EP1414853A2 - Methode pour etudier l'effet d'une flore microbienne commensale sur l'intestin d'un mammifere, et traitement d'une maladie gastro-intestinale fonde sur cette methode - Google Patents

Methode pour etudier l'effet d'une flore microbienne commensale sur l'intestin d'un mammifere, et traitement d'une maladie gastro-intestinale fonde sur cette methode

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Publication number
EP1414853A2
EP1414853A2 EP01995938A EP01995938A EP1414853A2 EP 1414853 A2 EP1414853 A2 EP 1414853A2 EP 01995938 A EP01995938 A EP 01995938A EP 01995938 A EP01995938 A EP 01995938A EP 1414853 A2 EP1414853 A2 EP 1414853A2
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European Patent Office
Prior art keywords
expression
protein
genes
gene
angiogenin
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EP01995938A
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German (de)
English (en)
Inventor
Jeffrey Ivan Gordon
Lora Virginia Hooper
Thaddeus F.IV Washington University STAPPENBECK
Per Falk
Lennart Hansson
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Washington University in St Louis WUSTL
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AstraZeneca AB
University of Washington
Washington University in St Louis WUSTL
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Publication of EP1414853A2 publication Critical patent/EP1414853A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods for investigating changes resulting from commensal microflora colonisation of mammalian intestine, and the use of information obtained in the production of agents and therapies useful in the modification of the digestive tract and in the treatment of gastrointestinal- associated disease.
  • Mammals generally and humans in particular are home to an incredibly complex and abundant ensemble of microbes. Contact with components of this microflora begin at birth.
  • the human intestine is more densely populated with microbes than any other mucosal surface. Therefore, this organ represents a site where the microflora are likely to have a pronounced influence on host physiology.
  • a method of investigating changes resulting from commensal microflora colonisation of mammalian intestine which comprises : a) measuring gene expression in commensal bacterium-colonized and germ-free intestine of at least one gene; and b) identifying a gene from a) that has at least a 2-fold difference in expression level between intestines colonised by at least one commensal bacterium and germ-free intestines.
  • multiple gene expression is measured by DNA icroarray analysis and/or quantitative RT-PCR as illustrated hereinafter, but other conventional methods may be applied. Examples of such methods include quantitative Northern blotting as well as Representational Differentiation Analysis (RDA) which is a PCR based assay which detects genes that differ between two samples (e.g., Odeberg J, et al . , Biomol. Eng. 17, 1-9, 2000).
  • RDA Representational Differentiation Analysis
  • the use of high-density oligonucleotide arrays is preferred for conducting a comprehensive analysis of the range of intestinal functions that are shaped by components of the microflora.
  • Microarray analysis can be used to measure host responses in a complex tissue composed of multiple cell types, as is found in the intestine.
  • the value. of an in vivo model for delineating host cellular responses to a given microbe is that, unlike cell culture-based models, the contributions of lineage and environmental factors to shaping the response are preserved, and may be studied in a germfree experimental system.
  • the responding cell population is recovered without perturbing its expressed mRNA population, so that its reaction to the microbe can be characterized in quantitative terms . This is suitably achieved by combining two techniques, laser-capture microdissection (LCM) , followed by quantitative analysis for example quantitative PCR and/or microarray analysis of the laser-capture samples.
  • LCM laser-capture microdissection
  • the germ-free intestine and the commensal bacterium- colonized intestine used in the method of the invention may be in any animal model, but in particular a simplified mouse model of intestinal-microbial interactions is used.
  • Genes identified in this way and their function may then be subject to further study in order to determine their effects, for example on the digestive process.
  • the further investigation may be carried out in for example using any of the conventional methods .
  • the method further comprises (c) the step of further investigating a gene identified in b) with regard to its function.
  • Suitable methods by which this can be achieved include work in in vitro cell culture assays or in lower eukaryotic model organisms as well as in animal models such as transgenic animal models.
  • Lower eukaryotic model organisms such as yeast (e.g., Saccharomyces cerevisae) , the fruit fly (Drosophila melanogaster) and the ne atode (Caenorhbditis elegans) are eukaryotic organisms with very well defined genomes and in many instances cellular function.
  • yeast e.g., Saccharomyces cerevisae
  • the fruit fly Drosophila melanogaster
  • the ne atode ne atode
  • These organisms are easier to handle than mammals .
  • the function can be studied using methods such as i) transgenic knockout; ii) dominant-negative experiments; iii) transgene overexpression; iv) antibody binding assay; v) by pharmacological intervention using defined chemical agents .
  • Transgenic knockout methods are based upon inactivation of a gene within an organism, for example using recombinant DNA technology to delete or mutate a gene such that the gene product is dysfunctional, or to introduce an antisense oligonucleotide to silence the gene.
  • antisense oligonucleotides which are complementary to the messenger RNA molecule (mRNA) of the gene are introduced in the organism.
  • the messenger RNA molecule (mRNA) that is -the result of transcription of a gene is single stranded and can not be translated into a protein sequence if it is double stranded. Double stranded RNA is formed with the complementary fragment and the resulting double stranded RNA fragment can not be further processed.
  • Classical transgenic knockout methods are based on introducing a mutated, and thereby dysfunctional gene into an embryonal cell line. This cell line is then introduced into a normal blastocyst, thereby creating a chimeric fetus consisting of germ cells from the normal background and from the embryonal stem (ES) cell line. Through several rounds of breeding the ES cell- derived mutant allele will be found in the germ cell and thus be transferable to offspring. New techniques now allow for such mutations to be inherited in a silent and inactive form, so that they can be activated in adult life when development is completed.
  • the classical transgenic experiment involving transgene overexpression includes introducing a gene normally absent from the model (e.g., a human-specific gene into mouse) to assess the effects of the gene on cellular function and/or physiology. If required, the transgene may be placed under the control of tissue- specific promoter sequence so that expression is directed to a cell population of choice. Promoters exist both for a generalised expression throughout the body and for very subtle distribution in specific minor cell population in defined areas of an organ. Other means of studying the effects of genes involve intervention at the protein level with the gene product.
  • antibody binding assays can be used both to determine whether a protein is present in a cell, model organism or mammal and/or to inhibit the function of the gene product by specifically binding to it and interfering with its capacity to interact with its intended molecular partners.
  • Pharmacological intervention using defined chemical agents may be particularly useful if the gene can be identified as belonging to a specific class of molecules, e.g., G-protein coupled receptors, proteases or nuclear receptors, its functions can be assessed by using chemicals that are known to act as agonists or antagonists for this type of molecules.
  • a specific class of molecules e.g., G-protein coupled receptors, proteases or nuclear receptors
  • Any commensal bacteria may be used in the method of the invention.
  • Particular examples include Bacteroides thetaiotaomicron, Escherischia coli , Bifidobacterium infant is, or mixtures thereof, or complete ileal and/or cecal microflora obtained from conventionally-raised species.
  • a particularly suitable bacterium for use in the method is B. thetaiotaomicron .
  • the applicants have found that different members of the commensal flora produce different results, indicating a high level of specificity amongst species.
  • the method of the invention has already revealed an unanticipated breadth of this commensal's impact on gut gene expression.
  • claim expression of a wide range of genes is measured in step (a) of the method of the invention.
  • genes include genes associated with the nutrient uptake and metabolism, hormone/maturational responses, mucosal barrier function, detoxification/drug resistance, xenobiotic metabolism, motility, enteric nervous system/muscular layer development or activity, angiogenesis, cytos eleton/extracellular matrix function or development, signal transduction and other essential cellular functions .
  • Nutrient uptake and metabolism genes which may be the subject of study include genes associated with carbohydrate uptake and metabolism such as Na+/glucose cotransporter (SGLTl) or lactase phlorizin-hydrolase genes, genes associated with lipid uptake and metabolism such as pancreatic lipase-related protein 2, colipase, liver fatty acid binding protein, fasting induced adipose factor (FIAF) , apolipoprotein A-IV, phospholipase B and CYP27 genes, metal uptake or sequestration genes such as high-affinity copper transporter, metallothionein I, metallothionein II or ferritin heavy chain genes, or cellular energy production such as isocitrate dehydrogenase subunit, succinyl CoA transferase, transketolase, malate oxidoreductase and aspartate aminotransferase genes.
  • SGLTl Na+/glucose cotransporter
  • genes associated with hormonal/maturational responses include adenosine deaminase, o ithine decarboxylase antizyme, 15-hydroxyprostaglandin dehydrogenase, GARG-16, FKBP51, androgen-regulated vas deferens protein, short chain dehydrogenase and heat-stable antigen genes.
  • genes associated with mucosal barrier function include decay-accelerating factor, polymeric Ig receptor, small proline-rich protein 2a, serum amyloid A protein, CRP-ductin ⁇ (MUCLIN) , zeta proteasome chain, and anti-DNA IgG light chain genes .
  • Suitable genes which are involved in detoxification/drug resistance include glutathione S-transferase, P-glycoprotein (mdrla) and CYP2D2 genes.
  • genes associated with enteric nervous system/muscular layer development or activity include L-glutamate transporter, L-glutamate decarboxylase, vesicle-associated protein- 33, cysteine-rich protein 2, smooth muscle (enteric) gamma actin and SM-20 genes.
  • RNAse super family members include several angiogenins . They also include the angiogenin-4, which the applicants have sequenced and found to be particularly interesting in that it is intestine- specific, epithelial-based and its expression appears to be regulated by components of the microbiota.
  • the sequence of murine angiogenin-4 has recently been published (D.E. Holloway et al . , Protein Expression and Purification, (2001), 22:307. The applicants have surprisingly found that one such gene, specifically the angiogenin-4 gene is expressed in intestinal epithelium and that expression levels are particularly affected by commensal bacteria.
  • genes associated with cytoskeleton/extracellular matrix function include gelsolin, destrin, alpha cardiac actin, endoB cytokeratin, fibronectin, proteinase inhibitor 6 and alpha 1 type 1 collagen genes .
  • Signal transduction genes include Pten, gplO ⁇ (TB2/DP1) , rac2, Semcap2, serum and glucocorticoid-regulated kinase, STE20- like protein kinase and B-cell myeloid kinase.
  • the signal pathway in which rac2 gene is associated in known to have an impact on the mucosal barrier function
  • genes associated with essential cellular functions include glutathione reductase, calmodulin, elF3 s ⁇ bunit, hsc70, oligosaccharyl transferase subunit, fibrillarin, HH—transporting ATPase and Msec23 genes.
  • step (a) at least 10 such genes are measured in step (a) .
  • expression of all the genes listed above are measured.
  • genes with at least a 2- fold difference in expression are identified and selected for further study.
  • genes for which at least a 4-fold difference, more suitably at least a 5-fold difference, yet more suitably a 7-fold difference and preferably a 9-fold difference in expression are identified in step (b) and are selected for further study.
  • genes which have already been identified using the method of the invention following colonisation with B. thetaiotaomicron and are of particular interest include colipase, decay-accelerating factor (DAF) , the polymeric IgA receptor, small proline-rich protein 2a (Sprr2a) , ⁇ angiogenin-3, ⁇ Pten, CYP2D2, Sprr2a, rac2, and Mdr-1. They also include angiogenin-4, a newly discovered protein which is related to angiogenin-3.
  • genes may have useful therapeutic functions and thus, the expression of one or more of these genes is preferably measured in the method of the invention in order to detect the impact of the particular commensal bacteria undergoing study on their expression.
  • the method of the invention has found that expression of colipase and angiogenins such as the angiogenin whose gene is amplifiable using primers such as SEQ ID NO 12 and 25 (see Table 3 hereinafter) which is angiogenin-4, as well as Sprr2a and rac2 should be subject to further investigation.
  • angiogenins such as the angiogenin whose gene is amplifiable using primers such as SEQ ID NO 12 and 25 (see Table 3 hereinafter) which is angiogenin-4, as well as Sprr2a and rac2 should be subject to further investigation.
  • the invention further comprises evaluation of the biochemical pathway in which the angiogenin whose gene is amplifiable using primers such as SEQ ID NO 12 and 25 (see Table 3 hereinafter) which is angiogenin-4, participates in the intestine.
  • the invention further comprises evaluation of the biochemical pathway in which colipase participates in the intestine.
  • the invention further comprises evaluation of the biochemical pathway in which Sprr2a participates in the intestine.
  • the invention further comprises evaluation of the biochemical pathway in which rac2 participates in the intestine. Evaluation can be carried out using any suitable method, including those described above.
  • This may be effected by administration of an appropriate commensal bacteria.
  • Increased populations of desirable microflora may be achieved by administration of the bacteria in oral form, such as in the form of tablets, pharmaceutical or nutriceutical compositions or even foodstuffs such as live yoghurt cultures
  • the invention provides a method of modulating epithelialy-expressed angiogenesis factor by colonisation with a commensal bacteria which effects said modulation.
  • Another particular example of such a methods is a method of modifying metabolism, in particular of dietary lipids, which method involves use of a commensal bacteria identified using a method as described above, as having an effect on said metabolism.
  • Yet another particular example is a method of modifying epithelial barrier function using a commensal bacteria identified using the above-described method.
  • Another example is a method of preventing or treating tumors of the intestine, by modifying the population of commensal bacteria present therein which bacteria have been identified using the method of the invention as modulating angiogenesis, or signal transduction .
  • the method of the invention may be useful in diagnosis of disease or conditions caused by inappropriate levels of gene expression in the gut.
  • Analysis of commensal microflora taken from a patient will show a high degree of natural variation in the populations of microflora as discussed above.
  • the detection of particularly elevated or reduced levels of commensal microflora identified using the method of the invention may indicate that a particular gene is being expressed at abnormal levels, giving rise to a disease state or condition.
  • Treatment of such conditions may be effected either by altering the levels of the commensal bacteria as appropriate and as discussed above, or by direct administration of a bacterial or human gene product or derivative, or of means to block the gene product at the protein level, such as using chemical or biological inhibitors or antagonists of the gene product.
  • the method of the invention can be used widely to address a question that applies to humans and innumerable other species that reside in our microbe-dominated world, namely how do bacteria contribute to and regulate the physiology and maturation of their hosts?
  • assembly and maintenance of a microflora undoubtedly involves intricate combinatorial regulatory mechanisms, developed over the course of a long selective process that involved co-evolution of our predecessors with their microbial partners.
  • the results presented below demonstrate the impact of an indigenous bacterial species on expression of genes that participate in vital physiologic functions, and emphasize the importance of viewing our biology as intertwined with the biology of our complex assemblies of resident bacteria.
  • results demonstrate the practicality of using defined in vivo models to deduce the responses of specified cellular populations within complex tissues to microbes, in a manner that preserves the influence of the surrounding cellular and environmental milieu.
  • the method has already revealed that a number of genes or gene products appear to have a significant effects in intestinal tissue, giving rise to the possibility that pharmaceuticals could be developed to target such genes or gene products in a manner which is beneficial to a patient. This can be done by screening for compounds which modulate the activity of the gene product .
  • a further aspect of the invention comprises a method for identifying genes that function as regulators of intestinal biology, said method comprising applying the method as described above and detecting expression genes which have not heretofore been associated with such function.
  • a further aspect of the invention comprises a method of screening compounds having a pharmaceutical application in a gastrointestinal disease, which method comprises assaying the compounds for their ability to modulate the activity of the product of a gene identified using a method described above.
  • Yet a further aspect of the invention is a method of treating or preventing gastrointestinal disease which method comprises administering a therapeutically effective amount of a compound which modulates the activity of the product of a gene identified using a method according to claim 1.
  • the invention further provides a method of screening for a compound potentially useful for treatment or prophylaxis of conditions characterized by a defect in intestinal barrier function which comprises assay of the compound for its ability to modulate the activity or amount of small proline-rich protein 2a (sprr2a) or rac2.
  • a defect in intestinal barrier function comprises assay of the compound for its ability to modulate the activity or amount of small proline-rich protein 2a (sprr2a) or rac2.
  • the compounds are useful in the treatment or prophylaxis of conditions in which intestinal barrier function is comprised.
  • a further aspect of the invention comprises the use of a compound able to modulate the activity or amount of small proline-rich protein 2a (sprr2a) or rac2 in preparation of a medicament for the treatment or prophylaxis of conditions characterized by a defect in intestinal barrier function.
  • small proline-rich protein 2a sprr2a
  • rac2 small proline-rich protein 2a
  • the compounds will be formulated as pharmaceutical compositions.
  • Novel compositions of this type and their preparation form a further aspect of the invention.
  • Figure 2 shows the results of real-time quantitative RT-PCR analyses of mRNA levels in isolated from laser-captured cell populations. Values are expressed relative to levels in germ-free mesenchyme using ⁇ CT analysis described below. Each gene product per sample was assayed in triplicate in 3-4 independent experiments. Representative results (mean ⁇ 1 S.D.) from pairs of germ-free and colonized mice are plotted.
  • FIG. 3 shows the results of an experiment to illustrate the specificity of host responses to colonization with different members of the microflora.
  • Germ-free mice were inoculated with one of the indicated organisms, or with a complete ileal/cecal flora from conventionally raised mice (Conv. microflora) ( 4) .
  • Figure 4 shows the nucleotide sequences of mouse angiogenin-4 and angiogenin-3 in alignment (SEQ ID NOS 29 and 30 respectively) .
  • Figure 5 illustrates the sequence alignment of the amino acid sequences of mouse angiogenin family members (SEQ ID NOS 31-34) .
  • Figure 6 shows the locations of primers specific for mouse angiogenin family members .
  • Figure 7 is a graph illustrating tissue distribution of angiogenin-4 mRNA, together with the results of an agarose gel analysis .
  • Figure 8 is a graph illustrating tissue distribution of angiogenin-1 mRNA.
  • Figure 9 is a graph illustrating tissue distribution of angiogenin-3 mRNA following quantitative real-time RT-PCR analysis.
  • Figure 10 shows the results of RT-PCR analysis showing the absence of angiogenin-related protein expression.
  • Figure 11 is a set of graphs showing the results of experiments on the microbial regulation of angiogenin-4 expression in the small intestine.
  • Figure 12 is a graph showing the regulation of angiogenin-4 expression during postnatal development.
  • Figure 13 is a block graph showing cellular localization of angiogenin-4 expression in small intestine: qRT-PCR analysis of cells isolated from the crypt base.
  • mice In order to study at a molecular level, the changes in the intestine orchestrated by commensal bacteria, germ-free mice were colonised with commensal bacteria including Bacteroides thetaiotaomicron, a prominent component of the normal mouse and human intestinal microflora.
  • B. thetaiotaomicron normally colonizes the distal small intestine (ileum) during the suckling-weaning transition, a time of rapid and pronounced functional maturation of the gut (W. E. C. Moore, L. V. Holdeman, Appl . Microbiol . 27, 961 (1974), T. Ushijima, M. Takahashi, K. Tatewaki, Y. Ozaki, Microbiol . Immunol . 27, 985 (1983) ) .
  • Colonization elicited a concerted response involving enhanced expression of four components of the host's lipid absorption machinery.
  • mRNAs encoding pancreatic lipase related protein-2 (PLRP-2) and colipase increased an average of 4- and 9- fold, respectively (Tables 1 and 2) .
  • PLRP-2 hydrolyzes tri- and diacylglycerols, phospholipids and galactolipids .
  • Colipase augments PLRP-2 activity (M. E. Lowe, M. H. Kaplan, L. Jackson- Grusby, D. D'Agostino, M. J. Grusby, J. Biol . Chem. 273, 31215 (1998) ) .
  • L-FABP mRNA which encodes an abundant cytosolic protein involved in fatty acid trafficking within enterocytes, and an induction of apolipoprotein AIV, a prominent component of triglyceride-rich lipoproteins (chylomicrons, VLDL) secreted from the basolateral surfaces of enterocytes (Table 1) .
  • B . thetaiotaomicron elicits an increased host capacity for nutrient absorption/processing and may provide a partial explanation as to why germ-free rodents require a higher caloric intake to maintain their weight than those with a normal microflora (B. S. ostmann, C. Larkin, A. Moriarty, E. Bruckner- Kardoss, Lab. Anim. Sci . 33, 46 (1983) .
  • colonisation produces changes in expression of four genes involved in dietary metal absorption.
  • a high affinity epithelial copper transporter (CRT1) mRNA was increased, while metallothionein-I, metallothionein-II, and ferritin heavy chain mRNAs were decreased (Table 1) .
  • CRT1 epithelial copper transporter
  • metallothionein-I, metallothionein-II, and ferritin heavy chain mRNAs were decreased (Table 1) .
  • Colipase plays a critical role in dietary lipid metabolism by stimulating the activity of both pancreatic triglyceride lipase and PLRP-2. Furthermore, proteolytic cleavage of procolipase yields a pentapeptide (enterostatin) that functions as a satiety signal for fat ingestion (S. Okada, D. A. York, G. A. Bray, Physiol . Behav. 49, 1185 (1991)).
  • entertatin pentapeptide
  • the significantly elevated expression found following colonisation with B. thetoaiotaomicron illustrated hereinafter are indicative of a previously unappreciated mechanism by which the intestinal epithelium, together with a resident gut bacterium, contributes to dietary lipid metabolism.
  • DAF decay accelerating factor
  • Colonization did not produce a notable change (i.e. two fold or more) , in the expression of genes encoding other proteins linked to desmosomes (desmoplakin, plakoglobin, plakophilin, plectin) , or tight junctions (ZO-1, occludin) .
  • Sprr2a expression in the intestine and its microbial regulation are novel findings.
  • the critical contribution of sprr2a to the squamous epithelial barrier and the dramatic response of sprr2a expression to B . thetaiotaomicron together suggest that this protein plays an important role in intestinal barrier function. It is therefore a particularly suitable target for further investigation in accordance with the invention, in particular by evaluating the biochemical pathway in which Sprr2a participates in the intestine.
  • LPH lactase-phlorizin hydrolase
  • enterocytic brush-border enzyme that hydrolyzes the principal milk sugar, lactose.
  • LPH mRNA levels rise throughout the small intestine of conventionally raised animals during the suckling period, and then fall in the ileum during weaning (S. D. Krasinski et al . , Am. J. Physiol . 267, G584 (1994) ) .
  • the effects of commensal bacteria on expression of these genes in particular may be of interest in determining whether the bacteria have signficant impact.
  • colonization results in increased expression of angiogenin-4 which resembles angiogenin-3, a secreted protein with demonstrated angiogenic activity (X. Fu, et al . , Mol . Cell Biol . 17, 1503 (1997), X. Fu, et al . , Growth Factors 17, 125 (1999)).
  • the 11-fold increase in expression of the angiogenesis factor recognizable by amplification using primers of SEQ ID NO 12 and SEQ ID NO 25, which is angiogenin-4 (Table 1,2) upon B .
  • thetaiotaomicron colonization represents a novel mode of regulation for an angiogenesis factor and so may be the subject of further investigation in accordance with the invention.
  • Pten is a member of a family of dual specificity protein phosphatases. PTEN haploinsufficiency in humans is associated with increased susceptibility to tumorigenesis (D. J. Marsh et al., Hum. Mol . Genet . 7, 507 (1998)). Furthermore,- Pten+/-**Pten+/+ chimeric mice develop colonic polyps and adenocarcinoma (A. DiCristofano, B. Pesce, C. Cordon-Cardo, P. P. Pandolfi, Na t . Genet . 19, 348 (1998)).
  • the human homolog of Gpl06, TB2/DP1 is a component of a locus which when mutated produces multiple intestinal adenomas (R. W. Burt, Adv. Exp. Med. Biol . 470, 99 (1999) ) .
  • the finding that a component of the microflora affects expression of genes such as the angiogenesis factor whose gene is amplifiable using primers such as SEQ ID NO 12 and 25 (see Table 3 hereinafter) which is angiogenin-4, Pten and Gpl06 highlights the importance of considering mechanisms by which intestinal bacteria may contribute to the initiation or progression of tumorigenesis within, or even outside, the gut.
  • the motility of the intestine is regulated by its enteric nervous system (ENS) .
  • ENS enteric nervous system
  • the relative contributions of intrinsic and extrinsic factors to ENS activity are poorly understood, despite the fact that irritable bowel syndrome, which involves dysregulated motor activity, is a major health problem.
  • commensal bacteria such as B. thetaiotaomicron on gut physiology extends to genes expressed in the enteric nervous system (ENS) and in the muscular layers.
  • mRNAs encoding the L- glutamate transporter and L-glutamate decarboxylase, which converts glutamate to GABA, are both increased, suggesting a colonization-associated effect on the glutamatergic neurons of the ENS (M. T. Liu, J. D. Rothstein, M.
  • thetaiotaomicron normally colonizes mouse and human intestine during weaning (W. E. C. Moore, L. V. Holdeman, Appl . Microbiol . 27, 961 (1974), T. Ushiji a, M. Takahashi, K. Tatewaki, Y. Ozaki, Microbiol . Immunol . 27, 985 (1983)).
  • This period is characterized by a dramatic shift in the composition of the microflora and by a series of critical developmental changes in the intestinal epithelium. It is unclear how many of these changes are regulated by intrinsic cellular mechanisms, and how many are controlled by extrinsic signals emanating from the mesenchyme, or from luminal (dietary, microbial) factors.
  • B. thetaiotaomicron colonization produces an increase in mRNAs encoding ADA and ornithine decarboxylase (ODC) antizyme but not a 5-fold increase.
  • ODC ornithine decarboxylase
  • a further aspect of the invention provides a protein of SEQ ID NO 29 as shown in Figure 4 hereinafter, or an allelic variant thereof or a protein which has at least 85% amino acid sequence identity with SEQ ID NO 29.
  • the invention provides a protein of SEQ ID NO 29.
  • the invention provides a nucleic acid which encodes a protein as described above.
  • Example 1 illustrates the invention.
  • mice Age-matched groups of 7-15 week-old germ-free NMRI/KI mice were maintained in plastic gnotobiotic isolators on a 12hour light cycle, and given free access to an autoclaved chow diet (B&K Universal) . Males were inoculated with wild-type B. thetaiotaomicron (strain VPI-5482) (L. Hooper et al (1999) supra) . Mice were sacrificed 10 days later, 2hours after lights were turned on. The distal 1 cm of the small intestine was used to define the number of colony forming units per ml of extruded luminal contents .
  • AGG liver fatty acid binding protein (L- 5'- CTCCGGCAAGTACCAATTGC 5'- TGTCCTTCCCTTTCTGGATGAG 16
  • FABP metallothionein I (MT-I) -, 5'- ATGTGCCCAGGGCTGTGT 4 5'- AACAGGGTGGAACTGTATAGGA 17
  • pigR decay accelerating factor
  • adenosine dearninase (ADA) 5'- GCGCAGTAAAGAATGGCATTC 11 5'- CTGTCTTGAGGATGTCCACAGC 24 angiogenin-4 5*- TCGATTCCAGGTCACCACTTG 12 5'- CACAGGCAATAACAATATATCT 25 o o GAAATCT glyceraldehyde 3 -phosphate 5'- TGGCAAAGTGGAGATTGTTGCC 13 5'- AAGATGGTGATGGGCTTCCCG 26 dehydrogenase
  • transcripts represented by 95 probe-sets were increased, while those represented by 23 probe-sets were decreased.
  • the genes represented by 84 of these probe sets were assigned to functional groups and these are set out in Table 1 hereinafter. In this table, results are presented as the fold-difference in mRNA levels between colonized and germ-free ileum and represent average values from duplicate microarray hybridizations. The average fold-changes for genes represented by 2 or more independent probe sets are listed separately.
  • genes which were found to have a difference in expression levels of 5-fold or more as a result of B. thetaiotaomicron colonisiation were colipase, liver fatty acid binding protein, fasting-induced adipose factor, metallothionein I and metallothionein II, malate oxidoreductase, Sprr2a, angiogenin-3, angiogenin-related protein, angiogenin family, gelsolin, gpl06 (TB2/DP1) and rac 2.
  • colipase, fasting-induced adipose factor, angiogenin 3 and Sprr2a genes showed a difference in expression levels of 9-fold or more.
  • a notable feature of the host response to B. thetaiotaomicron was the absence of detectable or changed expression of the many genes involved in immuno-inflammatory processes that are represented on the microarrays . These include genes involved in the NF-KB-regulated processes that are critical regulators of host responses to invasive pathogens (D. Elewaut et al . , J. Immunol . 163, 1457 (1999)). The absence of these responses can be contrasted to results obtained in a recent cDNA microarray analysis of the response of a human intestinal epithelial cell line to Salmonella , an invasive gut pathogen (L. Eckmann, J. R. Smith, M. P. Housley, M. B. Dwinell, M. F.
  • LCM laser-capture microdissection
  • Colipase is produced by the exocrine (acinar) cells of the pancreas. Expression in the intestine had not been reported previously. Therefore, LCM and real-time RT-PCR analysis were employed to delineate the cellular origins of its response to B. thetaiotaomicron .
  • proteolytic cleavage of procolipase yields a pentapeptide (enterostatin) that functions as a satiety signal for fat ingestion (S. Okada, et al . , Physiol . Behav. 49, 1185 (1991).
  • Entertatin a pentapeptide that functions as a satiety signal for fat ingestion
  • Angiogenin-3 was originally identified in NIH 3T3 fibroblasts (X. Fu et al., Mol . Cell Biol . 17, 1503 (1997), but little is known about its cellular origins or regulation. LCM and qRT-PCR revealed that the crypt epithelium is the predominant location of a gene amplifiable using primers such as SEQ ID NO 12 and 25 (see Table 3 hereinbefore) which are specific for angiogenin-3 mRNA, but also for the new protein which is angiogenin-4 mRNA and that colonization results in a 7-fold increase in its levels within this compartment. This increase accounts for the change in expression defined by microarray and qRT-PCR analyses of total ileal RNA (Tables 1,2).
  • the epithelial location of a secreted/RNAse/angiogenesis factor puts it in a strategic position to function as an effector of a number of host responses to microbial colonization (e.g., enhanced absorption/distribution of nutrients/augmented barrier function.
  • Example 2 The 11-fold induction of its mRNA seen in Example 1 was independently validated by real-time RT-PCR of total ileal RNAs (Table 2) .
  • Angiogenin-3 was originally identified in NIH 3T3 fibroblasts (X. Fu, M. P. Kamps, Mol . Cell Biol . 17, 1503 (1997) ) .
  • LCM and real-time RT-PCR analysis revealed that in colonized ileum, the levels of mRNA which are amplifiable using primers designed for angiogenin-3 are highest in crypt epithelium (values in the ileal villus epithelium and mesenchyme are 14- and 15-fold lower, respectively; Fig. 2) .
  • this mRNA is in fact, angiogenin-4 mRNA.
  • the 7-fold increase in these angiogenin-4 mRNA levels observed in the crypt epithelium after colonization account for the change defined by microarray and real-time RT-PCR analyses of total ileal RNA.
  • Bacterial modulation of epithelially-expressed angiogenin-4 represents a novel mode of regulation for an angiogenesis factor.
  • B. thetaiotaomicron strain VPI-5482 (L.V. Hooper, et al . , Proc. Na tl . Acad. Sci . U. S. A. 96, 9833 (1999)), (iii) E. coli K12 which was originally recovered from a normal human fecal flora, (iv) Bifidobacterium infantis (ATCC15697) , a prominent component of the pre-weaning human and mouse ileal flora and a commonly used probiotic.
  • a further control group comprised mice conventionally raised since birth.
  • mice were sacrificed lOdays later, 2hours after lights were turned on.
  • the distal 1 cm of the small intestine was used to define CFU/ml ileal contents.
  • the 3 cm of intestine just proximal to this segment was used to isolate total ileal RNA (Qiagen RNeasy kit) .
  • the Mdrla/GST responses provide direct evidence that components of the normal microflora can modulate host genes involved in drug metabolism, and suggest that variations in drug metabolism between individuals may arise, in part, from differences in their resident gut flora.
  • Example 4 Following the observation that a lOd colonization was associated with a 11-fold increase in ileal expression of a mRNA detected by an Affymetrix-designed probe-set designed from the published sequence of angiogenin-3, we designed primers specific for the 3' and 5' ends of the mouse angiogenin-3. There were:
  • RNA prepared from the ileums of ex-germ- free NMRI mice had been colonized , for lOd with a complete ileal/cecal flora harvested from conventionally-raised animals belonging to the same inbred strain.
  • the nucleotide sequence of the ORF was only 90% identical to that of mouse angiogenin-3. Since the primer sequences used in the PCR reaction (specific for angiogenin-3) were incorporated into the product, we used 5'- and 3' -RACE to (a) obtain accurate sequence at the 5' and 3' ends of the ORF of this new angiogenin, and (b) characterize the 5'- and 3'- untranslated regions of its mRNA. The results revealed only 88.3% nucleotide sequence identity with angiogenin-3 mRNA.
  • the nucleotide sequence which encodes the angiogenin-4 protein, aligned with the angiogenin-3 sequence is shown hereinafter in Figure 4 as SEQ ID NO 29 and 30 respectively.
  • - Angiogenin-4 has 74 to 81% amino acid sequence identity to the other 3 members of the mouse angiogenin family ( Figure 5) . It was found that the 5' and 3' -untranslated regions of angiogenin-4 are closely related to the corresponding regions of angiogenin-3 mRNA ( Figure 4) . Subsequently a comparative analysis of the tissue distribution of the various mouse angiogenin mRNAs, was conducted.
  • cDNA was synthesized from RNAs isolated from tissues harvested from conventionally raised adult (12-14 week old) male and female NMRI mice (25 tissues/mouse) .
  • primer sets specific for each of the four mouse angiogenin family members (Figure 6; Table 4) and used them for SYBR-Green-based real-time quantitative RT-PCR (qRT-PCR) analyses .
  • angiogenin-4 mRNA was restricted the intestine where it is expressed from the duodenum to the rectum ( Figure 7) .
  • angiogenin-1 expression is highest in liver, lung, and pancreas ( Figure 8)
  • angiogenin-3 is expressed primarily in liver, lung, pancreas, and prostate ( Figure 9) .
  • Angiogenin-related protein mRNA was undetectable in all tissues surveyed even after 40 cycles of PCR ( Figure 10) .
  • angiogenin-4-specific primers and qRT-PCR were used to compare angiogenin-4 mRNA levels along the length of the small intestine of germ-free NMRI mice and germ-free mice colonized for 10 d with an ileal/cecal flora harvested from conventionally raised NMRI animals. Pair-wise comparisons revealed that expression of angiogenin-4 is highest in the jejunum of colonized mice, and that conventionalization induces up to a 17-fold increase in angiogenin-4 expression in this region ( Figure 11) . Mono-association of germ-free NMRI mice with B. thetaiotaomicron for lOd resulted in a comparable induction of angiogenin-4 expression (data not shown) .
  • angiogenin-3 forward 37 5' CTGGCTCAGGATAACTACAGGTACAT reverse 38 5' GCCTGGGAGACCCTCCTTT angiogenin- 1 forward 39 5' AGCGAATGGAAGCCCTTACA reverse 40 5' CTCATCGAAGTGGACCGGCA
  • angiogenin- forward 41 5' GGTGAAAAGAAAGCTAACCTCTTTC related protein reverse 42 5' AGACTTGCTTATTCTTAAATTTCG
  • Example 2 The previous laser capture microdissection (LCM) /qRT-PCR study of the cellular origins of angiogenin protein expression (Example 2) used primers that recognize both angiogenin-3 and angiogenin-4, and RNAs that had been isolated from captured crypt epithelium, villus epithelium, or mesenchymal populations from the villus core.
  • the qRT-PCR analysis indicated that the icrobially-regulated angiogenin' was produced in epithelial cells located at the base of crypts of Lieberkuhn (Hooper et al., 2001) .

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Abstract

L'invention concerne une méthode qui permet d'étudier des modifications chimiques résultant de la colonisation de l'intestin d'un mammifère par une flore microbienne commensale. La méthode consiste à: a) mesurer une expression génique d'au moins un gène dans un intestin colonisé par des bactéries commensales et dans un intestin exempt de parasites; et b) identifier un gène de l'étape a) qui présente au moins une double différence quant au niveau d'expression entre un intestin colonisé par des bactéries commensales et un intestin exempt de parasites. La méthode de l'invention permet de sélectionner des gènes pour une étude ultérieure, et donne lieu à la mise au point de traitements prophylactiques.
EP01995938A 2000-11-27 2001-11-27 Methode pour etudier l'effet d'une flore microbienne commensale sur l'intestin d'un mammifere, et traitement d'une maladie gastro-intestinale fonde sur cette methode Withdrawn EP1414853A2 (fr)

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