Patent Application
20160120963
The invention relates to methods of regulating weight gain, serum cholesterol levels, and liver triglycerides in a mammal. In particular, the invention relates to a method of treatment of a disease or condition in a mammal that is associated with weight gain, serum cholesterol levels, and/or liver triglycerides in a non-obese mammal, for example obesity or hypercholesteremia.
The gastrointestinal microbiota exerts a major influence on host energy metabolism and adiposity however the precise microbial activities that influence lipid metabolism in the host remain largely unexplored. Large scale sequencing studies have catalogued the genetic composition of the human gut microbiota (the microbiome), aiding our understanding of core microbial genes whose products are predicted to influence host metabolism. However studies elucidating the influence of individual bacterial gene sets on systemic metabolic processes in the host are lacking There is currently a significant need for functional categorization of both gut-specific and gut-enriched microbial activities in order to determine the relevance of specific gene sets in a physiological or pathological context.
Bile acids are the main functional components of bile secretions that play a role in the emulsification of dietary lipids and also act as signalling molecules in the host, triggering cellular farnesoid X receptor (FXR)- and G-protein coupled receptor (TGR5)-mediated host responses. Bile acids influence the composition of the gastrointestinal microbiota and in turn are chemically modified by bacterial enzymes in the gut. Many consider bile acids as mediators of a reciprocal microbe-host crosstalk with the ability to influence host metabolic pathways and the potential to influence microbial community structure. Bile acids are synthesized in hepatocytes as cholesterol moieties conjugated to either a taurine or a glycine amino acid and are stored in the gallbladder prior to secretion into the duodenum via the common bile duct. Bacterial enzymes in the gut significantly modify bile acids, a process which in turn influences host bile acid synthesis through a feedback mechanism in which the hepatic enzymes involved in bile acid synthesis (including Cyp7A1 and Cyp27A1) are regulated.
In particular, bacterial bile salt hydrolase (BSH) enzymes in the gut catalyse an essential gateway reaction in the metabolism of bile acids. BSH enzymes cleave the amino acid side-chain of glyco- or tauro-conjugated bile acids to generate unconjugated bile acids (cholic and chenodeoxycholic acids), which are then amenable to further bacterial modification to yield secondary bile acids (deoxycholic and lithocholic acid). It has previously been shown that functional BSH activity is a conserved microbial adaptation that is unique to the gut associated microbiota and is distributed across the major bacterial divisions, as well as archaeal species in the GI tract. It has previously been demonstrated that BSH contributes to bile tolerance in gut bacteria and hypothesized that the evolution of BSH activity is governed by host-driven selection.
The Applicant has discovered that expression of certain cloned bacterial BSH enzymes in the mammalian GI tract significantly modifies plasma bile acid profiles in gnotobiotic mice and influences both local and systemic gene expression profiles in pathways governing lipid metabolism, metabolic signalling events, circadian rhythm and immune function (
Thus, in a preferred aspect, the invention provides a non-therapeutic method of reducing weight gain, serum cholesterol levels, or liver triglyceride levels, in a non-obese mammal, comprising the step of administering to the gut of a mammal an active agent comprising a bacteria that expresses BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof having at least 90% sequence identity with SEQUENCE ID NO: 1. Examples of bacteria that expresses BSH1 enzymes having at least 90% sequence identity with SEQUENCE ID NO: 1 are provided in Table 1 below.
In a another aspect, the invention relates to a method of reducing one, more or all of weight gain, serum cholesterol levels, and liver triglyceride levels, or modulating circadian rhythms, in a mammal, comprising the step of administering to the gut of a mammal an effective amount of a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof (hereafter “active of the invention”).
The active of the invention may be administered in the form of an enzyme, typically in a suitable formulation, for example a liposome or microcapsule formulation designed to release the active in the gut of the mammal. Such liposome or microcapsule formulations will be known to those skilled in the art, and are described in more detail below.
In another aspect, the invention relates to a method of reducing one, more or all of weight gain, serum cholesterol levels, and liver triglyceride levels, or regulating circadian rhythms, in a mammal, comprising the step of administering to the mammal an effective amount of bacteria, preferably a probiotic bacteria, that expresses a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof.
Thus, in an alternative embodiment, the active may be administered by administration to the gut of the mammal of a bacteria that expresses the active of the invention. The bacteria may be a bacteria that naturally expresses the active of the invention—an example of such a bacteria is Lactobacillus salivarius JCM1046 (Korean Collection of Type Cultures, KCTC 3156 http://www.straininfo.net/strains/171296) Alternatively, the bacteria may be genetically modified to express, ideally stably express, the active of the invention—an example of such a bacteria is the commensal Escherichia coli strain MG1655, which is genetically modified to express the BSH1 gene of SEQUENCE ID NO: 1. Typically, the bacteria is genetically modified using the mini-Tn7 transposon system. Suitably, the gene encoding the active of the invention is integrated into the host genome downstream of the glmS gene.
Preferably the bacteria is a bacteria that exhibits elevated expression of the active of the agent.
Suitably, the bacteria is a probiotic bacteria. Preferably, the bacteria is selected from the group consisting of APC1484 to APC1502.
In a third aspect, the invention relates to a bacteria, preferably a probiotic bacteria, that is genetically engineered to express a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof.
The invention also provides a recombinant vector comprising a nucleic acid encoding a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, optionally under the control of a constitutive promotor. Details of constitutive promotors will be well known to those skilled in the art.
The invention also relates to a host cell transformed by a recombinant vector of the invention (hereafter “host cell of the invention”).
The invention also relates to a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use as a medicament.
The invention also relates to a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use as an antibacterial agent or an antibiotic.
The invention also relates to a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing a disease or condition characterised by weight gain, elevated cholesterol levels, elevated liver triglyceride levels. Examples of such diseases include obesity, hypercholesterolemia, cardiovascular disease and metabolic disease.
The invention also relates to a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing a disease or condition characterised by disregulated circadian rhythm, for example sleep apnoea.
The invention also relates to a bacteria that expresses BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing a disease or condition characterised by disregulated circadian rhythm, for example sleep apnoea. The bacteria may be genetically modified to express the active of the invention. Preferably, the bacteria is a probiotic bacteria. Ideally, the bacteria exhibits elevated expression of the active of the invention.
The invention also relates to a pharmaceutical composition comprising BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, in combination with a suitable pharmaceutical excipient.
The invention also relates to a pharmaceutical composition comprising a bacteria that expresses, ideally exhibits elevated expression, of a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, in combination with a suitable pharmaceutical excipient. Preferably the bacteria is a probiotic bacteria.
The invention also relates to a formulation comprising a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, or a bacteria that expresses, ideally exhibits elevated expression, of a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof. Suitably, the formulation is a pharmaceutical formulation and additionally comprises a pharmaceutically acceptable carrier. Alternatively, the formulation may be a comestible product, for example a food product. Ideally, the food product is a fermented food, for example a fermented dairy product such as a yoghurt. The formulation may also be a hygiene product, for example an antibacterial formulation, or a fermentation product such as a fermentation broth. For formulations that comprise the BSH1 enzyme or variant thereof, it will be appreciated that the enzyme may be directly added to the formulation, or it may be produced in-situ in the formulation by a bacteria.
The invention also relates to BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing a metabolic disease or metabolic syndrome.
The invention also relates to BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing vascular dementia or multi-infarct dementia.
The invention also relates to BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing hypertension.
The invention also relates to BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing a disease or condition associated with local gastrointestinal inflammatory disease such as Crohn's disease and ulcerative colitis.
The invention also relates to BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing gastrointestinal cancer.
The invention also relates to BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing irritable bowel syndrome (IBS).
The invention also relates to BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, for use in treating or preventing diarrhoea associated with dysregulated microbiota.
The invention also relates to an isolated bacteria selected from the group consisting of:
a strain of Lactobacillus johnsonii, comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 8, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 7;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 4, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 3;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 6, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 5;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 10, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 9;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 12, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 11;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 14, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 13;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 16, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 15;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 18, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 17;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 20, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 19;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 22, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 21;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 24, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 23;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 26, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 25;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 28, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 27;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 30, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 29;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 32, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 31;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 34, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 33;
a strain of Lactobacillus salivarius comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 36, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 35;
a strain of Staphylococcus epidermidis, comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 38, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 37; and
a strain of Streptococcus salivarius, comprising a 16S ribosomal RNA sequence of SEQUENCE ID NO: 40, and expressing a BSH1 enzyme having a sequence of SEQUENCE ID NO: 39.
Typically, the Lactobacillus strains are isolated from pigs, typically pig faeces. Suitably, the Streptococcus and Staphylococcus strains are isolated from human faeces, preferably infant human faeces.
The bacteria employed in the methods of the invention are typically selected from the isolated bacteria of the invention.
SEQUENCE ID NO: 1 and 2 are the amino acid, and nucleic acid, sequences, respectively, of BSH1 enzyme from Lactobacillus salivarius JCM1046
Lactobacillus salivarius JCM1046 was obtained from the Korean Collection of Type Cultures, KCTC 3156 (open repository).
The term “functional variant thereof” should be understood to mean a bacterial BSH enzyme having at least 60% sequence identity with SEQUENCE ID NO: 1, and which is capable of displaying an ability to significantly decongugate bile acids in vitro as determined by the chemical analysis assays described below (ninhydrin assay and UPLC-MS analysis). Non functional variants lack the ability to significantly deconjugate bile acids in these analyses. In a preferred embodiment, the functional variant is capable of altering expression of loci associated with immune function, cholesterol transport, and lipid transport and synthesis, relative to the E. coli control, when expressed in the ileum of a mouse according to the methods described below. Suitably, the functional variant is capable of altering (increasing) expression of the gene encoding the hormone adipopnectin, the gene encoding the Angiopoietin-4, and preferably both, relative to the E. coli control, when expressed in the liver of a mouse according to the methods described below. Preferably, the functional variant is capable of regulating major metabolic pathways involved in triglyceride biosynthesis, bile synthesis, and fatty acid transport and synthesis, relative to the E. coli control, when expressed in the liver of a mouse according to the methods described below.
Preferably, the functional variant of the BSH1 enzyme of SEQUENCE ID NO: 1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO: 1. Thus, for example, the term should be taken to include enzymes that are altered in respect of one or more amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared with the BSH1 enzyme of SEQUENCE ID NO: 1. Preferably such alterations involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged. The variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted. Typically, the functional variant is an ortholog or paralog of BSH1 of SEQUENCE ID NO: 1. The term sequence identity comprises both sequence identity and similarity, i.e. a polypeptide sequence that shares 90% amino acid identity with SEQ ID NO: 1 is one in which any 90% of aligned residues are either identical to, or conservative substitutions of, the corresponding residues in SEQ ID NO: 1. The term “variant” is also intended to include chemical derivatives of the BSH1 enzyme of SEQUENCE ID NO: 1, i.e. where one or more residues of is chemically derivatized by reaction of a functional side group. Also included within the term variant are functional variant molecules in which naturally occurring amino acid residues are replaced with amino acid analogues. Details of amino acid analogues will be well known to those skilled in the art.
Examples of bacteria that express functional variants of BSH1 of SEQUENCE ID NO: 1 are Strains APC1484 to APC1502 described in Tables 1, 2 and 3 below. All of the strains are available within the Alimentary Pharmabiotic Centre (APC) culture collection, University College Cork, Cork, Ireland (http://www.ucc.ie/research/apc/content/)
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
johnsonii
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus
Lactobacillus
salivarius
salivarius:
Lactobacillus salivarius
Lactobacillus salivarius
Staphylococcus
epidermidis
Streptococcus
salivarius partial
The BSH1 gene sequences and 16s rRNA sequences for the strains referenced in Tables 1-3 are provided in the Appendix below.
Proteins (including variants thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. The proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984)).
In this specification, the term “elevated expression” as applied to the level of expression of the active of the invention in a bacterial host should be understood to mean an expression level that is greater than the expression level of BSH1 in the genetically modified Escherichia coli strain MG1655 (ECBSH1).
In this specification, the term “probiotic” as applied to a bacteria should be understood to mean a live microorganism that confers a health benefit on the host.
In this specification, the term “obesity” should be understood to mean a body mass index of greater than 30 kg/m2.
In this specification, the term “hypercholesteremia” should be understood to mean total cholesterol of greater than 5 mmol/L, and low-density lipoprotein cholesterol (LDL) of greater than 3 mmol/L. For people at high risk of cardiovascular disease, the recommendation for total cholesterol is 4 mmol/L or less, and 2 mmol/L or less for LDL.
In this specification, the term “metabolic disorder” should be understood to mean a disease or condition that disrupts normal metabolism in a mammal. Examples include: pre-diabetes, diabetes; Type-1 diabetes; Type-2 diabetes; metabolic syndrome; obesity; diabetic dyslipidemia; hyperlipidemia; hypertension; hypertriglyceridemia; hyperfattyacidemia; hypercholerterolemia; MODY; HNF1A-MODY; and hyperinsulinemia. Preferably, the metabolic disorder is selected from MODY; HNF1A-MODY; pre-diabetes, and diabetes (including Type-1 diabetes or Type-2 diabetes).
The invention also relates to a recombinant vector comprising a nucleic acid encoding a BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, optionally under the control of a constitutive promotor. Typically, the nucleic acid is cloned into a recombinant vector (for example a plasmid) which is capable of replicating in the host bacteria. Typical plasmids contain, in addition to the cloned insert, a selection gene (i.e. antibiotic resistance, a dye etc) and an origin of replication effective in the host bacterium. The plasmid may also comprise regulatory sequences, for example promoters, terminators and/or enhancers. Examples of such vectors include pBKminiTn7GM2 (Koch, B., Jensen, L. E., and Nybroe, O. (2001). A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site. J Microbiol Methods 45, 187-195) or pNZ44 (McGrath, S., Fitzgerald, G. F., and van Sinderen, D. (2001). Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis. Appl Environ Microbiol 67, 608-616.)
The nucleic acid may also be cloned into an integrative cassette suitable for integration into the genome of suitable host bacteria. Such an integrative cassette typically comprises a nucleic acid encoding the BSH1 enzyme of SEQUENCE ID NO: 1, or a functional variant thereof, linked to (or flanked by) one or several sequences allowing integration, preferably site-specific integration. Such sequences may be for instance nucleic acid sequences homologous to a targeted region of the genome, allowing integration through crossing over. Various techniques can be used to insert a nucleic acid into a host bacteria, for example through natural transformation or electroporation.
The host bacteria suitable for cloning the active of the invention may be selected from any host bacteria known to a person skilled in the art such as, for example, Bifidobactrium (B. adolescentis, B. animalis, B. breve, B. infantis, B. longum, B. sp), Lactobacillus (L, acidophilus, L. casei, L. feermentus, L. gasseri). Preferably, the host bacteria is a probiotic bacteria.
In the specification, the term “mammal” or “individual” as employed herein should be taken to mean a human; however it should also include higher mammals for which the method, prophylaxis, therapy or use of the invention is practicable, for example, pigs. The term “animal” should be understood to include any animal including humans.
In this specification, the term “administering” should be taken to include any form of delivery that is capable of delivering the enzyme or bacteria, including local delivery, intravenous delivery, oral delivery, intranasal delivery, intramuscular delivery, intrathecal delivery, transdermal delivery, inhaled delivery and topical delivery. Methods for achieving these means of delivery will be well known to those skilled in the art of drug delivery.
In this specification, the term “pharmaceutical composition” should be taken to mean compositions comprising a therapeutically effective amount of the active of the invention, that in one embodiment are produced in-situ in the composition by a bacterial strain, and a pharmaceutically acceptable carrier or diluent. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the bacterial strain and/or active of the invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
“Effective amount” refers to the amount or dose of the active of the invention upon single or multiple dose administration to the patient, which provides the desired effect in the patient under treatment. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of enzyme or bacterial strain expressing the enzyme administered, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
The term “comestible product” should be understood to include products that are intended to be consumed by ingestion by humans or animals, such as foods and drinks. In particular, the comestible product is a food or drink product intended for consumption by humans, for example a fermented product or a diary product, especially a fermented dairy product such as a yoghurt.
E. coli colonization in the gastrointestinal tract of conventional C57Bl/6J mice administered streptomycin ad libitum. E. coli was enumerated by standard plate counts from faeces on the days indicated.
Bile salt hydrolases from Lactobacillus salivarius strains (Fang et al., 2009) were cloned independently into pBKminiTn7GM2 (Koch et al., 2001) under the control of the P44 promoter (McGrath et al., 2001) using splicing by overlap extension (SOE) PCR. Transposon integration was carried out as described previously (Koch et al., 2001). PCR downstream from the glmS region confirmed constructions as did sequence analysis (GATC Biotech).
EC, ECBSH1 and ECBSH2 were examined for their ability to deconjugate bile in vitro using the ninhydrin assay for free taurine (Lipscomb et al., 2006) and by co-incubation for 90 minutes in murine gall bladder BA followed by UPLC MS analysis. Protein concentrations were measured with the Biorad Protein Assay (Biorad, Hercules, Calif.), and bovine serum albumen (BSA) (Sigma) was used as standard.
Germ free Swiss Webster mice were maintained in the germ-free unit in the Alimentary Pharmabiotic Centre. Monocolonisation experiments were initiated by oral dosing of appropriate strains at 1×109 CFU per mouse. Monocolonised mice were housed in relevant groups in individual germ free isolators for the duration of the experiment. For analysis of conventional mice C57Bl/6J male mice were purchased from Harlan (Oxon, UK) and housed under barrier maintained conditions at University College Cork. 6 week old male C57Bl/6J mice were fasted for 24 hours and immediately supplied with Streptomycin treated drinking water (5 mg ml−1 final concentration) for the duration of the experiment. After 24 hours mice were fed either a low fat diet ((n=20) 10% calories from fat Research Diets International, New Jersey, USA D12450B) or a high fat diet ((n=20) 45% calories from fat Research Diets International, New Jersey, USA D12451) for 10 weeks. These two groups were further divided into parallel groups (n=5 for each group) and were inoculated with relevant strains in PBS at 1×106 CFU per mouse by oral gavage (inoculations on two consecutive days). Body weight and food intake was assessed weekly. Faecal samples were taken from each individual on a weekly basis and used for bacterial enumeration. At the end of the study mice were sacrificed and internal organs (liver, spleen, intestine) and fat pads (reproductive, renal, mesenteric and inguinal) were removed, weighed and stored at −80° C. The experiments outlined were approved by the University Animal Experimentation Ethics Committee.
Mice were fasted for 5-6 hours and blood glucose was measured using a Contour glucose meter (Bayer, UK) using blood collected from the tip of the tail vein. Blood was collected by cardiac puncture and plasma was extracted. Plasma insulin concentrations were determined using an ELISA kit (Mercodia, Uppsala, Sweden), plasma and liver triglyceride levels were determined using infinity triglyceride liquid stable reagent (Thermoscientific) and cholesterol levels were determined from plasma Cholesterol quantification kit (BioVision, CA, USA). Inflammasome activation was assessed using 7-plex MesoScale Discovery Kit (Gaithersburg, Md., USA) directly from plasma and from liver extracts.
Standard C-BAs and BAs were purchased from Sigma Aldrich or Steraloids and are listed in supplementary information (Table S1). Deuterated cholic acid (D-2452) and deuterated chenodeoxycholic acid (D-2772) were purchased from CDN Isotopes Inc. HPLC-grade methanol, acetonitrile, water, ammonium acetate, ammonium formate, ammonium hydroxide, formic acid, and acetic acid and water were obtained from Fisher Scientific (Fair Lawn, N.J.). Standards were constituted as 1 mg/ml stock solutions of individual sulfated BAs were prepared in water:MeOH (1:1) and combined to a final volume of 1.0 ml in water to give a concentration of 40 mg/ml for each. Subsequent dilutions were made as necessary to create a standard curve for each bile acid.
Bile acids were extracted from 100 μl of plasma spiked with internal standards added to 50% ice-cold methanol. The extract was mixed then centrifuged at 16,000×g for 10 minutes at 4° C. The supernatant was retained and further extracted by addition of ACN (5% NH4OH). The resultant supernatant was dried under vacuum and reconstituted in 50% MeOH. The extracted bile acids were resuspended in 150 ml of ice cold 50% MeOH.
UPLC-MS was performed using a modified method of Swann et al. (Swann et al., 2011). 5 μL were injected onto a 50 mm T3 Acquity column (Waters Corp.) and were eluted using a 20-min gradient of 100% A to 100% B (A, water, 0.1% formic acid; B, methanol, 0.1% formic acid) at a flow rate of 400 μL/min and column temperature of 50° C. Samples were analyzed using an Acquity UPLC system (Waters Ltd.) coupled online to an LCT Premier mass spectrometer (Waters MS Technologies, Ltd.) in negative electrospray mode with a scan range of 50-1,000 m/z. Bile acids ionize strongly in negative mode, producing a prominent [M-H]− ion. Capillary voltage was 2.4 Kv, sample cone was 35 V, desolvation temperature was 350° C., source temperature was 120° C., and desolvation gas flow was 900 L/h.
PCA analysis was performed in Markerlynx (Waters) by limiting the number of elements (N, H, S, C) to be detected in individual analytes. Furthermore a template of defined known masses to allow bile acid detection only was applied to generate a table of markers and their retention time. Group Differences were detected using the pareto scaling in OPLS-DA. Here weighted averages provide a summary of the X variables. In addition, these scores of PLS-DA display the separation of the groups. The scores t[1] and t[2] summarize separating the data. The plot of t[1] vs. t[2] shows a picture of the data. The groups (types) are shown in different colours, and the separation of the groups is easily visible. Each analyte was identified according to its mass and retention time. Standard curves were then performed using known bile acids and each analyte was quantified according to the standard curve and normalized according to the deuterated internal standards.
Tissues were stored in RNA-later (Qiagen) prior to RNA extraction using the RNAeasy plus universal kit (Qiagen). Microarrays were carried out using mouse Exon ST1.0 arrays (Affymetrix) by Almac Group, Craigavon, Northern Ireland. Analysis and pathway mapping was carried out using Subio Platform software (Subio Inc) and Genesis Software. Microarray data will be deposited on the Gene Expression Omnibus website.
qRT-PCR utilised RNA to generate cDNA. Universal ProbeLibrary (Roche) designed primers and pairs were used for qPCR with the LightCycler 480 System (Roche). The 2−ΔΔC method (Livak and Schmittgen, 2001) was used to calculate relative changes in gene expression.
Data for all variables were normally distributed and therefore allowed for parametric test of significance. Data is presented as mean values and their standard deviation is indicated. Statistical analysis was performed by analysis of variance and students t test.
Pig samples were taken from the porcine facility in the biological services unit in UCC and human faeces was from a 2 year old female infant donor. Samples of porcine or human faeces were sieved, serially diluted (in phosphate buffered saline, PBS) and plated onto MRS plates under anaerobic conditions. Single colonies were grown anaerobically in MRS broth in 96-well plates for further characterisation. 960 putative Lactobacillus species isolates were isolated for further characterisation. Isolates were screened using PCR for the presence of BSH1 (Seq ID No: 1) based upon the presence of known regions using the following primer pairs:
The F1/R detects the full length BSH1 sequence whereas the F2/R primer set detects the presence of a unique 24 nt region. We sequenced BSH genes from 17 isolates from pigs (labelled as APC1484 to APC1500) and 2 isolates from human faeces (labelled APC1501 and APC1502) (see Table). We generated PCR products using 16s primers (F-DG74-AGGAGGTGATCCAACCGCA (SEQ ID 45) and R-RW01-AACTGGAGGAAGGTGGGGAT (SEQ ID 46)) which were sequenced in each case to determine the closest homologues in the NCBI database. This allowed identification of strains to species level (see Table).
Two porcine strains APC1486 (Lactobacillus. salivarius APC1486) and APC1488 (Lactobacillus johnsonii APC1488) and a type strain expressing Seq ID No: 1 activity (Lb. salivarius JCM1046) were incubated separately with a human bile extract (0.5% w/v in MRS broth) (obtained from clinical cholesystectomy from Cork University Hospital) for 90 mins anaerobically at 37 degrees C. Subsequently either untreated or bacterially treated human bile was subjected to chemical analysis using UPLC-MS (see below).
Standard C-BAs and BAs were purchased from Sigma Aldrich or Steraloids. Deuterated cholic acid (D-2452) and deuterated chenodeoxycholic acid (D-2772) were purchased from CDN Isotopes Inc. HPLC-grade methanol, acetonitrile, water, ammonium acetate, ammonium formate, ammonium hydroxide, formic acid, and acetic acid and water were obtained from Fisher Scientific (Fair Lawn, N.J.). Standards were constituted as 1 mg/ml stock solutions of individual sulfated BAs were prepared in water:MeOH (1:1) and combined to a final volume of 1.0 ml in water to give a concentration of 40 mg/ml for each. Subsequent dilutions were made as necessary.
Bile acids were extracted from 100 μl of plasma added to 50% ice-cold methanol. The extract was mixed then centrifuged at 16,000×g for 10 minutes at 4° C. The supernatant was retained and further extracted by addition of ACN (5% NH4OH). The resultant supernatant was dried under vacuum and reconstituted in 50% MeOH. The extracted bile acids were resuspended in 150 ml of ice cold 50% MeOH.
UPLC-MS was performed using a modified method of Swann et al. (5). 5 μL were injected onto a 50 mm T3 Acquity column (Waters Corp.) and were eluted using a 20-min gradient of 100% A to 100% B (A, water, 0.1% formic acid; B, methanol, 0.1% formic acid) at a flow rate of 400 μL/min and column temperature of 50° C. Samples were analyzed using an Acquity UPLC system (Waters Ltd.) coupled online to an LCT Premier mass spectrometer (Waters MS Technologies, Ltd.) in negative electrospray mode with a scan range of 50-1,000 m/z. Bile acids ionize strongly in negative mode, producing a prominent [M-H]− ion. Capillary voltage was 2.4 Kv, sample cone was 35 V, desolvation temperature was 350° C., source temperature was 120° C., and desolvation gas flow was 900 L/h.
Bile acid deconjugation profiles were highly similar to those of a type strain expressing Seq ID No: 1 activity BSH activity (Lb. salivarius JCM1046) (see Figure outlining in vitro bile acid profiles) and exhibited ability to deconjugate conjugated bile acids and to generate cholic acid (CA) and chenodeoxycholic acid (CDCA) in the sample mixture.
Strains are available upon request from the Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland (http://www.ucc.ie/research/apc/content/)
The wide variation in BSH enzymes within the gut microbiota suggests that different BSH alleles may have differing impacts upon in vivo bile metabolism and downstream responses. To compare different BSH enzymes using an isogenic delivery system, bsh genes were expressed in Escherichia coli MG1655, a K-12 strain which lacks BSH activity and colonises both conventional and germ-free (this study) mice at high levels. To achieve stable expression in long-term colonisation experiments we utilised the mini-Tn7 transposon system for the cloning of bsh genes in single copy into the region downstream of glmS in the E. coli host (
In order to analyse the physiological effects of bile hydrolysis in a controlled system which lacks extant bile modification systems, gnotobiotic mice were monocolonised with our E. coli strains expressing BSH activity (ECBSH1 or ECBSH2). Colonisation of germ-free mice with BSH− E. coli MG1655 (EC) resulted in a significant elevation of total plasma bile acids to levels similar to those of conventionalised mice (CONV-D) (
Overall, the data indicate that the induction of in situ BSH activity in the model system significantly redirected the plasma bile acid signature (
The expression patterns of over 23,000 genes in the liver and ileum in GF, monocolonised (EC, ECBSH1 or ECBSH2) and CONV-D mice were examined. Overall there were significant changes in host gene expression patterns induced by BSH1 and BSH2 relative to EC colonised mice, in both the ileum and the liver. Gene annotation and pathway mapping were employed using Subio software to examine the primary functional groups of host genes regulated through in situ expression of BSH enzymes in the host GI tract. Due to the potent activity of BSH1 in vitro and in vivo herein we focus primarily upon the influence of BSH1 expression in our system. However many of the loci influenced by BSH1 are also influenced by BSH2 activity (
In addition BSH1 activity was a potent local trigger of the gene encoding the hormone adiponectin (adipoQ) as well as the gene encoding Angiopoietin-4 (also known as fasting induced adipose factor (FIAF)). Also note was the significant alteration of pathways regulated by circadian rhythm that have previously been implicated in energy metabolism and obesity (Costa et al., 2011). Genes encoding proteins with a known function in epithelial homeostasis and differentiation (EGFr, RegIIIg) were also strongly induced by BSH1 activity in our system. Gene expression profiles for a number of target genes were verified using qRT-PCR (
Given the influence of bacterial BSH on host energy pathways under controlled conditions in gnotobiotic mice, it was examined whether modulation of gastrointestinal BSH activity could form the basis of an intervention strategy for the control of host weight gain and metabolic processes in conventionally raised animals. In order to obtain consistent, high level expression of gastrointestinal BSH we again utilised the E. coli MG1655 gut colonisation model in which conventional streptomycin-treated mice were significantly colonised for over 70 days with strepR E. coli alone (EC) or E. coli expressing BSH1 or BSH2 enzymes (
Colonisation of conventional mice by ECBSH1 resulted in significantly decreased weight gain (46% reduction) relative to mice colonised by E. coli alone (EC) in animals fed either a normal fat (
The Applicant has identified, using mono-colonised gnotobiotic mice, a number of host pathways that are clearly affected by gastrointestinal BSH activity (
The invention is limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13171762.1 | Jun 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/062294 | 6/12/2014 | WO | 00 |
