained from an outbreak of human giardiasis. Trophozoites were cultured in axenic conditions grown in Keiser's modified TYI-S-33 medium (KM) adjusted at pH 6.0 and supplemented with heat-inactivated fetal calf serum (10%) (FCS, reference A15-101, PAA laboratories, GE Healthcare) as recently described (Travers et al., 2016). *In vitro* experiments were performed with or without bovine bile (Difco, DB Diagnostic System, reference 212820) supplementation (0.6 g/L).

## Anti-giardial Activity Assays

Increasing concentrations of rBSHs were co-incubated with fresh cultures of G. duodenalis WB6 trophozoites ($2 \times 10^5$ parasites/ml) in KM medium supplemented with 10% FCS in a final volume of 480 µl, at 37°C in anaerobic conditions for 22 h. Experiments were performed with or without bovine bile (0.6 g/L) supplementation. BSH from C. *perfringens* (1U, Sigma-Aldrich, reference C4018) was used as a positive control. Trophozoites were detached from tubes by chilling on ice for 10 min and the parasite load was measured by using hemocytometer (flagella mobility was used as viability criteria). The inhibition levels were determined in comparison with values of non-treated trophozoite cultures (percentage of growth). Three biological replicates were performed, each in duplicates. The half maximal inhibitory concentrations (IC₅₀) were calculated using Prism 5 software (GraphPad).

## G. duodenalis Viability Assays on Cell Cultures

Caco-2 epithelial cells (human colonic adenocarcinoma, ATTC HTB-37) were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 200 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, and 10% fetal bovine serum (FBS) (Gibco, reference 12484-028) at 37°C and 5% CO₂. Caco-2 cells

were cultured (passage 28-32) at 80% confluence with trypsin-EDTA and seeded at $10^5$ cells/mL onto 12-wells plates (Caco-2 growth medium). Cells were cultured until the monolayer was confluent (3-4 days) with medium changes every 48 h. 3 days prior to co-incubation, cultures of G. duodenalis NF strain trophozoites were axenically cultured in KM medium supplemented with 10% heat-inactivated FBS at 37°C to confluence. Parasites were ice-chilled for 15 min, harvested by centrifugation for 10 min at 1,300 × g (4°C), and resuspended in Caco-2 growth medium supplemented with bovine bile (0.6 g/L). For co-culture experiments, trophozoites were seeded at a multiplicity of infection (MOI) of 10:1. Recombinant BSHs were then added to co-cultures at different concentrations and the plates were incubated at 37°C and 5% CO₂. After 20 h of incubation, trophozoites were collected after chilling of plates on ice and the parasite load was determined using hemocytometer (flagella mobility was used as viability criteria).

## Scanning Electron Microscopy

For scanning electron microscopy (SEM), fresh cultures of G. duodenalis trophozoites WB6 strain were treated with either rBSH47 (0.5 µg/ml), rBSH56 (0.08 µg/ml), or DCA (0.1 g/L and 0.2 g/L) in KM supplemented with 10% heat-inactivated FCS (10%), with or without bovine bile (0.6 g/L) supplementation. Cultures of treated and untreated Giardia trophozoites were subsequently seeded in 12 wells plates on poly-lysine glass coverslips placed at the bottom, and parasites were let to settle on the glass coverslips. After 16 h incubation, the supernatants were removed gently and cells were fixed on the glass coverslips with cacodylate 0.1 M and glutaraldehyde 2.5% (pH 7.2) overnight at 4°C. After two washing steps with 0.1 M cacodylate (pH 7.2), cells were dehydrated in a graded ethanol series (50, 70, 90, and 100%) and critical point-dried in liquid CO₂ (Emitech K850, Quorum Technologies). Coverslips were then mounted onto holders and coated with 20 nm of gold (JEOL Fine Coater JFC-1200). The samples were then examined with a Hitachi Scanning Electron SU3500 Premium.

## Experimental Infection Model

OF1 mice were obtained from Charles River (Saint-Germain-Nuelles, France). Mice were housed in pathogen-free conditions and all experiments were performed under a laminar flow hood. Neonatal (suckling) mice were challenged with $10^5$ G. duodenalis WB6 trophozoites at day 10 by intragastric gavage (100 µl). Recombinant BSH47 was diluted in DMEM with NaHCO₃ 16.4% (vehicle) and daily administered by intragastric gavage to neonatal mice from days 10 to 15. Control animals received vehicle instead of rBSH47. Animals were sacrificed by cervical dislocation at day 16 (peak of infection, as determined in parallel assays) and assayed for the presence of G. duodenalis trophozoites in the small intestine. Small intestines were resuspended in 5 ml of cold PBS, incubated on ice for 10 min, and mixed thoroughly. The parasite load was estimated using hemocytometer chambers. Mice with no detectable trophozoites (threshold: <$10^3$ parasites/5 ml intestine suspension) were considered as parasite-free. All protocols were carried out in accordance with the institutional ethical guidelines
of the ethics committee ANSES's Animal Health Laboratory at Maisons-Alfort on the campus of the French National Veterinary School of Alfort (ENVA), which approved this study.

## Statistical Analysis

Data analysis was performed with Prism 5 software (GraphPad). One-way ANOVA, Mann-Whitney, and t-test were used to evaluate difference between means. Results were presented as means ± standard error of the mean (SEM). Statistical significance was calculated at a P value of 0.05 and 95% confidence interval.

## RESULTS

### In Silico Analysis of L. johnsonii La1-BSHs Protein Sequences

The amino acid sequences of L. johnsonii-BSH12, BSH47, and BSH56 enzymes were blasted against reported sequences from several Gram-positive bacteria using Blastp. For the three L. johnsonii La1 BSH, results indicated high identity levels with BSH of different Lactobacillus species ranging from 54 to 100% but lower levels of identity (less than 54%) with BSH from Bifidobacterium and Clostridium species. In particular, the L. johnsonii-BSH12 shared 54, 57, 60, 79-84, and 60-100% identities with BSHs from C. perfringens, L. acidophilus, L. reuteri, L. gasseri, and L. johnsonii, respectively. L. johnsonii-BSH47 shared 54-55, 56-58, 60-66, 57, 70, and 97-100% identities with BSHs from L. crispatus, L. reuteri, L. gasseri, L. acidophilus, L. amylovorus, and L. johnsonii, respectively. Finally, L. johnsonii-BSH56 showed 94 and 99% identity with BSHs from L. gasseri and both L. acidophilus and L. johnsonii, respectively.

The 3D structures of CBAH-1 from C. perfringens (Rossocha et al., 2005) and BIBSH from B. longum (Kumar et al., 2006) have been determined (PDB: 2BJF and PDB: 2RF8, respectively), revealing the presence of key residues in the enzymatic active site (Cys-2, Arg-18, Asp-21, Asn-82, Asn-172, and Arg-225; numbering referring to CBAH-1). In addition, experimental studies validated the importance of Arg-18 in the catalytic site (Fang et al., 2009; Lin, 2014; Lin et al., 2014). Therefore, multiple amino acid sequence alignments of BSH12, BSH47, and BSH56 with CBAH-1 and BIBSH were performed using ClustalO program (GONNET PAM 250 matrix), thereby indicating that these key residues were indeed highly conserved in all three L. johnsonii La1-BSHs (Figure 1). Moreover, motifs surrounding these key amino acid positions were also found to be well conserved such as ¹⁶FGRNXD, ⁷²NEXGLXXAGLNF, ¹⁷⁰VXXLTNXPFX, and ²¹³GXGXGXXGXPGD, a point that has been also reported in other studies (Elkins et al., 2001; Kim and Lee, 2008). However, residues, which are predicted to be involved in the substrate-binding site based on the 3D structure of C. perfringens, did not appear to be conserved in either L. johnsonii BSH enzyme (Ridlon et al., 2006).

Predicted tridimensional structures of L. johnsonii BSH12, BSH47, and BSH56 were modeled with I-TASSER software, using existing 3D structures (Figure S1, BSH47 and BSH56 and Figure S2, BSH12). Clostridium perfringens CBAH-1 was

used as a template for BSH47 modeling (RMSD: 0.64; TM-score: 0.991; Identity, 35.4%) and BSH12 (RMSD: 1.5; TM-score: 0.967; Identity: 37.3%). Bifidobacterium longum BIBSH was used as a template for BSH56 modeling (RMSD: 1.16; TM-score: 0.966; Identity: 43.8%). A superimposition of BSH47 and BSH56 models revealed very similar structures. In particular, the distinctive αββα-folding pattern is conserved in both proteins (Patel et al., 2010; Lin et al., 2014). In addition, the residues involved in the catalytic site are superimposed which confirms a conservation of 3D structure despite a high variability of amino acid sequence among BSHs. Similar results were obtained with BSH12 (Figure S2).

### Heterologous Expression and Purification of BSHs in E. coli

To study the biochemical and enzymatic characteristics of L. johnsonii La1-BSHs, bsh genes (i.e., bsh12, bsh47, and bsh56) were cloned in E. coli CYS21 strain and expressed in E. coli SE1 strain. Western blot analysis from E. coli SE1 cells expressing His-tagged BSHs showed an efficient production of BSH12, BSH47, and BSH56, respectively (Figure 2A). High yields of heterologous proteins were produced from 1.5 L of recombinant E. coli cultures upon 1 mM IPTG induction. No cytotoxic effects were observed during bacterial growth. C-terminal His-tagged rBSHs were subsequently purified using Ni-NTA agarose affinity chromatography and desalted. The purity of BSHs was assessed by Coomassie-blue staining of SDS-PAGE (Figure 2B). The molecular weights observed on SDS-PAGE corresponded with those expected (based on theoretical predictions) for rBSH 47 (37.1 kDa) and rBSH56 (35.8 kDa). However, the molecular weight for rBSH12 appeared slightly higher than theoretically expected (37.4 kDa). Nanodrop quantifications of desalted proteins showed that 35 mg of rBSH47 and 28 mg of rBSH56 were successfully purified from 1.5 L of culture. However, only 3 mg of rBSH12 could be recovered.

### L. johnsonii La1 BSH Activities and Substrate Specificities

The substrate specificities of the L. johnsonii BSHs were assayed by two approaches. Enzymatic activities were monitored in solution, using recombinant enzymes, by measuring amino acids released from the hydrolysis of conjugated bile salts as described in materials and methods. These enzymatic activity assays revealed a slight activity toward glycocholic acid, but a much higher level of activity toward taurocholic acid, for both rBSH47 and rBSH56 (Table 2). No significant enzymatic activity was detected with the purified rBSH12 with any substrate (Table 2). In parallel, the substrate specificities of the three L. johnsonii BSHs (produced in E. coli) were determined using LB agar supplemented with either taurodeoxycholic (0.3%) or glycodeoxycholic (0.3%) acids. A white and iridescent precipitate around colonies is indicative of BSH hydrolytic activity. E. coli SE1 strain expressing rBSH47 efficiently hydrolyzed tauro-conjugated bile salts, whereas no BSH activity was detected for glyco-conjugated bile salts (Figure 3A). E. coli SE1 strain expressing rBSH56 efficiently hydrolyzed both tauro- and glycol-conjugated bile salts (data not shown). A slight deconjugation
BSH12 MCTSIVYSSN-NHYFGRNLDDLEISFGEHPVITPRNYEFQYRKLPS-KKAKYAMVGMAIV 58
BSH47 MCTSILYSPK-DNYFGRNLDDYEIAVQKQVVITPRNYQLNYRHLPT-QDTHYAMIGVSVV 57
CBAH-1 MCTGLALETKDGLHLFGRNMDIEYSFNQSIIFIPRNFKCVNNSKKELTTKYAVLGMGTI 60
BSH56 MCTGLRFTDDQGNLYFGRNLDDVQDVGEGVIIIPRNYPLPYKFLDN-TTTKKAVIGMGIV 59
B1BSH MCTGVRFSDDEGNTYFGRNLDDWSFSYGETILVTPRGYHYDTVFGAGGKAPNAVIGVGVV 60
**: . . ****: : : . **: *: *: .

BSH12 ENNYPLYFDAANEEGLGIAGLNFDPGCHYPFPEN-AEKNNVTPFELIPYLPSQCTTVAEVK 117
BSH47 ANDYPLYCDAINEKGLGIAGLNFNTGPGKYFSVD-ESKKNVTSFELIPYLPSNCETIEDVK 116
CBAH-1 FDDYPTFADGMNEKGLGCAGLNFVPVYVSYSKEDIEGKTNIPVYNFLLWVLANFSSVEEVK 120
BSH56 VDGYPSYFDCYNEDGLGIAGLNFPHFAKFSDDGPIDGKINLASYEIMLWVTQNFTHVSEVK 119
B1BSH MADRPMYFDCANEHGLAIGLNFPGYASFVHEPVEGTENVATFEFPLWVARNFDSVDEVE 120
* : * **.**. ****: : . *: ::: :: : : : *:

BSH12 DALKDVSLVNINESEKLPLSPWHLMADKTGESIVVESTLSGLHVYDNPVHVLTNNPEFP 177
BSH47 KLLSETNITDEFSKDLPVTTLHWLMGDKSGKSIVIESTETGLHVYDNPVNTLNTNPVFP 176
CBAH-1 EALKNANIVDIPISENIPNTTLEHWMISDITGKSIVVEQTKEGLNVFDDNIGVLTNSPTEFD 180
BSH56 VDGYPSYFDCYNEDGLGIAGLNFPHFAKFSDDGPIDGKINLASYEIMLWVTQNFTHVSEVK 119
B1BSH ETLRNVTLVSQIVP-GQQESLHWFIGDGKRSIVVEQ-MADGMHVHHDDVDVLTNQPTFD 178
. * : . . . . . : ****: * : * : . * . . : **** *

BSH12 GQLRNLANYSNIAPAQPKNTLVPGVDLNLYSRGLGTHFLPGGMDSASRFVKIAFRAHSP 237
BSH47 AQVETLANFASVSPAQPKNTLVPNADINLYSRGLGTHHLPGGTDSNSRFIKASFVLAHSP 236
CBAH-1 WHVANLNQYVGLRYNQVPEFKLGDQSLTALGQGTGLVGLPGDFTPASRFIRVAFLRDAMI 240
BSH56 WHTLNGLNYTGLNPHDATAQSWNGQKVAPWGVGTGSLGLPGDISIPADRFVKAAAYLNVNYP 239
B1BSH FHMENLRNYMCVSNEMAEPTSWGKASLTAWGAGVGMHGIPEGDVSSPSRFVRVAYTNAHYP 238
* : . * : : : . : . * * : ** : **: : :

BSH12 QGNNELSSVTNYFHILHSVEQPKGTDEVGPNSEYETIYSDGTNLETGTFYTYNYENNQIN 297
BSH47 KGNDEVENVTNFFHVILHSVEQAKGTDEVEDNVFEFTMYSDCMNLDKGILYFTTYDNNQIN 296
CBAH-1 KNDKDSIDLIEFFHILNNVAMVRGSTRTVEEKSDLQTQYSCMCLEKGIYYNYTENNQIN 300
BSH56 TAKGEKANVAKFFNILKSVAMIKGSVVNDQGKDEYTVTACYSSGSKTYYCNFEDDFELK 299
B1BSH QQNDEAAVNSRLFHTLGSVQMVDVGMAKMGDGDQFERTLFTSGYSSKTNNTYMMNTYDDPAIR 298
. . : . . . . * : . * : : : . . . . : .

BSH12 AIELNKENLNGDELTDYKLIEKQTINYQN 326
BSH47 AVDMNNEDLGTSSDLITYELFKDQAIKFEN 325
CBAH-1 AIDMNKENLDGNEIKTYKYNKTLSINHVN 329
BSH56 TYKLDDHTMNSTSLVTY---------- 316
B1BSH SYAMADYDMDSSELISVAR---------- 317
: . : . : . :

**FIGURE 1** | *L. johnsonii* La1 BSH possess conserved key amino acids in their predicted active sites. Multiple sequence alignments of BSH were performed with the ClustalO program (http://www.ebi.ac.uk/Tools/msa/clustalo/) using GONNET 250 matrix. An "*" (asterisk) indicates positions of fully conserved residues in all five sequences; a ":" (colon) indicates conservation of amino acid with strongly similar chemical properties (Gonnet PAM 250 matrix score > 0.5); a "." indicates conservation of amino acid with similar chemical properties (Gonnet PAM 250 matrix score ≤ 0.5). Residues highlighted in light gray correspond to the predicted key active site amino acids, based on the 3D structures of BSHs from both *C. perfringens* (CBAH-1, PDB: 2BJF) (Rossocha et al., 2005) and *B. longum* (B1BSH, PDB: 2RF8) (Kumar et al., 2006). Residues highlighted in dark gray indicate amino acids putatively involved in substrate binding based on CBAH-1 3D structure (Rossocha et al., 2005; Ridlon et al., 2006). Boxes indicate conserved signatures, i.e., <sup>16</sup>FGRNXD, <sup>72</sup>NEXGLXXAGLNF, <sup>170</sup>VXXLTNXPXF, and <sup>213</sup>GXGXGXXGXPGD (CBAH-1 numbering).

was observed with *E. coli* strain producing rBSH12 against glyco-conjugated bile salts. However, BSH12 was not further tested in this study.

Phylogenetic relationship among selected BSH sequences and related substrate predictions were represented on a neighbor-joining tree, constructed using amino acid sequences of BSHs whose substrate specificities have been previously characterized, with 500 bootstrap replications using MEGA5 software (http://www.megasoftware.net/). Such a phylogenetic analysis showed that *L. johnsonii* La1-BSH12 is more closely related to *L. johnsonii* 100-100-BSH-α (Figure 3B), an enzyme which is able to hydrolyze both tauro- and glyco-conjugated bile salts. The *L. johnsonii* La1-BSH56 is phylogenetically related to a compact cluster including *L. johnsonii* PF01-BSHB (LjBSHB),

*L. acidophilus* PF01-BSH (LaBSH), and *L. johnsonii* 100-100-BSH-β (LjBSH-β). Both BSH56 and LjBSH-β display broad substrate specificity, with a slight preference for tauro-conjugated over glyco-conjugated bile salts, whereas LaBSH and LjBSHB exclusively hydrolyze tauro-conjugated bile salts (Chae et al., 2013). Finally, *L. johnsonii* La1-BSH47 was more closely related to *L. johnsonii* PF01-BSHC (LjBSHC) hydrolyzing only glyco-conjugated bile acids, whereas BSH47 displays a preference for tauro-conjugated substrates. These observations suggest that substrate specificities are not systematically conserved among lactobacilli BSHs, despite a good conservation of their 3D-structures and of key amino acids in their active sites, which makes substrate specificity prediction based on phylogenetic analysis not straightforward for the moment.
<figure><img src="image_4.png" /></figure>

**FIGURE 2** | Expression and purification of L. johnsonii La1 BSHs. (A) Detection of heterologous production of BSH in E. coli by Western Blotting using anti-His tag antibody. Production of BSH was done under the control of pStaby system in E. coli (induced with 1 mM IPTG). Cytoplasmic fractions were extracted from E. coli SE1 harboring pLB490 plasmid (E. coli BSH12), pLB491 plasmid (E. coli BSH47) or pLB492 plasmid (E. coli BSH56). (B) SDS-PAGE analysis of purified BSHs. Purification steps of rBSH12, rBSH47, and rBSH56 with Ni-NTA affinity chromatography. Lanes 1–3 correspond to eluted fractions of rBSH12 with imidazole gradient (25, 75, and 500 mM). Lane 4 is rBSH12 after desalting. Lanes 5–7 correspond to eluted fractions of rBSH47 (imidazole gradient: 25, 75, and 500 mM); lane 8 is rBSH47 after desalting step. Lanes 9–11 correspond to eluted fractions of rBSH56 (imidazole gradient: 25, 75, and 500 mM); lane 12 is rBSH56 after desalting step.

<table><caption>**TABLE 2** | Activities of L. johnsonii BSH against tauro- and glyco-conjugated bile salts.</caption><thead><tr><th rowspan="2">Enzyme</th><th rowspan="2">TDCA<br>hydrolase<br>activity<sup>a</sup></th><th colspan="2">Relative<br>activity<sup>b</sup></th><th rowspan="2">GDCA<br>hydrolase<br>activity<sup>c</sup></th><th colspan="2">Relative<br>activity<sup>d</sup></th></tr><tr><th>U/g*</th><th>%</th><th>U/g*</th><th>%</th></tr></thead><tbody><tr><td>BSH12</td><td>–</td><td>–</td><td>Nd</td><td>+</td><td>–</td><td>Nd</td></tr><tr><td>BSH47</td><td>++</td><td>720</td><td>100</td><td>+</td><td>65</td><td>9</td></tr><tr><td>BSH56</td><td>++</td><td>2600</td><td>100</td><td>++</td><td>530</td><td>21</td></tr><tr><td>BSH C. perf</td><td>Nd</td><td>150</td><td>26</td><td>Nd</td><td>580</td><td>100</td></tr></tbody></table>

Besides, enzymatically active BSHs from L. johnsonii La1 that could be measured here showed a higher activity for tauro-conjugated than glyco-conjugated substrates, whereas a clear preference has been observed for glyco-conjugated bile salts among other lactobacilli-BSHs characterized so far (Tanaka et al., 1999).

## Enzymatic Activities of L. johnsonii La1 BSH47 and BSH56 Display Anti-giardial Effects

To evaluate the anti-giardial potential of purified L. johnsonii La1-BSHs, G. duodenalis WB6 trophozoites were incubated for 22 h in the presence of increasing concentrations of rBSH47 and rBSH56 from $2 \times 10^{-5}$ to 17.4 µg/ml (rBSH12 was not tested for anti-giardial activity due to weak BSH-activity). Positive controls with C. perfringens BSH (Sigma-Aldrich) and negative experimental controls were set up in each independent inhibition

<footnote>*µmol/5 min per g of protein.</footnote>

<footnote><sup>a</sup>Based on activity on taurodeoxycholic acid (plate assay).</footnote>

<footnote><sup>b</sup>Based on activity on taurocholic acid (enzymatic assay).</footnote>

<footnote><sup>c</sup>Based on activity on glycodeoxycholic acid (plate assay).</footnote>

<footnote><sup>d</sup>Based on activity on glycocholic acid (enzymatic assay).</footnote>

<footnote>Nd, Non-detected. One unit of BSH activity was defined as the amount of enzyme that can liberate 1 µmol of amino acid from a given substrate in 5 minutes.</footnote>
A **E. coli WT** **E. coli rBSH47**

TDCA 0.3%

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

GDCA 0.3%

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

B

**FIGURE 3** | Substrate specificities of *L. johnsonii* La1 BSHs. (A) Hydrolase activity of rBSH47 produced in *E. coli* tested on LB plates. *E. coli* SE1 wild type or *E. coli* SE1 expressing rBSH47 were plated in the presence of taurodeoxycholic acid 0.3% (TDCA) or glycodeoxycholic acid 0.3% (GDCA) and incubated for 48–72 h. Activity is detected when a whitish halo forms around colonies. Data with *E. coli* SE1 expressing rBSH12 and rBSH56, respectively, are not shown. (B) Phylogenetic relationship among selected BSH sequences and substrate prediction. Programme MEGA5 was used for the phylogenetic tree and for the 500-replication bootstrap analysis. The following predicted amino acid sequences were obtained from UniProtKB/Swiss-Prot databases: BSH12, *L. johnsonii* La1 (Q74IV4_LACJO); BSH47, *L. johnsonii* La1 (Q74JG0_LACJO); BSH12, *L. johnsonii* La1 (Q74LX7_LACJO); CBAH-1, *C. perfringens* strain 13 (P54965.3); BSHA, *L. acidophilus* LA4 (ACL98173.1); BSHB, *L. acidophilus* LA4 (ACL98173.1); BSHA, *L. acidophilus* LA11 (ACL98175.1); BSHB, *L. acidophilus* LA11 (ACL98176.1); BSHA, *L. acidophilus* NCFM (YP_193782.1); BSHB, *L. acidophilus* NCFM (AAV42923.1); BSH, *L. gasseri* AM1 (ACL98172.1); BSH-α, *L. johnsonii* 100-100 (AAG22541.1); BSH-β, *L. johnsonii* 100-100 (AAC34381.1); BSHA, *L. johnsonii* pf01 (EGP12224.1); BSHB, *L. johnsonii* pf01 (EGP13287.1); BSHC, *L. johnsonii* pf01 (EGP12391.1); pCBH1, *L. plantarum* 80 (AAB24746.1); BSH1, *L. plantarum* WCSF (CCC80500.1); BIBSH, *B. longum* subsp. *longum* (2HF0); BSH, *L. acidophilus* PF01 (ABQ01980.1); BSH, *L. plantarum* CK 102 (Ha et al., 2006); Penicillin V Acylase (PVA), *Bacillus sphaericus* (3PVA). Substrate specificity, when known (see text for referenced literature) is indicated for each BSH enzyme (in brackets): (TC), specificity for tauro-conjugated bile salts; (GC), specificity for glyco-conjugated bile salts; (TC/GC), specificity for both tauro and glyco-conjugated bile salts.

assay. Treatments of *Giardia* trophozoites with BSHs showed a dose-dependent inhibition of the parasite growth in presence of bile, when compared to the controls without bile (**Figures 4A,B**).

The $IC_{50}$ of rBSH56 ($IC_{50}\text{BSH56} = 0.018 \pm 0.002$ µg/ml) was slightly lower than that of rBSH47 ($IC_{50}\text{BSH47} = 0.030 \pm 0.003$ µg/ml). Concentrations higher than 1 µg/ml of either
A

<figure><img src="image_9.png" /></figure>

B

<figure><img src="image_10.png" /><figcaption>**FIGURE 4** | Anti-giardial activity of *L. johnsonii* La1 BSHs *in vitro*. Inhibition of *G. duodenalis* WB6 strain growth by enzymatically active rBSH47 (A) and rBSH56 (B) from *L. johnsonii* La1 with and without bovine bile supplementation (0.6 g/L) after 22 h of incubation. Increasing doses of BSH were tested ranging from 2 × 10⁻⁵ to 17.4 µg/ml. Values are in percentage ± SEM (standard error of the mean). Growth inhibition curves of *G. duodenalis* were calculated using Prism 5 software (GraphPad). IC₅₀<sub>BSH56</sub> = 0.018 ± 0.002 µg/ml; IC₅₀<sub>BSH47</sub> = 0.030 ± 0.003 µg/ml.</figcaption></figure>

rBSH47 or rBSH56, respectively, were sufficient to kill 100% of trophozoites in 22 h. Co-cultures of trophozoites and rBSHs in a growth medium without bile supplementation did not exhibit any toxic effects, further supporting the fact that the anti-giardial effect is mediated by BSH activity and requires the presence of an appropriate substrate (bile).

To better characterize the damages induced by BSHs activity, the morphology of *G. duodenalis* trophozoites WB6 was analyzed by SEM after 16 h of treatment with either rBSH56 (0.08 µg/ml), rBSH47 (0.5 µg/ml), or deoxycholic acid (DCA, 0.1 and 0.2 g/L), which is a major product of bile hydrolysis. SEM analysis of non-treated trophozoites showed the characteristic giardial tear-drop shape with no apparent sign of morphological alteration (Figures 5a,b). Trophozoites treated with either DCA, or rBSH56 or rBSH47 in presence of bile revealed significant structural damage when compared to controls (Figures 5c-h). BSH-treated parasites displayed several alterations such as protrusions and perforations at the surface of their plasma membrane (Figures 5d,f-h). In DCA-treated trophozoites, membrane and median body were dramatically disrupted (Figures 5g,h). In contrast, with both treatments, the ventral disk microtubule array was still observable.

## Assessment of *in Vivo* Anti-giardial Activities of *L. johnsonii* La1 BSH47

Numerous studies have reported that bile acids conjugated to taurine are predominant in mice (Claus et al., 2011). Recombinant BSH47 efficiently hydrolyzed tauro-conjugated bile acids, and its efficacy against *Giardia* has been demonstrated *in vitro*. Since this enzyme was available in larger quantities compared to rBSH56 and rBSH12, rBSH47 was selected to evaluate the potential of rBSH to treat giardiasis in a murine model (Figure 6A). OF1 suckling mice were divided into four

groups (n = 7-12). Mice were challenged with *G. duodenalis* WB6 trophozoites (1 × 10⁵) at day 10 by intragastric gavage. Increasing doses of rBSH47 corresponding to 0.5, 5, and 50 µg (50 µl, diluted in NaHCO₃ 16.4%) were thawed and daily administered by intragastric gavage to neonatal mice from day 10 to 15. The control group received vehicle (PBS + NaHCO₃ 16.4%). Animals were sacrificed at day 16, corresponding to the peak of trophozoite colonization, and small intestinal contents were sampled and analyzed. Six days after inoculation, trophozoites were able to efficiently colonize and persist in the small intestine with a parasite load 20-fold higher than the inoculum (Figure 6B). In groups treated with rBSH47, the parasite burden decreased in a dose-dependent manner (Figure 6B). Interestingly, the highest dose of rBSH47 (50 µg daily for 5 days) induced a significant reduction of 68.8% of *G. duodenalis* trophozoites compared to the control group.

## DISCUSSION

*L. johnsonii* La1 is a probiotic strain with pathogen inhibition and host immunomodulation properties (Vidal et al., 2002; Cruchet et al., 2003; Pridmore et al., 2008). The activity of BSH and lactobacilli's bile resistance have been widely accepted as key factors for gut persistence and colonization by these bacteria (Tannock et al., 1994; Tanaka et al., 2000; Begley et al., 2006). Three *bsh* and two bile acid transporters genes were identified in the genome of *L. johnsonii* La1 (Pridmore et al., 2004). In this study, we cloned, purified, and characterized these 3 BSH enzymes in order to assess their antiprotozoal effect on *Giardia*. Nucleotide homology comparisons previously highlighted the similarities between *L. johnsonii* La1 *bsh*12 and *bsh*56 with *cbsHα* and *cbsHβ* from *L. johnsonii* 100-100, respectively (Elkins and Savage, 1998; Elkins et al., 2001; Pridmore et al., 2004). A
<figure><img src="image_11.png" /><figcaption>**FIGURE 5** | Morphological alterations following *in vitro* treatments of G. duodenalis by BSH or deoxycholic acid (DCA). Scanning electron microscopy of G. duodenalis trophozoites WB6 strain treated with either BSH47 (0.5 µg/ml), BSH56 (0.08 µg/ml) or DCA (0.1 and 0.2 g/L). (a) KM control (KM+ 10% FCS) and (b) KM control with bile (bovine bile 0.6 g/l) show the characteristic pear-shaped of trophozoites. (c) G. duodenalis treated with rBSH47 and (d) G. duodenalis treated with rBSH47 with bile reveal altered morphology and plasma membrane disruption in presence of bile. (e) G. duodenalis treated with rBSH56 and (f) G. duodenalis treated with rBSH56 with bile showed similar cell lysis. (g,h) DCA-treated (0.1 and 0.2 g/l, respectively) *Giardia* present similar alterations and a disruption of plasma membrane exposing cell interior. Scale bar = 5 µm (b,d,f,g) or 10 µm (a,c,e,h).</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**FIGURE 6** | Anti-giardial activity of rBSH47 *in vivo*. (A) Experimental design of *G. duodenalis* infection in OF1 suckling mice model. Increasing doses of rBSH47 were daily administered by intragastric gavage (0.5, 5, 50 µg) to OF1 suckling mice from day 10 to 15. Control animals received PBS+ NaHCO₃ 16.4% (vehicle). Mice were challenged with *G. duodenalis* WB6 trophozoites (10⁵) at day 10 by intragastric gavage. Animals were euthanized by cervical dislocation at day 16. (B) *G. duodenalis* trophozoites enumeration in small intestine after rBSH47-treatment. Group I (control): mice received vehicle (n = 12); Group II: mice received 0.5 µg of BSH47 daily for 5 days (n = 10); Group III: mice received 5 µg of BSH47 daily for 5 days (n = 9); mice received 50 µg of BSH47 daily for 5 days (n = 7). Small intestines were resuspended in PBS and trophozoites were counted using a hemocytometer. Values are mean ± SEM; *p* < 0.0001.</figcaption></figure>

neighbor-joining tree of various BSHs protein sequences from several lactic acid bacteria confirmed that *L. johnsonii* La1-BSH enzymes are phylogenetically related to BSHs from other species. In addition, they share a high degree of similarity to various subgroups of BSHs; for instance, BSH12 shares 99% identity with *L. johnsonii* 100-100 cbsHα and *L. johnsonii* PF01 BSHA at amino acid level, and BSH56 shares 99% identity with *L. johnsonii* 100-100 cbsHβ and 98% with *L. johnsonii* PF01 BSHB at amino acid level. *L. johnsonii* La1 and *L. johnsonii* PF01 both possess a third BSH gene, *bsh47* and *bshC*, respectively, which is absent from *L. johnsonii* 100-100. Besides, the close relationship among *L. johnsonii* La1 BSH47, *L. johnsonii* PF01 BSHC, and *L. acidophilus* NCFM BSHB, at amino acid level, suggests that these enzymes likely share a common ancestor. These observations contribute to the idea that BSH might have been acquired through horizontal gene transfer from microorganisms sharing the same intestinal environment (Corzo and Gilliland, 1999; Franz et al., 2001; McAuliffe et al., 2005; Begley et al., 2006), although this latter hypothesis remains to be tested.

Multiple amino acid sequence alignment of *L. johnsonii* La1 BSHs indicated a high variability among characterized BSHs; however, well-conserved motifs were observed around residues involved in the active site (Cys-2, Arg-18, Asp-21, Tyr-82,

Asn-172, and Arg-225). These observations are in agreement with previous studies (Rossocha et al., 2005; Fang et al., 2009; Lin et al., 2014) and highlight that the biological functions of BSHs are strictly conserved despite sequence/phenotypic variabilities. BSHs (EC 3.5.1.24) belong to N-terminal nucleophilic (Ntn) hydrolases, a superfamily of enzymes containing N-terminal cysteine residue involved in the catalytic site (Suresh et al., 1999; Kim et al., 2004). Penicillin V acylases (EC 3.5.1.11), which are closely related to BSH, also belong to Ntn hydrolases and share similar structures. *In silico* modeling, BSH47, BSH56, and BSH12 showed that the typical αββα tertiary structure arrangement, which is characteristic of Ntn superfamily, is conserved (Oinonen and Rouvinen, 2000; Patel et al., 2010; Lin et al., 2014). Taken together, these structural observations indicate that the folding of BSHs remained stable during evolution despite low sequence identity, which suggest a high evolutionary pressure to maintain their functionality (Chothia and Lesk, 1986; Sander and Schneider, 1991; Krieger et al., 2003).

Experimental determination of BSH activities showed that at least 2 of the 3 BSHs from *L. johnsonii* La1 have broad substrate specificities. The enzyme BSH56 exhibited high hydrolysis activities toward tauro- and glyco-conjugated bile salts, with a preference toward tauro-conjugated substrates.
Interestingly, BSH56 belongs to a phylogenetic cluster of BSH
enzymes exhibiting activity exclusively directed toward tauro-
conjugated bile acids. Similarly, BSH47 was more efficient
at hydrolyzing tauro-conjugated bile salts and exhibited a
very low relative activity toward glyco-conjugated bile salts.
Surprisingly, the amino acid sequence of BSH47 is more
closely related to that of *L. johnsonii* BSHC that has been
reported to hydrolyze glyco-conjugated bile salts (Chae et al.,
2013). Our results, therefore, contrast with previous studies
reporting that most BSH enzymes isolated from lactobacilli
are very efficient at hydrolyzing glyco-conjugated bile salts
(Coleman and Hudson, 1995; Liong and Shah, 2005). In addition,
these observations point out the limits of substrate predictions
based on phylogenetic relationships considering whole enzyme
sequences. The mechanisms responsible for substrate specificity
of BSH enzymes are still unclear. Putative amino acids have
been associated with substrate binding pockets in *C. perfringens*
(Rossocha et al., 2005; Ridlon et al., 2006), but these residues
are different in all 3 BSHs from *L. johnsonii*. Several studies
emphasized that BSH preferably recognize conjugated substrates
at amino acid moieties (Coleman and Hudson, 1995; Tanaka
et al., 2000; Kim et al., 2004; Rossocha et al., 2005), whereas
others still suggest that steroid moieties are recognized in priority
(Moser and Savage, 2001; Begley et al., 2006; Patel et al., 2010)
which could explain broad substrate specificities. Experimentally
solving the 3D structures of rBSH47 and rBSH56 harboring
appropriate substrates would certainly provide invaluable
information regarding this important question.

The third BSH isolated from *L. johnsonii* La1, BSH12, was
successfully expressed in *E. coli* and clearly observed in SDS-
PAGE and by Western blotting, but no specific activity could
be detected toward either substrate in liquid tests. However, a
slight positive signal was observed with *E. coli* strain-expressing
rBHS12 on agar plates supplemented with glyco-specific bile
salts, suggesting a weak glyco-specific activity. Similar difficulties
to express recombinant BSHs have been observed with BSHs
from *L. plantarum* JPP2 (Ren et al., 2011) and BSH2 from *L.
plantarum* WCFS1 (Lambert et al., 2008). Proteolytic degradation
and misfolding of the recombinant protein produced in *E. coli*
may have affected the enzyme's function (Baneyx and Mujacic,
2004). The functionality of BSH12 is, therefore, still under
investigation.

As mentioned previously, the putative natural role of BSHs is
to decrease the toxicity of conjugated bile salts for bacterial cells
(De Smet et al., 1995). In this work, we assessed the anti-parasitic
activity of recombinant BSHs and we demonstrated that rBSH47
and rBSH56 were highly active against viable trophozoites of G.
duodenalis strains WB6 and NF. The minimum concentration
found to kill 100% of G. duodenalis WB6 trophozoites *in vitro* was
1 µg/mL for both rBSH47 and rBSH56. When tested on human
enterocyte Caco-2 cells, both rBSH47 and rBSH56 inhibited the
growth of G. duodenalis NF in the presence of bile (0.6 g/L) in
a dose-dependent fashion and prevented the attachment of the
parasites to the cell monolayers (Figure S3). The rBSH56 was
more effective than rBSH47 in killing both G. duodenalis WB6
and NF strains, although it induced more cell damages to Caco-2
cells at high concentrations (Figure S3). Recombinant BSH47

was, therefore, chosen to assess the *in vivo* effectiveness of BSH
to treat *Giardia* infection in a suckling murine model. Moreover,
given that bile acids are predominantly conjugated to taurine in
mice (Claus et al., 2011), the ability of BSH47 to preferentially
hydrolyze tauro-conjugated bile salts seemed more appropriate.
We observed that rBSH47 inhibited *Giardia* growth in a dose-
dependent manner *in vivo*, in keeping with the data obtained *in
vitro*. At the highest dose (50 µg/mice/day) administered daily
for 5 days, the parasite (trophozoite) burden was significantly
reduced by 68.8% in the small intestine of the mice. Despite
this important decrease of the parasite load, none of the treated
mice were free of parasites at 16 days post-inoculation. The
anti-giardial effects mediated by rBSH47 *in vivo* were, therefore,
modest compared to their efficacy in the *in vitro* assays. This
can be explained by a partial degradation of BSHs by acidic
proteases and peptidases in the stomach. The activity of BSHs
correlates with the amount of conjugated bile salts released in
the duodenum, which is highly variable in neonates (Heubi et al.,
2007). The production of deconjugated bile salts at inhibiting
levels is, therefore, dependent to the bioavailability of BSH in the
small intestine.

It has been reported that bile salts, and more specifically
conjugated bile salts, have growth promoting effects on G.
duodenalis *in vivo* (Halliday et al., 1988). Indeed, bile uptake
contributes to cholesterol and exogenous phospholipids needs,
which are essentials for parasite growth (Yichoy et al., 2011).
So far, to our knowledge, there is no evidence showing that
*Giardia* is able to deconjugate bile salts. Conjugated bile salts
are, thus, directly consumed by *Giardia* without being detoxified
(Farthing et al., 1985). In contrast, bacterial deconjugation
aims at reducing the detergent properties of conjugated bile
salts, which are more toxic for bacterial cells than secondary
bile salts, i.e., cholic acid (CA), deoxycholic acid (DCA), and
chenodeoxycholic acid (CDCA) (De Boever and Verstraete,
1999). It is likely that detoxification of bile salts by the duodenal
microbiota has a collateral effect on parasite survival. Earlier
work carried out in our lab showed that DCA and CDCA exerted
cytotoxic effects on G. duodenalis trophozoites *in vitro* at non-
micellar concentrations (Travers et al., 2016). Therefore, we
investigated the morphological perturbations induced by DCA
and BSHs on trophozoite cultures and we noticed both induced
degenerations and perforations of the parasite plasma membrane.
It has been established that DCA perturbs eukaryotic membranes
structure by altering the membrane lipid microdomains (Jean-
Louis et al., 2006). Furthermore, secondary bile salts induced
a redistribution of cholesterol and decrease membrane fluidity.
Hence, we hypothesized that secondary bile salt, and more
specifically DCA, would kill *Giardia* trophozoites by damaging
the cell structure. In the upper parts of the small intestine, where
bile salt deconjugation occurs at high rate, the Gram-positive
bacteria might be protected against secondary deconjugated bile
salts by cell wall peptidoglycan.

A major side effect of BSH-based treatment would be a shift
of bile salt balance in the gut. It has been reported in previous
studies that an enhancement of BSH activity might impact host
physiology by disturbing fat digestion and lipid metabolism
(Begley et al., 2006; Lin et al., 2014). Moreover, secondary bile
acids resulting from the deconjugation of bile salts have also been linked to DNA damage in bacterial and host cells, colon cancer, and inflammation (Cheah and Bernstein, 1990; Moser and Savage, 2001; Bernstein et al., 2005). On the other hand, BSH activity is a natural process that plays a central role in the reduction of cholesterol (Jones et al., 2013).

The aim of this study was to evaluate the anti-giardial potential of BSHs against *G. duodenalis*. We expressed for the first time the BSHs isolated from the probiotic strain *L. johnsonii* La1 and showed that rBSH47 and rBSH56 exhibited high specific activity and broad substrate specificities. Antiprotozoal assays demonstrated that BSHs were highly effective against *G. duodenalis* in vitro and in vivo and represent a promising therapeutic strategy based on their natural catalytic activity. Future studies will determine whether such treatment approaches should be of short duration in order to avoid putative side effects related to the enhancement of bile salt deconjugation. Besides, this anti-giardial effect can be extended to any BSH activity as long as it efficiently converts conjugated bile salts into their deconjugated counterparts. However, further works are still needed to investigate the positive impact of such treatment on the pathophysiology of giardiasis, including protective effects on epithelial permeability, mucosal injury, and malfunction. Moreover, newer galenic formulations are needed in order to provide a better persistence of rBSHs *in vivo*, which can improve health outcomes in routine clinical and veterinary usages.

## AUTHOR CONTRIBUTIONS

IF, PG, BP, TA, and LB-H conceived and designed the study. TA, IF, and LB-H produced and isolated the recombinant BSH, performed the biochemical characterizations with SC and the *in silico* analyses. TA, SC, AB, and IF performed the *Giardia* assays *in vitro* and SEM. TA, MT, and BP performed the *Giardia* assays in the suckling mice model. IV, PL, and PG discussed the experiments and results. TA, IF, and LB-H wrote the manuscript with contributions from all authors.

## FUNDING

This work was partially funded by Region Ile de France-DIM (Maladies Infectieuses Parasitaires et Nosocomiales Emergentes, programm n° 120092).

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## ACKNOWLEDGMENTS

We deeply thank Geraldine Toutirais (PTME, Plateau Technique de Microscopie Électronique et de Microanalyses du Museum National d'Histoire Naturelle, Paris) for assistance with SEM imaging. We thank Jasmine Kirati (UMR BIPAR, Anses, ENVA, INRA) for help with *in vivo* assays and Olivier Berteau (Micalis Institute, INRA) for assistance in protein purifications.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2017.02707/full#supplementary-material

**Figure S1** | Protein structure prediction of *L. johnsonii* La1 BSHs. Protein structures were modeled for *L. johnsonii* BSH56 (a) and BSH47 (b) using I-TASSER software (http://zhanglab.ccbmb.med.umich.edu/I-TASSER/). CBAH-1 (PDB: 2BJF) from *C. perfringens* (Rossocha et al., 2005) and BIBSH from *B. longum* (Kumar et al., 2006) (PDB: 2HF0) were used as templates for modeling BSH47 and BSH56, respectively. Best values of the C-score were chosen for model structure prediction. Visualization of predicted structures for BSH47 (red, a) and BSH56 (blue, b) and superimposition (c,d) were performed using Pymol software (https://www.pymol.org/).

**Figure S2** | Protein structure prediction of *L. johnsonii* La1 BSH12. Protein structure was modeled for *L. johnsonii* BSH12 using I-TASSER software (http://zhanglab.ccbmb.med.umich.edu/I-TASSER/). Bile salt hydrolase (PDB: 2RF8) from *C. perfringens* was used as a template for modeling. Best value of the C-score was chosen for model structure prediction. Visualization of predicted structure was performed using Pymol software (https://www.pymol.org/).

**Figure S3** | Anti-giardial effect of BSH in co-culture with Caco2 cells. To further investigate the effect of *L. johnsonii* La1-BSHs on the adherence of *Giardia* enterocytes, differentiated Caco-2 cells were co-incubated with fresh trophozoites cultures of *G. duodenalis* NF strain and treated with either rBSH47 or rBSH56, over a range of concentrations from 0.005 to 1 µg/ml. *G. duodenalis* NF strain trophozoites were inoculated to Caco2 cells at a multiplicity of infection (MOI) of 10:1 in the presence of increasing doses of rBSH47, rBSH56 or DMEM. Anti-giardial activity assays were performed with bovine bile (0.6 g/l) added to the medium. The results are expressed as relative percentage of growth compared to untreated trophozoites as negative control. Data are presented as mean ± SEM and correspond to triplicates. As expected from previous experiments done with the *G. duodenalis* WB6 strain, both BSHs triggered a growth inhibition of *G. duodenalis* NF trophozoites, in a dose-dependent fashion, after 20 h of exposure (this figure). In contrast, high concentrations of rBSH in assays induced cell damages on Caco-2 monolayer (data not shown). This phenomenon might be due to a putative cytotoxicity of deconjugated bile salt. Interestingly, neither rBSH47 nor rBSH56 displayed cytotoxic effects when tested in absence of bile (data not shown).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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