Patent Application
20170216415
The present invention relates to a composition exhibiting a bile-salt hydrolase activity for its use for the treatment or the prevention of giardiasis, said composition comprising a bile-salt hydrolase (BSH) enzyme, a bacterium able to secrete a BSH, a recombinant host cell able to secrete a BSH, or a combination thereof. The present invention also relates to the use of a composition exhibiting a BSH activity for the treatment or the prevention of giardiasis, and to a pharmaceutical composition or a food composition comprising, as an active principle, a BSH, a bacterium able to secrete a BSH, or a recombinant host cell able to secrete a BSH.
Giardia lamblia is a parasitic protozoan responsible for giardiasis, a disease characterized by acute or chronic intestinal malabsorption, diarrhea, weight loss, dehydration and abdominal pain in humans and a variety of animals. It is one of the most common intestinal parasites in the world. Giardiasis has high veterinary impact and high impact on public health, is responsible for important human morbidity, especially causing nutritional deficiencies in children in developing countries (Ali and Hill, 2003). Developed countries are also concerned by giardiasis and outbreaks have been associated with drinking water contamination resulting from runoff of contaminated soils by rain falls, agricultural practices and sewage treatment plant dysfunctions (Mons et al., 2009).
G. lamblia enters vertebrates when cysts are ingested with food, water or fomites contaminated by feces from infected hosts. Cysts remain infective for months in environmental waters. They are highly resistant to chlorinated disinfectants used in drinking water treatments and infectious doses are low (10 to 100 cysts). After ingestion, infective forms excyst, releasing trophozoites that establish infections in the upper small intestine (duodenum) where they interact with the intestinal barrier. Duodenum is a very specific environment where food from the stomach, bile from the gall bladder and digestive enzymes from the pancreas pour in. There, G. lamblia divides by binary fission and eventually exits the host with the feces once trophozoites have re-formed cysts. The drug of choice for treating giardiasis remains metronidazole, a 5-nitroimidazole, which is reduced into toxic compounds by the parasite enzyme pyruvate:ferredoxin oxido-reductase. Side effects of drug treatment are reported and metronidazole resistance appears in vitro and in clinical settings (up to 20% of environmental strains were reported as resistant) (Uperoft and Uperoft, 2001).
How Giardia pathology is produced is still unclear but it is probably multifactorial (Farthing et al., 1997). Patho-physiologies observed comprise damages of the host mucosal surface by microvillus and crypt atrophy, decreased epithelial permeability and impairment of the activity of digestive enzymes. These changes may be due as much to factors of the host as to those of the parasite.
It is now widely recognized that intestine microbiota plays a role in the protection of the host against gut colonization by pathogens (Travers et al., 2011). Different mechanisms may be responsible for the protective effects: competition for pathogen receptor sites, barrier for pathogen access, production of antimicrobial compounds, competition for nutritional substrates and enhancement of the innate and adaptive host immune responses (Tancrede et al., 1992). Nutritional strategies are based on an increase of the components of the gut microbiota that are associated with the protective activity (Brassard D. and Schiffrin E J., 1997). Colonization of the intestine by G. lamblia strongly depends on the gut microbiota (Singer and Nash, 2000). Evidences strongly suggest the involvement of the bacterium Enterococcus faecium in the host immune response against G. lamblia (Benyacoub et al., 2005). Extracellular factors of L. johnsonii La1 block the in vitro growth of G. lamblia in G1 phase indicating that bacteria may directly affect parasite development (Perez et al., 2001). It was shown that the probiotic bacteria Lactobacillus johnsonii La1 antagonizes G. lamblia establishment in rodent model (Humen et al., 2005).
The increasing numbers of outbreaks of giardiasis due to drinking water contamination, the side effects of available anti-Giardia drugs as well as, the increasing emergence of resistance to these drugs, render giardiasis a worrying subject, and there is a real need for alternative therapeutic strategies.
The present invention now provides compositions exhibiting a bile-salt hydrolase (BSH) activity as effective inhibitors for Giardia lamblia, for the prevention or the treatment of giardiasis. The inventors have indeed shown that parasite growth inhibition is mediated by secreted bacteria bile-salt hydrolase activity(ies) that produce(s) deconjugated bile salts from bile present in the culture medium, and that, unlike normal conjugated bile salts which are the main components of the bile, deconjugated bile salts are found toxic for Giardia.
Anti-Giardia activity was studied in vivo and in vitro for L. johnsonii La1 (LjLa1) (Perez et al. 2001, Humen et al. 2005). The genomes of L. johnsonii La1 and G. lamblia were sequenced (Pridmore et al. 2004, Morrison et al. 2007). Halliday et al (1995) disclose that G. lamblia internalizes conjugated bile salts, however this document does not mention a role for unconjugated bile salts. Shukla et al. (2011, 2012, 2013) describe that the administration of Lactobacillus rhamnosus lessens the severity of giardiasis. U.S. Pat. No. 6,905,679 discloses a method for the treatment of a disorder associated with the colonization of gut by Giardia intestinalis comprising the administration of Lactobacillus acidophilus La10 bacteria (CNCM reference number 1-2332), of Bifidobacterium bifidum (1-2333) or of Bifidobacterium infantis (1-2334). The inhibitory effect of lactic acid bacteria is possibly attributed to particular organic acids secreted by said lactic acid bacteria.
However, none of these documents disclose the role of bile-salt hydrolase activity, in the presence of bile salts, for the inhibition of Giardia lamblia.
A composition according to the present invention represents a new therapeutic strategy against Giardia lamblia, based on the natural catalytic activity of bile-salt hydrolases, in order to prevent or treat giardiasis. A composition according to the invention furthermore represents a public health interest by providing a means to counteract the emergence of drug resistant Giardia strains.
The invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the invention.
The present invention first relates to a composition exhibiting a bile-salt hydrolase activity for the treatment or the prevention of giardiasis.
The term “bile-salt hydrolase activity” or “BSH activity” refers to the ability of a composition according to the invention to hydrolyse conjugated bile-salts to generate deconjugated bile salts, and glycine or taurine. BSH cleave the peptide linkage of bile acids, which results in removal of the amino acid group from the steroid core and in the production of deconjugated, or unconjugated, bile acids, which precipitate at low pH.
The expression “treatment or prevention of giardiasis” refers to the treatment or the prevention of disorders associated with the presence in the gut of Giardia lamblia or of Giardia intestinalis, and in particular disorders associated with the infestation and/or colonization of the gut by said parasites.
Conjugated bile salts, or bile acids, are glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA), glycochenodeoxycholic acid (GCDCA) and taurochenodeoxycholic acid (TCDCA). Unconjugated bile-salts, also called deconjugated bile salts are: cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA).
BSH activity is assessed by the detection the products generated by the enzymatic reaction, in particular the detection:
A specific method for detection of BSH activity includes a method such as described in the present specification, using mass spectrometry analysis (LC/ESI-MS analysis) and proteomic analysis of bile components. It may also be detected, in bacteria, by a method comprising the growth of said bacteria in an adapted broth, the streaking of bacteria onto medium supplemented, or not, with 0.2% (wt/vol) glycodeoxycholic acid and anaerobic incubation for 48 h. The white precipitates around colonies and the clearing of the medium are indicative of BSH activity.
Therefore, the present invention relates to a composition exhibiting a bile-salt hydrolase activity for the treatment or the prevention of giardiasis, said composition comprising at least one of the following elements, or a combination thereof:
The present invention also relates to a composition exhibiting a bile-salt hydrolase activity for the treatment or the prevention of giardiasis, said composition comprising at least one of the following elements, or a combination thereof:
The BSH activity of a BSH enzyme is expressed as enzymatic units (U) when tested using a taurodeoxycholic and/or a glycodeoxycholic substrate. An example of this determination is described in Example 13 of the present patent application. The level of the activity of a BSH, in a composition according to the invention, can be expressed by reference to the activity of commercial C. perfringens BSH, and possibly using a test for said activity as described in the C. perfringens manufacturer's notice.
In a particular embodiment, the present invention relates to a composition exhibiting a BSH activity for use for the treatment or prevention of giardiasis, wherein said BSH activity is comprised between 0.0001 U and 100 U, between 0.001 U and 10 U, between 0.01 U and 1 U, between 0.1 U and 0.5 U.
In a first particular embodiment, the present invention relates to a composition exhibiting a bile-salt hydrolase activity for the treatment or the prevention of giardiasis, wherein the bile-salt hydrolase activity is associated with the presence of at least one bile-salt hydrolase (BSH) enzyme.
In another particular embodiment, the present invention relates to a composition exhibiting a BSH activity for use for the treatment or prevention of giardiasis, wherein said composition comprises a BSH enzyme, or is a able to secrete at least one BSH enzyme, wherein the concentration of BSH present in said composition is comprised between 0.01 μg/ml and 10 mg/ml, between 0.1 μg/ml and 1 mg/ml, between 0.5 μg/ml and 200 μg/ml, between, between 1 μg/ml and 100 μg/ml, between 1 μg/ml and 50 μg/ml or between 5 μg/ml and 20 μg/ml. In a more particular embodiment, a composition according to the invention exhibits a bile-salt hydrolase activity associated with the presence of at least one bile-salt hydrolase (BSH) enzyme, said enzyme being chosen among prokaryotic BSH, or BSH synthesized by bacteria. In a more particular embodiment, a composition according to the invention exhibits a bile-salt hydrolase activity associated with the presence of at least one bile-salt hydrolase (BSH) enzyme synthetized by a lactic acid bacterium.
A lactic acid bacterium is defined as gram positive bacteria sharing common metabolic and physiological characters, wherein lactic acid is produced as a major metabolic end product of carbohydrate fermentation. The group of lactic acid bacteria includes in particular Lactobacillus, Lactococcus and Bifidobacterium. In a more particular embodiment, a composition according to the invention exhibits a bile-salt hydrolase activity associated with the presence of at least one bile-salt hydrolase (BSH) enzyme which amino acid sequence comprises an amino acid sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with, or which is a natural variant of, an amino acid sequence chosen in the group consisting of:
Any BSH enzyme suitable in a composition according to the present invention is easily found by a man skilled in the art, and may be chosen, in particular, among the following:
The present invention comprises the presence of variants of BSH enzymes having at least 80%, at least 85%, preferably 90%, at least 90%, more preferably 95%, at least 95% and even more preferably 98%, at least 98% or at least 99% identity with a sequence chosen in the group consisting of SEQ ID No 1 to SEQ ID No 7. As used herein the term “identity” herein means that two amino acid sequences are identical (i.e. at the amino acid by amino acid) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size) and multiplying the result by 100 to yield the percentage of sequence identity. The percentage of sequence identity of an amino acid sequence can also be calculated using BLAST software with the default or user defined parameter. As applied to polypeptides, the term substantial identity means that two peptide sequences, when optimally aligned, share at least about 80% sequence identity, preferably at least 85% identity, preferably at least about 90% sequence identity, more preferably at least about 95% sequence identity, at least 98% sequence identity or more (e.g., 99% sequence identity). As used herein, a “derivative” or “sequence derived from”, or “natural variant” refers to an amino acid sequence having at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity and even more preferably at least 95% identity or more, such as at least 98% sequence identity or 99% identity with said reference amino acid sequence.
More particularly, the present invention refers to a composition exhibiting a bile-salt hydrolase activity associated with a BSH enzyme, for its use in the prevention of the treatment of giardiasis, wherein the amino acid sequence of said enzyme is chosen among the following:
In another particular embodiment, the invention relates to a composition exhibiting a bile-salt hydrolase activity for its use in the prevention of the treatment of giardiasis, wherein said composition comprises a lactic acid bacteria able to secrete a BSH, said lactic acid bacteria being chosen among: Lactococcus, Lactobacillus, with the exception of the bacterial strains Lactobacillus johnsonii La1 (NCC533, CNCM I-1225) and of Lactobacillus acidophilus (NCC90, CNCM I-2332), and Bifidobacterium, with the exception of the bacterial strains Bifidobacterium bifidum (NCC189, CNCM I-2333) and of Bifidobacterium infantis (NCC200, CNCM I-2334).
The term “lactic acid bacteria” refers to the presence of at least one micro-organism, wherein the micro-organism is preferably present of about 106 to 1012 cfu (colony forming unit). The microorganisms may be present as such or optionally after a step of purification from a culture medium. In another embodiment, a composition according to the invention comprises the supernatant of a culture medium of said microorganism, which may be concentrated by any method known by a man skilled in the art.
Therefore, the invention relates to a composition exhibiting a bile-salt hydrolase activity for its use in the prevention of the treatment of giardiasis, said composition comprising a Lactobacillus, with the exception of the bacterial strains Lactobacillus johnsonii La1 (NCC533, CNCM I-1225) and of Lactobacillus acidophilus (NCC90, CNCM I-2332).
The invention also relates to a composition exhibiting a bile-salt hydrolase activity for its use in the prevention of the treatment of giardiasis, said composition comprising a Bifidobacterium, with the exception of the bacterial strains Bifidobacterium bifidum (NCC189, CNCM I-2333) and of Bifidobacterium infantis (NCC200, CNCM I-2334).
In a more particular aspect, a composition according to the invention comprises a Lactobacillus chosen in the group consisting of:
The following bacterial strains have shown a particular activity in a composition according to the invention, as shown in the “Examples” part of the specification.
In a particular embodiment, the invention relates to a composition exhibiting a bile-salt hydrolase activity for its use in the prevention of the treatment of giardiasis, said composition comprising a lactobacillus chosen in the group consisting of the bacterial strains referenced as:
In a particular embodiment, the present invention relates to a composition exhibiting a BSH activity for the prevention or the treatment of giardiasis, said composition comprising a lactic acid bacteria able to secrete a BSH, wherein said bacteria are present in an amount comprised between 106 to 1012 cfu/g of bacteria.
In another particular embodiment, the present invention relates to a composition exhibiting a bile-salt hydrolase activity for its use in the prevention of the treatment of giardiasis, said composition comprising a host cell comprising a heterologous nucleic acid sequence encoding for at least one bile-salt hydrolase enzyme, said host cell being able to secrete said bile-salt hydrolase enzyme.
The term “host cell” refers to a living cell able to receive heterologous nucleic acid, and able to synthesize at least one protein encoded by said heterologous nucleic acid. In a preferred embodiment, said heterologous nucleic acid is an expression vector containing nucleic acid sequences able to encode a protein of interest and all necessary regulatory elements for the production of said protein in said host cell.
In a more particular embodiment, a composition according to the invention comprises a host cell comprising at least one nucleic acid sequence chosen among the following:
Any BSH enzyme suitable in a composition according to the present invention can be selected by a man skilled in the art, and a nucleotide sequence encoding for said enzyme can be found in public databases, it may be chosen, in particular, among the following:
In a more particular embodiment, a composition according to the present invention comprises a host cell comprising at least one nucleic acid sequence encoding for a BSH, said host cell being chosen in the group of lactic acid bacteria. More particularly, said lactic acid bacterium is chosen among lactobacilli and lactococci. In a particular embodiment of the invention, said lactic acid bacterium is Lactococcus lactis. In another particular embodiment of the invention, said lactic acid bacterium is a lactobacilli chosen among the group consisting of:
In another aspect, the present invention relates to the use of a composition exhibiting a bile-salt hydrolase activity for the treatment or the prevention of giardiasis, wherein said composition comprises:
In another more particular embodiment, the present invention relates to the use of a composition exhibiting a bile-salt hydrolase activity wherein said composition comprises: a bile-salt hydrolase (BSH) enzyme, a bacteria able to secrete at least one BSH, with the exception of the strain Lactobacillus johnsonii La1, a recombinant host cell able to secrete at least one heterologous BSH, or a combination thereof for the treatment or the prevention of giardiasis in animals. Indeed, a composition according to the invention may be used for the treatment or the prevention of giardiasis in pigs and in cows, therefore, in a particular embodiment, a composition according to the invention is for use in the treatment or prevention of giardiasis in pigs or in cows.
In a further aspect, the present invention relates to a pharmaceutical composition comprising, as an active principle, a BSH, a lactic acid bacteria able to secrete at least a BSH, a host cell able to secrete a BSH, or a combination thereof, and a pharmaceutically acceptable carrier.
A pharmaceutical composition according to the invention comprises an effective amount of a BSH, a lactic acid bacteria able to secrete at least a BSH, a host cell able to secrete a BSH, or a combination thereof, and a pharmaceutically acceptable carrier, wherein said effective amount is defined as catalytic units (U), for BSH enzyme or recombinant BSH enzyme, or as a number of colony forming units (cfu), for living bacteria.
A pharmaceutical composition according to the invention is selected from an orally administrable composition, including, with no restriction, tablets, liquid bacterial suspension, dried oral supplement, wet oral supplement and tube feeding compositions.
Other pharmaceutical or nutritional preparations suitable for oral administration are hard or soft gelatin capsules made from gelatin and a plasticizer such as glycerol or sorbitol. Hard capsules may include the inventive compound in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and if desired, stabilizers. In soft capsules, the inventive compound is preferably dissolved or suspended in a suitable liquid, such as fatty oil, paraffin oil or a liquid polyethylene glycol, to which a stabilizer can be added.
In a particular embodiment, a pharmaceutical composition according to the invention comprises an effective amount of a BSH enzyme and a pharmaceutically acceptable carrier for an administration to animals. In a more particular embodiment, a pharmaceutical composition from the invention comprises a BSH-12, a BSH-47 and/or a BSH-56 enzyme, and a carrier comprising a vehicle for oral administration of molecules, mixed with a cell culture buffer.
A person skilled in the art is able to choose an adapted vehicle, as an example, the SYRSPEND® can be cited, and to prepare and adapted mixture for its oral administration, for example by mixing said vehicle with a diluting solution and/or with a buffer. As an example, a pharmaceutical composition according to the invention comprises an effective amount of BSH-12, a BSH-47 and/or a BSH-56 enzyme and a carrier comprising a mixture of SYRSPEND® NaHCO3 16.4%, DMEM (Dilbecco Modified Culture Medium).
Coated tablet cores can be provided with suitable coatings, which if appropriate are resistant to gastric juices, using, inter alia, concentrated sugar solutions which may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, shellac solutions in suitable organic solvents or solvent mixtures or, for the preparation of coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments can be added to the tablets or coated tablets, for example, to identify or indicate different doses of the active compound ingredient.
In a more particular embodiment, the present invention relates to a pharmaceutical composition comprising a BSH, a lactic acid bacterium able to secrete at least a BSH, a host cell able to secrete a BSH, or a combination thereof as an active principle, wherein said active principle is encapsulated.
In another particular embodiment, the present invention relates to a process for the preparation of a composition exhibiting a bile-salt hydrolase activity for the treatment or the prevention of giardiasis, said composition comprising a carrier and at least one of the following elements, or a combination thereof:
In a more particular embodiment, the present invention relates to a process for the preparation of a composition exhibiting a bile-salt hydrolase activity for the treatment or the prevention of giardiasis, said composition comprising a pharmaceutically acceptable carrier and at least one of the following elements, or a combination thereof:
In another more particular embodiment, the present invention relates to a process for the preparation of a composition exhibiting a bile-salt hydrolase activity for the treatment or the prevention of giardiasis, said composition comprising a carrier acceptable for a food composition or a dietary supplement and at least one of the following elements, or a combination thereof:
A process according to the invention comprises at least a step of contacting said carrier with an effective amount of said enzyme and/or said bacterium and/or said host/cell, wherein said and a step of mixing said elements in order to prepare a composition suitable for its intended use.
The present invention also relates to a method for the prevention or for the treatment of giardiasis, said method comprising the administration, to a patient in need thereof, of a composition comprising at least one of the following elements, or a combination thereof:
In a further aspect, the present invention relates to a food composition or a dietary supplement comprising, as an active principle, a BSH, a lactic acid bacterium able to secrete at least a BSH, a host cell able to secrete a BSH, or a combination thereof, and optionally a carrier.
A food composition or a dietary supplement according to the invention comprise an effective amount of a BSH, a lactic acid bacteria able to secrete at least a BSH, a host cell able to secrete a BSH, or a combination thereof, and a pharmaceutically acceptable carrier, wherein said effective amount is defined as catalytic units (U), for BSH enzyme or recombinant BSH enzyme, or as a number of colony forming units (cfu), for living bacteria.
A food composition or a dietary supplement according to the invention may be provided as a dry or a wet composition, and may be selected, with no restriction, among milk, yogurt, cheese, milk powder and pet food.
Bovine bile solutions (Sigma and Difco) were prepared at 100 mg/ml stock solution in distilled water, filtered at 0.2 μm and stored at −20° C. Pure bile salts (Sigma): glycocholate (GC), taurocholate (TC), glycodeoxycholate (GDC), taurodeoxycholate (TDC), glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC), bile salt mix (Sigma) or pure corresponding bile salts (Sigma): cholate (C), deoxycholate (DC) and chenodeoxycholate (CDC), and fusidic acid (Sigma) were dissolved in distilled water to 12 mg/ml stock solution, filtered at 0.2 μm and kept at −20° C. Choloylglycine Bile Acid Hydrolase (or Bile Salt Hydrolase, BSH, EC 3.5.1.24) from Clostridium perfringens (Sigma) was prepared at 10 U/ml in distilled water (milli-Q grade) and stored at −20° C. Iodoacetic acid (Sigma) was prepared as 0.5 M stock solution in distilled water and frozen at −20° C. Fetal calf serum was from PAA Laboratories, GE Healthcare. The NEFA-C kit used for quantitative determination of non-esterified fatty acids (NEFAs) was from Biolabo (WAKO Diagnostics).
Cell Culture of Giardia lamblia
Trophozoites of G. lamblia strains WB (clone C6, ATCC30957) and HP1 (Portland-1, gift of J. Tackezy) were grown as previously described in TYI-S-33 Keiser's medium (KM) with some modifications. TYI-S-33 was adjusted at pH 6.0, supplemented with 10% heat-inactivated fetal calf serum (Paget et al., 2004) and 0.6 mg/ml bovine bile (Carnaby et al., 1994). They were regularly subcultured at a density of 5×104 cells per tube (12 ml) from log phase parasites chilled on ice for 10 min and centrifuged at 700×g, 5 min.
Culture of Lactobacillus johnsonii and Production of Bacterial Supernatant
L. johnsonii La1 (LjLa1), CNRZ 1897, NCC533, was kindly provided by Pascal Quénée (INRA Jouy en Josas, Equipe Atalis) and has been isolated from LC1 product in 1996 (Chambourcy, France). LjLa1 stock cultures were kept frozen in MRS Broth media with 15% glycerol. Bacteria were subcultured in MRS Broth medium (Sigma) or modified TYI-S-33 medium (MTYI) (Pérez et al. 2001) and incubated anaerobically for 12-18 h at 37° C. Bacteria were subsequently grown in MTYI or KM medium supplemented or not with 10% heat-inactivated fetal calf serum for 12-16 h in the presence or absence of 0.6 mg/ml bovine bile. After centrifugation (3,000×g, 10 min) and 0.2 μm filtration, the pH of the bacterial supernatant was adjusted to 6.0, 6.2, 6.7, 6.9 or 7.2 with 5 N NaOH. Appropriated controls were prepared as follows: lactic acid produced during growth was quantified from aliquots of supernatants (Enzytec™ kit, R-Biopharm) and equivalent amount of lactic acid was added to fresh medium before pH adjustment.
In Vitro G. lamblia Growth Inhibition Assay
One milliliter of trophozoites suspension (1×105 parasites/ml in KM supplemented with 10% fetal calf serum but without bovine bile) was mixed with either 500 μl of bacterial supernatant or bile salt hydrolase from C perfringens (BSH, 0.2, 1 or 2 U) or appropriate controls in KM or MTYI, in the presence of different concentrations of either bovine bile (0 to 0.6 mg/ml), mixed bile salts (0 to 0.2 mg/ml), conjugated bile salts (0 to 0.2 mg/ml) or deconjugated bile salts (0 to 0.2 mg/ml). Samples were incubated for 24 h at 37° C. and then chilled on ice for 10 min to dislodge trophozoites from tube wall. Living trophozoites (parasites with pear shape showing signs of flagella mobility) were counted using Malassez cell chamber and/or using an hemocytometer. Multiplication factor (i.e. number of total trophozoites at the end of the experiment/number of trophozoites at time zero), survival rate (i.e. (number of living cells/total number of trophozoites)×100) and inhibition percentage (i.e., 100−(number of living cells in the presence of tested compounds/number of living cells in control)×100) were calculated.
Partial Purification of Active Fractions from Bacterial Supernatants by Gel Filtration
Supernatants from LjLa1 cultures in MTYI or KM, adjusted to pH 6.0, were concentrated up to 30-fold by ultrafiltration using 10 kDa Centriprep centrifugal filter unit (Millipore). After 0.2 μm filtration, the concentrated supernatants were placed on a Sephacryl 5300 column 16/100 (GE Healthcare) previously equilibrated with 20 mM ammonium sulfate, pH 6.0, in a cold room and were eluted with the same buffer at a flow rate of 1.8 to 2.0 ml/min. Twelve effluent fractions of 12 ml were obtained, concentrated 4-fold by ultrafiltration on a 10 kDa Centriprep as described above, and tested for Giardia inhibition by classical in vitro growth inhibition assays in the presence of bovine bile or bile salts. Fractions obtained by similar processing of elution buffer alone and control media containing lactic acid (see above) were used as controls. Column calibration was carried out with ribonuclease A (13,700 Da) and bovine serum albumin (67,000 Da).
Molecular size of active molecule(s) present in the bacterial supernatant was assessed by ultrafiltration using 10 kDa, 30 kDa and 50 kDa Centriprep centrifugal filter units. Thermal stability was tested by heating bacterial supernatant at 90° C. for 10 minutes. Preservation of the LjLa1 supernatant activity upon dialysis was checked by dialyzing twice (for 2 h and 15 h at 4° C., respectively) the supernatant against 100 volumes of KM medium supplemented with 10% FCS or against GKN solution (Perez et al., 2001) (NaCl, 8 g/l; KCl, 0.4 g/l; glucose, 2 g/l; NaH2PO4, H2O, 0.69 g/l; Na2HPO4, 1.57 g/l; pH 7.2 to 7.4) using a MWCO 3.5 kDa Spectra-Por dialysis membrane. The dialyzed supernatant was then sterilized by filtration through 0.2 μm membrane and kept frozen at −80° C. before G. lamblia inhibition assays.
Finally, biochemical nature of active molecules was determined by preincubating 5-fold concentrated bacterial supernatant obtained by ultrafiltration (>10 kDa) with different proteases coupled to beads. Briefly, proteinase K (Invitrogen) and pronase (Merck) were coupled to CNBr-activated Sepharose™ 4B (GE Healthcare) following manufacturer's instructions. Five milliliter of 5-fold concentrated bacterial supernatant or 5-fold concentrated fresh control medium were incubated for 6 h at room temperature in presence of 100 μl of packed beads previously coupled with 1 mg of each protease. Before growth inhibition assays, beads were removed by centrifugation (4,000×g 5 min). To assess proteases ability to digest proteins from bacterial supernatant, protein content before and after incubation with proteases, was estimated by SDS-PAGE after trichloroacetic acid (TCA) precipitation.
To assess the presence of free fatty acids in the complex medium inducing Giardia growth inhibition, FCS, bile and LjLa1 supernatant (LjLa1sn) were analyzed for non-esterified fatty acids (NEFAs) content either alone (FCS, LjLa1sn, bile) or in combination (FCS-bile, FCS-LjLa1sn, bile-LjLa1sn, FCS-bile-LjLa1sn). Concentration of each component was that in the inhibition assay. Samples (0.5 ml) were prepared and kept on ice before being incubated for 24 h at 37° C. in the presence of 4.8×104 trophozoites or without cells. At the end of the incubation period, tubes were chilled on ice, centrifuged at 700×g, 10 min at room temperature, then the supernatant was taken and frozen at −80° C. before NEFAs measurement. Numbers of living and dead trophozoites were determined using a Malassez cell chamber. NEFAs were quantified by using the NEFA-C kit, following manufacturer's instructions. Oleic acid was used as standard and NEFAs were expressed as oleic acid equivalents (Eq).
After gel filtration, the eluted fractions were concentrated 10-fold by dialysis against 20 mM ammonium acetate buffer containing 2 M sucrose, pH 6.0, using a 3.5 kDa MWCO membrane (Spectrum Laboratories). Glycodeoxycholate (GDC) was used to perform growth inhibition assays and enzymatic assays. BSH activity was monitored by measuring glycine liberation from conjugated bile salt, following the protocol described by Grill et al., (2000). Briefly 100 μl of effluent fractions or BSH 1 unit or elution buffer were mixed with 100 μl of 2.4 g/l of GDC and incubated 24 h at 37° C. Controls were performed in the absence of bile salt or by pre-incubating effluent fractions with 2 mM iodoacetic acid or 30 min at 37° C. To stop the enzymatic reaction, an equal volume of 15% TCA (200 μl) was added and proteins were precipitated by centrifugation at 20,000 g for 15 minutes. 680 μl of 0.3M borate buffer, 1% SDS, pH 9.5 and 80 μl of 0.3% picrylsulfonic acid solution (Sigma) were added to 80 μl of supernatants. Mixtures were incubated for 30 min in the dark and 800 μl of 1 mM HCl were added to stop the reaction. Glycine concentration was measured at 416 nm using an Uvikon spectrophotometer 930 (Kontron Instruments). Standard curve was established with free glycine.
125 μl of bile-containing culture media of G. lamblia (KM, pH 6.0, with 2 g/L bovine bile) and 125 μl of LjLa1 supernatants prepared from a bacterial culture in KM medium supplemented with 10% fetal bovine serum, adjusted at pH 6.0 were mixed and incubated overnight at 37° C. Two different bile batches (B1 and B2) and two different bacterial supernatants (S1 and S2) were tested. The samples were diluted 4-fold in Milli-Q water and subjected to solid-phase extraction (SPE) using Oasis® HLB cartridges (30 mg solid phase). After conditioning with 3 ml methanol and 3 ml Milli-Q water, the cartridges were loaded with 1 ml of 4-fold diluted sample, washed with 2 ml Milli-Q water and eluted with 2 ml methanol. The eluted fractions were dried under vacuum and resuspended in 500 μl Milli-Q water/acetonitrile 90:10 (v/v). Five μl of each resuspended sample was analyzed by LC/ESI-MS on a Ultimate U3000 chomatographic system (Thermo) connected to a Q-STAR Pulsar Qq-TOF mass spectrometer equipped with an ionspray source (AB Sciex). The LC separation was achieved on a Interchrom Strategy C18-2 micro column (5 μm, 150×1 mm, 100 Å, Interchim). The elution gradient was 10% mobile phase B (acetonitrile) to 70% B against mobile phase A (5 mM ammonium formate/formic acid, pH 6) over 45 min, at a flow rate of 40 μl/min. The MS data were acquired in negative mode, in the range m/z 250-1200. Each LC/ESI-MS experiment was conducted twice. Data-dependant LC/ESI-MS/MS experiments were also conducted on each sample, alternating 1-second full-scan MS followed by two 2-second product ion collision induced dissociation (CID) of the major ions detected at the first step, using a −50 V collision voltage. Each raw LC/ESI-MS data was converted into Network Commun Data Form (NetCDF) using the translation tool provided by AB Sciex. The data were processed with XCMS (Smith et al. 2006), a software implemented in the freely available R environment (www.r-project.org), which allows automatic retention time alignment, matched filtration, peak detection and peak matching.
0.1% of sodium lauroyl sarcosinate (NLS, Sigma) was added to bacterial supernatants. After mixing, TCA (trichloroacetic acid) was added to a final 7.5% concentration, and the solution was precipitated on ice overnight. The mixed protein-detergent precipitate was collected by centrifugation (10,000×g, 10 min, 4° C.). The supernatant was carefully removed and the pellet washed twice with 2 ml of precooled tetrahydrofuran (Sigma). Finally, the pellet was dissolved in 0.4 ml extraction solution (7 M Urea, 2 M Thiourea, 4% CHAPS, 5 mM Tris(carboxyethyl)phosphine) (Rabilloud et al., 2009).
TCA precipitated LjLa1 supernatant was resuspended in 8 M urea in 20 mM TEAB (triethylammonium bicarbonate) and incubated for 1 h at room temperature (RT) with 20 mM DTT (Dithiothreitol), then with 50 mM IAA (iodacetamide) and incubated for a further hour at RT in the dark. The sample was incubated with 0.05 UA of endoproteinase Lys-C (Wako Pure Chemical Industries, Osaka, Japan) for 18 h at RT. Trypsin (Promega) digestion was performed with 2 μg of enzyme during 4 h at RT and terminated with TFA (trifluoroacetic acid), final concentration of 0.5%. The sample was passed sequentially through two home-made Poros Oligo-R3 (PerSeptive Biosystems, Framingham, USA) microcolumns packed (±1 cm) on p200 tips over 3 MM C18 material plug. Loaded resin was washed with 100 μl 0.1% TFA and peptides were eluted with 100 μl 70% acetonitrile (ACN)/0.1% TFA, then 20 μl 100% ACN. The sample was desalted, dried down, resuspended in 50% ACN and 10% was collected to amino acid analysis using a Biochrom 30 amino acid analyzer (Biochrom, Cambridge, U.K.), then dried and stored at −80° C. again until analysis.
Samples (3 μg per run) were analyzed by an EASY-nano LC system (Proxeon Biosystems, Odense, Denmark) coupled online to an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Waltham, USA). Peptides were loaded onto a 18 cm fused silica emitter 75 μl in inner diameter) packed in-house with reverse phase capillary column ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH, Germany) and eluted using a gradient from 100% phase A (0.1% formic acid) to 35% phase B (0.1% formic acid, 95% acetonitrile) for 180 min, 35% to 100% phase B for 5 min and 100% phase B for 8 min (a total of 23 min at 250 nl/min). After each run, the column was washed with 90% phase Band re-equilibrated with phase A. Mass spectra were acquired in positive mode applying data-dependent automatic survey MS scan and tandem mass spectra (MS/MS) acquisition. Each MS scan in the orbitrap (mass range of m/z of 400-1800 and resolution 100,000) was followed by MS/MS of the fifteen most intense ions in the LTQ. Fragmentation in the LTQ was performed by collision-induced dissociation and selected sequenced ions were dynamically excluded for 25 s. Raw data were viewed in Xcalibur v.2.1 (Thermo Scientific, Waltham, USA) and data processing was performed using Proteome Discoverer v.1.3 (Thermo Scientific, Waltham, USA). Generated raw files were submitted to searching using Proteome Discoverer with an in house Mascot v.2.3 algorithm against database of proteins predicted from the L. johnsonii isolates NCC533 and FI9785 and the L. johnsonii prophage Lj965. Contaminant proteins (several types of human keratins, BSA and porcine trypsin) were also added to the database and all contaminant proteins identified were manually removed from the result lists. The searches were performed with the following parameters: ms accuracy 10 ppm, MS/MS accuracy 0.6 Da, trypsin digestion with one missed cleavage allowed, fixed carbamidomethyl modification of cysteine and variable modification of oxidized methionine and N-terminal protein acetylation. Number of proteins, protein groups and number of peptides were filtered for False Discovery Rates (FDR) less than 1% and only peptides with rank 1 and minimal of 2 peptides per proteins were accepted for identification using Proteome Discoverer (Charneau et al., 2007); Queiroz et al. 2013).
Most media previously described to support G. lamblia growth commonly contains bile as a supply for parasite cholesterol and fatty acids requirements (Farthing et al., 1985, Gillin et al., 1986, Halliday et al., 1995).
In our hands, G. lamblia trophozoites growth can be observed in a common medium (KM-FCS) in the absence of bovine bile. Moreover, recommended bile concentrations (0.75 mg/ml, Perez et al., 2001) appeared to affect trophozoites growth when added to the common KM-FCS (
Maximum inhibitory effects of LjLa1 supernatant on parasite growth was observed either in the presence of 0.6 g/L complete bovine bile or in the presence of 0.032 g/L bile salts (96.3% and 75% of inhibition, respectively, by comparison with controls containing similar amount of lactic acid and bile or bile salts,
To biochemically characterize the inhibitory compounds present in the LjLa1 supernatant, this supernatant was treated with immobilized enzymes prior to contact with parasites. Parasite growth inhibition was abolished by proteinase K and pronase treatments of the supernatant, suggesting involvement of inhibitory factor(s) of peptidic nature (
Moreover, as Pridmore et al. (2004) demonstrated that the LjLa1 anti-Salmonella activity is mediated by the toxic hydrogen peroxide molecule H2O2 and can be abolished by a pretreatment with catalase, we checked whether treatment of LjLa1 supernatant with catalase might prevent its anti-Giardia effect. As it can be seen in
To assess whether G. lamblia growth inhibition by LjLa1 supernatant might be due to toxic free fatty acids as demonstrated in previous reports (Rohrer et al. 1986), FCS, bile and LjLa1 supernatant were analyzed for non-esterified fatty acids (NEFAs) content either alone or in combination, using the NEFA-C methodology (WAKO diagnostics). The different samples were incubated for 24 h at 37° C. with or without G. lamblia trophozoites and the cell supernatants were analyzed for NEFAs. Survival and growth of the parasites in those different conditions were determined.
The following Table 1 represents the analysis of non-esterified fatty acids involvement in Giardia inhibition. The different components of the Giardia culture medium (FCS 10% and/or bile 0.5 g/L), as well as LjLa1sn, were analyzed either alone or in combination for NEFA content after 24 h of incubation at 37° C. Incubation was performed with or without G. lamblia trophozoites (9.6×104/ml). Survival and multiplication of G. lamblia after 24 h in those different media were determined. Parasite multiplication was expressed as the number of total trophozoites after 24 h of incubation/number of trophozoites at time zero of incubation (i.e. multiplication factor). Trophozoite survival rate after 24 h of incubation was expressed as the (number of living cells/total number of cells)×100. ±SD (triplicate).
Supernatants with FCS, LjLa1sn or bile alone displayed NEFAs concentrations of 40.9, 36.3 and 4.5 μM, respectively (Table 1).
The amount of NEFAs was doubled (95.4 μM) by co-incubation of the bile with serum suggesting enzymatic release of NEFAs. However, equal amounts were found at time zero and time 24 h of the co-incubation (not shown), indicating that fatty acids were not released in a time-dependent manner. It was hypothesized that the high amount of NEFAs might result from a detergent-like action of the bile on serum lipids, possibly improving accessibility of NEFAs to the NEFA-C kit reagents. The highest amount of NEFAs (140.9 μM) was measured upon co-incubation of FCS with bile and LjLa1sn, as could be expected by summing their respective NEFAs contents. Incubation with G. lamblia had no noticeable effect on the NEFA content of the various samples (Data not shown).
Normal growth of G. lamblia (multiplication factor: 3.34, Table 1) was observed in the presence of 10% FCS alone. Trophozoites did not survive in KM medium containing only bile, whereas most of them (˜70%) were still alive although they did not develop when incubated in KM with LjLa1 supernatant, most probably due to the low concentration of FCS in LjLa1 supernatant. Remarkably, trophozoites survival was abolished upon addition of bile to LjLa1 sn although the same concentration of FCS is present, indicating the presence of inhibitory elements. By comparing NEFAs contents of the different media, it appeared that NEFAs could hardly be responsible for parasite death. Indeed, more NEFAs were found in the KM+FCS+bile or KM+FCS+LjLa1sn media (95.4 and 68.2 μM of NEFAs respectively), which allowed parasite survival, than in the KM+bile+LjLa1sn medium (50.0 μM of NEFAs) which induced the death of all the parasites. This indicated that the concomitant addition of bile and bacterial supernatant rather than high level of NEFAs is lethal to Giardia and is responsible for the killing effect observed in the presence of FCS, bile and LjLa1 supernatant.
Fractionation experiments indicated that the inhibitory activity in LjLa1 supernatant was due to molecule(s) bigger than 10 kDa, since fraction >10 showed a high G. lamblia inhibitory activity compared to the fraction <10 kDa (
It has been reported that molecule(s) smaller than 1 kDa (Perez et al., 2001) would be involved in the giardiacidal activity of the LjLa1 supernatant, in contradiction with our findings of a ≧30 kDa molecule(s). The possibility that dialysis against GKN buffer as used in Perez's report might somehow inhibit the activity has been checked. Several points were checked. At first, because the pH of the GKN buffer is 7.4 and the LjLa1sn inhibitory activity is inactivated at pH above 7.0 (
This indicates on one side that no element crucial to activity is lost upon dialysis and on the other side that the GKN buffer is not inhibitory to the LjLa1 supernatant activity. Also, it can be noticed from Table 2 that addition of 25% GKN buffer to KM-FCS does not affect parasite survival but slows down its development (multiplication rate of 2.60 in the presence of GKN versus 3.82 in the absence of GKN).
Since concomitant addition of bile and LjLa1sn to the culture medium leads to inhibition of G. lamblia growth, we assessed whether bile composition might be modified by LjLa1 supernatant. Bile composition after 24 h of incubation with LjLa1sn was investigated by mass spectrometry (
The inhibitory effects toward G. lamblia growth of pure bile salts (cholate, desoxycholate and chenodesoxycholate) conjugated to glycine or taurine, or their deconjugated counterparts were investigated in the presence or the absence of LjLa1 supernatant (Table 3,
The following table 3 shows conjugated bile salts in association with LjLa1 supernatant (LjLa1sn) as well as deconjugated bile salts prevent the growth of G. lamblia. Various concentrations of taurine- and glycine-conjugated and unconjugated C, DC and CDC bile salts were tested for G. lamblia trophozoites growth inhibition in KM-FCS in the presence or absence of LjLa1sn (bacterial spent culture medium). IC50 values were determined from drug-response curves and expressed as mean+/−SD of at least three independent experiments.
These results suggested that a deconjugating process mediated by LjLa1sn component(s) and producing deconjugated bile salts might be responsible for the inhibitory effect of the association of bile and LjLa1sn. Such hypothesis is in line with the previous observation that fusidic acid, an antibiotic with a bile salt-like chemical structure is toxic to Giardia (see Table 3, IC50 value=26 μM) unless conjugated to taurine or glycine (Farthing et al., 1986).
It is known that bile salt deconjugating process is mediated by 3-alpha, 7-alpha, 12-alpha-trihydroxy-5-beta-cholan-24-oylglycine/taurine amidohydrolases (EC 3.5.1.24), also named choloylglycine/taurine hydrolases or conjugated bile acid hydrolases (CBAH) or bile salt hydrolases (BSH). These enzymes act on carbon-nitrogen bonds other than peptide bonds, specifically in linear amides, releasing glycine and taurine from conjugated bile salts. In L. johnsonii genome, three genes encoding BSH-like enzymes have been predicted (LJ0056 (BSH-56), LJ1147 (BSH-47) and LJ1412 (BSH-12) genes). They are also predicted as secreted proteins by SecretomeP, with theoretical full sizes of 34.9 kDa, 36.3 kDa and 36.6 kDa and secreted sizes of 29.8 kDa, 31.0 kDa and 31.3 kDa (data not shown).
To test the hypothesis of bacterial BSH(s) involvement in G. lamblia growth inhibitory activity by LjLa1sn, the bacterial supernatant was fractionated by gel filtration chromatography on Sephacryl S300 column 16/100, and eluted fractions were assayed for parasite inhibition and bile salt deconjugating activity.
A typical elution profile is shown in
To assess capability of BSH enzymes to promote bile-mediated anti-Giardia effect, BSH from the bacteria C. perfringens was tested for G. lamblia growth inhibition in the presence of bile or pure conjugated bile salts. As noted above (Table 3), the glycine or taurine conjugated bile salts, TDC, TCDC, GDC and GCDC have no inhibitory activity on G. lamblia growth in KM-FCS culture medium. In contrast, the addition of 2 units of C. perfringens BSH to the culture in the presence of those bile salts led to a remarkable parasite growth inhibition within the 24 h of the assay, with inhibition ranges of 95 to 100%, depending upon the conjugated bile salt tested (
It was then searched whether BSH-like enzymes annotated from L. johnsonii La1 genome, and predicted by SecretomeP to be secreted, are indeed released in the extracellular medium. High-resolution mass spectrometry-based proteomic analysis of the LjLa1 extracellular proteins was performed. 3 μg of proteins of LjLa1 supernatants from two independent culture replicates allowed identification of over a hundred of secreted protein groups (data not shown). Amongst them, two of the three predicted BSH were clearly identified in both replicates: LJ1412 (gi|41583570) and LJ1147 (gi|41583360). The following Table 4 shows the identification of two conjugated bile salt hydrolases of Lactobacillus johnsonii NCC533 by MS/MS peptide fragmentation using MASCOT stringent search.
aAccession number in the NCBI protein database. All accession numbers refer to sequences from Lactobacillus johnsonii NCC 533;
bProbability-based Mowse score of MASCOT software that evaluates if the peptides subjected to search are the same as those found in the database originated by in silico digestion of a known protein;
cCoverage is the percentage of predicted protein sequence covered by matched peptides via MASCOT;
dPeptide sequences identified via MASCOT following the experimental peptide masses after parental ion fragmentation. It was required that at least one spectrum should be matched with score of 50 or better to considered the hit.
eDoubly- to quintuply-charged ions of selected peptides were analyzed.
fTheoretical molecular masses of proteins calculated from amino acid sequences;
gTheoretical isoelectric points of proteins calculated from amino acid sequences;
The inhibitory effect of L. johnsonnii on G. lamblia was measured by flow cytometry, as shown in
The inhibitory effect on G. lamblia growth of different lactobacilli strains was screened (cf. table 5). Bacterial supernatant was co-incubated for 24 h at 37° C. with Giardia trophozoite cultures (triplicates), with or without bovine bile (0.6 g/L). Different species of lactobacilli were identified as potent inhibitors of Giardia lamblia growth, as Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus reuteri. 14 lactobacilli strains showed significant inhibitory effects (ANOVA statistic tests) in presence of bile (
L. johnsonii
L. gasseri
L. johnsonii
L. johnsonii
L. johnsonii
L. johnsonii
L. johnsonii
L. johnsonii
L. johnsonii
L. johnsonii
L. johnsonii La1
L. gasseri
L. gasseri
L. acidophilus N
To study the properties of the LjLa1 BSHs, we cloned the BSH genes into a pStaby vector (StabyExpress, Delphigenetics) in order to purify BSHs using His-Tag system. E. coli expressions cells (E. coli SE, Delphigenetics) were then transformed The recombinant BSH-47 protein was produced in E. coli SE cells and purified using a His-Tag system (Sephadex Ni-NTA column, GE Healthcare). The activity of recombinant BSH-47 was tested using taurodeoxycholic (0.3%) and glycodeoxycholic (0.3%) LB agar (Chae et al 2013). After 48 h of incubation at 37° C., we observed that the recombinant BSH-47 is taurospecific (
Furthermore, the effect of recombinant LjLa1 BSH-47 has been studied on Giardia trophozoïtes cultures, in the presence or not of bovine bile (0.06 g/L). The results show that recombinant BSH-47 (0.96 U, with “U” designing enzymatic units) exhibits an inhibiting effect, the level of this inhibiting effect is equivalent to the inhibiting effect of C. perfringens BSH (1 U) (
The effects of recombinant LjLa1 BSH-47 and BSH-56 were studied on cultures of G. lamblia trophozoites in either presence or absence of bovine bile (0.06 g/L). Recombinant BSH-56 was produced according to the process described for the production of BSH-47, such as described in Example 13. Several BSH concentration were tested ranging from 0.0001 μg/ml to 10 μg/ml. Interestingly, both BSH-47 (
Previous experiments allowed us to determine the substrate specificity of newly purified BSH-47 and BSH-56 and their specific activities had been determined. BSH-47 is able to deconjugate taurospecific bile salts while BSH-56 is able to deconjugate both taurospecific and glycospecific bile salts.
C. perfringens
L. johnsonii La1 and L. gasseri CNCM I-4884 strains (L. gasseri CNCM I-4884 is also designated as L. gasseri ATCC29601 in
Solutions of recombinant BSH-47 solutions (1 μg/ml, 10 μg/ml and 100 μg/ml diluted in NaHCO3 16.4%) were thawed and daily administered by intragastric gavage to neonatal mice (dose were respectively 0.05 m, 0.5 μg and 5 μg per mice) from day 10 to day 15 (day of sacrifice). Control animals received PBS instead of BSH. All groups treated with BSH-47 showed a reduction in trophozoites load in the small intestine compared with the control (
| Number | Date | Country | Kind |
|---|---|---|---|
| 14306265.1 | Aug 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/068334 | 8/10/2015 | WO | 00 |
