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WO2004076615A2 - Composes prtobiotiques immunomodulateurs - Google Patents

Composes prtobiotiques immunomodulateurs Download PDF

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Publication number
WO2004076615A2
WO2004076615A2 PCT/DK2004/000138 DK2004000138W WO2004076615A2 WO 2004076615 A2 WO2004076615 A2 WO 2004076615A2 DK 2004000138 W DK2004000138 W DK 2004000138W WO 2004076615 A2 WO2004076615 A2 WO 2004076615A2
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seq
lactobacillus
polynucleotide
polypeptide
microbial cell
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PCT/DK2004/000138
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English (en)
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WO2004076615A3 (fr
WO2004076615A9 (fr
Inventor
Hans Israelsen
Soeren Michael Madsen
Jacob Glenting
Astrid Vrang
Mette Rindom Noerrelykke
Anne Maria Hansen
Siv Elsa Ingegerd Ahrne
GÖran MOLIN
Peter Ravn
Hans Christian Beck
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Bioneer A/S
Probi Ab
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Publication of WO2004076615A2 publication Critical patent/WO2004076615A2/fr
Publication of WO2004076615A9 publication Critical patent/WO2004076615A9/fr
Publication of WO2004076615A3 publication Critical patent/WO2004076615A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01012—Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0067—Oxidoreductases (1.) acting on hydrogen as donor (1.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02003—Phosphoglycerate kinase (2.7.2.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01011—Phosphopyruvate hydratase (4.2.1.11), i.e. enolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C12Y503/01001—Triose-phosphate isomerase (5.3.1.1)

Definitions

  • the present invention relates to methods for modulating i) an immune response and/or ii) the amount and/or composition of mucosal mucins, by contacting a cell forming part of mucosal-associated lymphoid tissue (MALT), or an epithelial cell, with a microbial cell surface polypeptide.
  • MALT mucosal-associated lymphoid tissue
  • the modulation of the immune response preferably involves the induction of one or more cytokines.
  • the microbial cell surface polypeptide is preferably a polypeptide obtained from probiotic species of Lactobacillus or Bifidobacterium. It has surprisingly been found that intracellular enzymes acting in metabolic pathways in Lactobacillus and Bifidobacterium, or polypeptides substantially identical with such intracellular enzymes, are transported lo the surface of the cell where they may become at least partially exposed to the extracellular medium.
  • preferred cell surface polypeptides have intracellular (i.e. cytoplasm associated) equivalents acting in metabolic pathways, such as e.g. glycolysis, in probiotic species of Lactobacillus and/or Bifidobacterium.
  • the surface associated polypeptides and their intracellular equivalents share an extended stretch of consecutive amino acid residues, but are located in different parts of a cell.
  • the cell surface polypeptide can be administered in isolated form, or associated covalently or non-covalently with the surface of the cell having produced the polypeptide.
  • the polypeptide can furthermore be modified, e.g. by post-translational modification, as compared to its intracellular equivalent.
  • the invention also relates to species of Lactobacillus and Bifidobacterium having an altered expression of at least one cell surface polypeptide.
  • the altered expression can be generated by mutagenising an expression signal directing the expression of a gene encoding a cell surface polypeptide.
  • the altered expression can be generated by fusing a gene encoding a cell surface polypeptide to a heterologous expression signal not natively associated with said gene.
  • the altered expression can be an increased expression or a decreased expression.
  • the altered expression is an increased expression.
  • the invention is useful in the treatment of clinical conditions in an individual which responds to modulation of the mucosal immune system, including modulations involving one or more of e.g. the synthesis and/or secretion of cytokines, the stimulation of IgA antibodies, the inhibition of IgE antibodies, the regulation of the Th1/Th2 response, the stimulation of macrophage function, the stimulation of natural killer cell synthesis, and the general activation of the mucosa-associated lymphoreticular tissue system.
  • the invention further relates to methods for probiotic strain development and methods for performing a quality control procedure ensuring that a strain have desired probiotic qualities.
  • Probiotic microorganisms are defined as microorganisms that are beneficial to ani- mal or human health. This invention pertains to the field of immunomodulatory and mucin modulatory compounds produced by probiotic microorganisms.
  • paracasei 8700:2 and Lb. paracasei 02A (Ahrne et al.; 1998, Molin et al.; 1993, Hessle et al.; 1999, and Antonsson M.; 2001) can be used as standard indicators for probiotic potential as they have been shown to have probiotic properties such as e.g. survival in the gastrointestinal tract, adherence to intestinal mucosa, and induction/modification of cytokine release.
  • probiotic properties such as e.g. survival in the gastrointestinal tract, adherence to intestinal mucosa, and induction/modification of cytokine release.
  • HAV human immunodeficiency virus
  • Mucosal surfaces of the lungs and the Gl tract have several tasks that include absorption, macromolecule transport, barrier and secretory functions.
  • the large mucosal surfaces are continuously exposed to millions of more or less harmful antigens from the environment, food and microorganisms.
  • the mucosa possesses an immune system that tightly controls the balance between responsiveness and tolerance.
  • the immune system of the mucosa is part of the entire immune system and, consequently, immune responses in the mucosa are reflected in the entire body. It con- sists of an integrated network of tissues, lymphoid and non-lymphoid cells and effector molecules such as antibodies and cytokines.
  • the interaction between antigen-presenting cells (APCs), T lymphocytes and cytokines is the key for providing the correct specific immune response.
  • T-cell response may lead to immunological diseases such as allergy, inflammation and a number of autoimmune diseases.
  • Means to control T-cell differentiation and cytokine signaling will be essential for the prevention of or the development of therapeutics against such diseases.
  • MALT mucosa-associated lymphoreticular tissue
  • APC antigen presenting cells
  • DCs dendritic cells
  • B-cells capable of producing antigen-specific antibodies.
  • the B-cells can reach the mucosal tissues where they differentiate into plasma cells.
  • a second outcome of the entry of antigen and antigen presentation by DCs is the activation and differentiation of T cells that subsequently can migrate out of the
  • the T cells can secrete cytokines, such as IL10 and TGF ⁇ , which are essential for the induction of suppressive T cell responses i.e. an anti-inflammatory response.
  • cytokines such as IL10 and TGF ⁇
  • mucosal T helper cells can produce pro-inflammatory cytokines such as TNF ⁇ and IL1 ⁇ .
  • CD4+ T cells are termed naive T precursor cells, which are functionally immature.
  • the activation and further differentiation of naive T precursor cells requires at least two separate signals provided by APCs.
  • the first signal is delivered by the T cell receptor/CD3 complex after the T cell's interaction with antigen/major histocompati- bility complex on APCs.
  • the second signal is produced by a number of co- stimulatory or accessory molecules on the APC that interact with their ligands on T cells.
  • Cytokines play the most critical role in this so-called Th1/Th2 polarization. IL12 and IL4 are essential in the control of the differentiation of precursor T cells into Th1 and Th2, respectively. Cytokines induce the generation of their own T-helper subset and, simultaneously, inhibit the generation of the other subset.
  • the cytokine IL18 Besides cytokines such as IL12 and IL4, the cytokine IL18 also favors Th1 development. Although IL18 alone cannot induce Th1 cell differentiation it strongly enhances IL12-dependent Th1 cell development. Th1 cells secrete the cytokines interferon- ⁇ (INF ⁇ ), and TNF. The cytokine IL13 plays an important role in the Th2 development and its function is partly overlapping that of IL4.
  • the cytokine signaling in T cells as carried out by INF ⁇ , IL12 and IL4 occurs through the binding to the membrane-located cytokine receptors IFN- ⁇ R, IL12R and IL4R, respectively. The binding conducts activation of the transcription factors STAT1 , STAT4 and STAT6, respectively.
  • STAT1 activates the master transcription factor T- bet for Th1 cells.
  • T-bet induces Th1 cytokine production and IL12R ⁇ 2 chain expression while it simultaneously suppresses Th2 cytokine production.
  • STAT6 leads to the activation of the master transcription factor GATA-3 for Th2 cells. This activation directs Th2 cytokines production through activation of a number of other activators.
  • Th1 development eventually leads to a cell mediated immunological response while a Th2 development leads to a humoral response.
  • Some infections require Th1 response while others require Th2.
  • uncontrolled responses can result in significant tissue and organ damage, which eventually may result in the death of the host. Examples of diseases resulting from uncontrolled responses include inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, arteriosclerosis, allergy and diabetes. The inflammatory responses also have an essential role in the protection against growth and development of tumors.
  • the bacterial cell surface is the immediate object for interaction with or binding to eukaryotic host cells. It has been shown that, in general, Gram-negative bacteria induce a pro-inflammatory response while certain Gram-positive induce an anti- inflammatory response (Maasen et al.; 2000, Christensen et al.; 2002). It is speculated that Gram-negative bacteria contains surface located compounds, such as specific lipopolysaccharides or flagellin, which induce the pro-inflammatory response (Liaudet et al.; 2003). Gram-positive bacteria are generally believed to harbour anti- inflammatory inducing compounds although exceptions have been described (Han- age & Cohen; 2002).
  • GAPH glyceraldehydephosphate dehydrogenase
  • GAPDH of group A streptococci also functions as an ADP- ribosylating enzyme, which in the presence of NAD is auto-ADP-ribosylated (Pancholi & Fischetti 1993). Since ADP-ribosylation is involved in signal transduction events, this activity of GAPDH may be involved in the communication between the bacterium and a eukaryotic host cell. This is supported by the finding that the
  • GAPDH of group A streptococci is involved in the activation of protein tyrosine kinase and protein kinase C of human pharyngeal cells (Pancholi & Fischetti 1997).
  • Strepto- coccal surface enolase displays strong plasmin and plasminogen binding, which could be a virulence factor.
  • Group A streptococci may thus bind to plasminogen) via SEN and subvert the fibrinolytic activity of human plasmin(ogen) to their own advantage for tissue invasion.
  • enolase on the surface of streptococci and also on a variety of mammalian tissues including the brain provides new insight in the role of SEN-specific antibodies in post-streptococcal autoimmune diseases. Additionally, SEN has been shown to bind to the extracellular matrix, which makes it plausible to speculate if enolase is involved in bacterial signal trig- gering or transduction in eukaryotic host cells.
  • Lactobacillus and Bifidobacterium compounds capable of either modulating an immune response and/or modulating the amount and/or composition of mucosal mucins would be of great interest in the prevention and treatment of e.g. immuno-dependent diseases and infectious diseases.
  • intracellular Lactobacillus enzymes acting in metabolic pathways or polypeptides being substantially identical with such intracellular enzymes as described herein below, are transported to the cell surface and optionally becomes at least partially exposed to the extracellular medium.
  • methods for modulating an immune response and/or modulating the amount and/or composition of mucosal mucins exploit Lactobacillus and/or Bifidobacterium cell surface polypeptides having substantially identical intracellular equivalents acting in metabolic pathways, such as e.g. the glycolytic pathway, in Lactobacillus and/or Bifidobacterium.
  • the cell surface polypeptides are capable of contacting an animal or human cell forming part of the mucosa-associated lymphoid tissue (MALT) system and/or an animal or human epithelial cell, including MALT cells and epithelial cells of the gastro-intestinal (Gl) tract.
  • MALT mucosa-associated lymphoid tissue
  • Gl gastro-intestinal
  • This invention in one aspect relates to methods for i) induction of gene expression in an animal or human host cell and, subsequently, ii) increased or decreased production of compounds such as e.g. cytokines and/or mucins, wherein the production of e.g. cytokines and/or mucins result from the contacting of a microbial cell surface polypeptide and the animal or human host cell.
  • compounds such as e.g. cytokines and/or mucins
  • cytokines and/or mucins are according to one presently preferred hypothesis believed to be the result of the binding of a Lactobacillus or Bifidobacterium surface polypeptide to an epithelial cell, or a cell forming part of mucosa-associated lymphoid tissue (MALT).
  • MALT mucosa-associated lymphoid tissue
  • the binding of the surface polypeptide can be direct or indirect, i.e. additional binding factors can be involved in order for the Lactobacillus or Bifibobacterium cell surface polypeptide to bind an epithelial cell and/or a cell of the MALT system.
  • the binding of the Lactobacillus or Bifibobacterium cell surface polypeptide alone can mediate signal transduction, or signal transduction (ultimately resulting in cytokine modulation and/or modulation of the amount and/or composition of mucosal mucins) can require additional factors which may also need to bind the epithelial cell and/or the MALT and/or the surface polypeptide.
  • Signal transduction can also occur because the binding of a Lactobacillus or
  • Bifidobacterium surface polypeptide to an epithelial cell, or a cell forming part of MALT can prevent a pathogen microbial cell from gaining access to the site of binding of the Lactobacillus or Bifidobacterium surface polypeptide.
  • self-antibodies present in e.g. auto-immune diseases can be titrated by binding to a Lactobacillus or Bifidobacterium surface polypeptide according to the invention, thereby at least alleviating the auto-immune disease.
  • the present invention in a particularly preferred aspect relates to methods for modulating an immune response, such as a cytokine response, in an animal or human host cell, such as an epithelial cell or a cell of the MALT-system in an animal or human individual, by contacting said cell with a microbial cell surface polypeptide preferably obtained from a probiotic strain of Lactobacillus or Bifidobacterium.
  • a microbial cell surface polypeptide preferably obtained from a probiotic strain of Lactobacillus or Bifidobacterium.
  • the cell surface polypeptide can be in isolated form or associated with the surface of the cell having produced the polypeptide.
  • the cell surface polypeptide can be modified as compared to a substantially identical intracellular/cytoplasmatically located equivalent/homolog of the cell surface polypeptide.
  • the intracellular equivalent acts in a metabolic pathway and comprises an enzymatic activity.
  • the cell surface polypeptide can comprise an enzymatic activity, but need not have any enzymatic activity.
  • the modification of the cell surface polypeptide can be any post-translational modification, including ribosylation, phosphorylation, methylation acetylation, alkylation, glycosylation, sulfation, amidation, proteolytic processing, and the cell surface polypeptide can form oligomeric or mullimeric complexes with itself or other polypeptides, and attain a different tertiary structure as a result of the cell surface association or the association with e.g. molecular chaperones.
  • Cell surface polypeptides and their substantially identical cytoplasmic equivalents/homologs share an extensive stretch of consecutive amino acid residues, such as e.g. at least 20 amino acid residues, for example at least 40 amino acid residues, such as e.g. at least 50 amino acid residues, for example at least 60 amino acid residues, such as e.g. at least 70 amino acid residues, for example at least 80 amino acid residues, such as e.g. at least 90 amino acid residues, for example at least 100 amino acid residues, such as e.g. at least 120 amino acid residues, for example at least 140 amino acid residues, such as e.g. at least 160 amino acid residues, for example at least 180 amino acid residues, such as at least 200 amino acid residues.
  • amino acid residues such as e.g. at least 20 amino acid residues, for example at least 40 amino acid residues, such as e.g. at least 50 amino acid residues, for example at least 60 amino acid residues, such as e.g
  • Cell surface polypeptides and their substantially identical equivalents/homologs preferably comprise amino acid sequences which are e.g. at least 80% identical, such as at least 85% identical, for example at least 90% identical, such as at least 95% identical, for example at least 98% identical, such as completely (100%) identical amino acid sequences.
  • cell surface polypeptides and their equivalents share extensive stretches of amino acids they are likely also to share some secondary and/or tertiary structure and they can in some embodiments be identified by the same antibody, such as a polyclonal antibody or a monoclonal antibody.
  • substantially identical can be determined e.g. on the basis of the above characteristics. “Substantially identical” as used herein does not exclude differences between cell surface polypeptides and their intracellular/ cytoplasmatically located equivalents/homologs such as e.g. that one of the aforementioned having an enzymatic activity while the other does not (preferably the intracellular equivalent/homolog exerts an enzymatic activity), as well as differences resulting from post-translational modifications, and differences in secondary and/or tertiary structure resulting from different folding reactions or folding pathways.
  • equivalents/homologs can share homologous enzymatic activities, but do not need to do so as the cell surface polypeptides of the invention do not always (need to) have the activity of its intracellular equivalent in order to bind an epithelial cell or a cell of the MAST.
  • Equivalents/homologs can furthermore be encoded by the same or different gene(s) and/or regulated by the same or different regulator(s).
  • Gl gastro-intestinal
  • additional compounds such as e.g. one or more of mannose binding polypeptides, S-layer proteins, carbohydrates, lipotachoic acid as well as lipids, is believed to be responsible for signal triggering and/or signal transduction in the host cell. It is therefore also believed that signal triggering and/or signal transduction can be performed by the presence and/or binding to an animal or human cell of one or more additional compounds following the initial binding of the probiotic microbial cell to the host cell.
  • the invention in presently preferred embodiments relates to methods employing species of Lactobacillus and/or Bifidobacterium, as well as to species of Lactobacillus and/or Bifidobacterium having an altered expression of at least one cell surface polypeptide capable of exerting an immunomodulating effect when binding an epithelial cell or a cell of the mucosa-associated lymphoid tissue (MALT).
  • MALT mucosa-associated lymphoid tissue
  • the methods and microbial cells are directed to Lactobacillus species and/or Bifidobacterium species harbouring on their cell surface an enzyme also capable of acting in the glycolytic pathway, i.e. an enzyme the activity of which catalyses a reaction in the glycolytic pathway.
  • an enzyme also capable of acting in the glycolytic pathway, i.e. an enzyme the activity of which catalyses a reaction in the glycolytic pathway.
  • Particularly preferred examples are the surface located polypeptides Enolase and GAPDH from Lactobacillus plantarum. A surface localisation of an Enolase enzyme in a Lactic Acid Bacteria has not previously been described.
  • the invention further relates to isolated polynucleotides and isolated cell surface located polypeptides.
  • isolated polynucleotides and isolated cell surface located polypeptides have been isolated by cloning and characterisation of e.g. genes encoding Enolase, GAPDH, phospho- glycerate kinase (PGK) and triose phosphate isomerase (TPI) from Lb plantarum.
  • PGK phospho- glycerate kinase
  • TPI triose phosphate isomerase
  • Enolase, GAPDH, PGK and TPI are all candidate compounds for acting on epithelial cells, or cells of the mucosa-associated lymphoid tissue, and thereby modulating mucosa-associated cytokine production and/or cytokine secretion, and/or modulating the amount and/or composition of mucosal mucins in an animal or human individual.
  • the observed effect is possibly exerted through M cells and/or dendritic cells (DCs), and/or antigen presenting cells (APCs), and/or T cells, and/or B cells, and/or natural killer (NK) cells, and/or macrophages, and/or further mucosal associated cells.
  • DCs dendritic cells
  • APCs antigen presenting cells
  • NK natural killer cells
  • the above candidate compounds in another preferred embodiment also act as a signal transducer of the animal or human cell being contacted by the compound.
  • GRE regulator protein
  • the cell surface location of e.g. Enolase, GAPDH, PGK, TPI and GRE of Lactobacillus and Bifidobacterium cells, or their modified equivalents, or genes encoding such proteins, or polypeptides involved in production, secretion and/or modification thereof, is believed to be important markers for probiotic activity and would, therefore, serve as an indicator for optimisation of the probiotic strains.
  • the optimisation could be carried out using classical screening methods, by using recombinant DNA techniques, or by using and optimising growth conditions, storage conditions and formulation techniques.
  • the isolated and/or purified Enolase, GAPDH, PGK and TPI could be provided alone or in combination with the probiotic microorganisms producing the compounds in methods for modulating immune responses and/or for modulating the mucin composition of the mucosa.
  • the markers can also serve as important probiotic indicators during production processes and/or concomitant or subsequent quality control.
  • probiotic strains in another preferred embodiment there is provided methods for the construction of probiotic strains and methods for the production of the above-mentioned candidate compounds for use in an analysis of immuno-modulatory and/or mucin modulating effects.
  • the analyses comprise e.g. using one or more of 0-mutants (null-mutants, i.e. a probiotic strain not expressing one or more candidate compounds), or mutants defective in secretion and/or post-translational modification, the isolated compounds, and combinations thereof.
  • the wild type strain Lb plantarum 299v can be used as a standard indicator for probiotic potential.
  • the analysis can e.g. be carried out in in vitro models using cell cultures and in animals using colitis models.
  • the ultimate goal is to demonstrate the immuno- modulatory and mucin modulating effects in human trials.
  • the present invention relates to the following aspects: A microbial cell comprising at least one microbial cell surface polypeptide and a substantially identical intracellular equivalent thereof,
  • the microbial cell is selected from the group consisting of Lactobacillus species and Bifidobacterium species, and
  • the microbial cell comprises an altered polynucleotide sequence as compared to a reference microbial cell comprising a reference polynucleotide sequence without said alteration
  • the activity of the intracellular equivalent is capable of converting a substrate in a Lactobacillus metabolic pathway and/or a Bifidobacterium metabolic pathway, and
  • altered polynucleotide sequence results in an altered, preferably increased, production and/or secretion and/or post-translational modification in the microbial cell of the at least one microbial cell surface polypeptide as compared to the production and/or secretion and/or post-translational modification of the cell surface polypeptide in a reference microbial cell comprising said reference polynucleotide sequence without said alteration.
  • a method for determining the probiotic potential of a candidate microbial cell preferably selected from the group consisting of Lactobacillus species and Bifidobacterium species, such as, but not limited to, a microbial cell described herein, said cell comprising a microbial cell surface polypeptide and a substantially identical intracellular equivalent capable of converting a substrate in a metabolic pathway of the candidate microbial cell, said method comprising the steps of i) providing a candidate microbial cell for which the probiotic potential is to be determined, ii) performing a qualitative and/or quantitative determination of the production and/or secretion and/or post-translational modification in the candidate microbial cell of said microbial cell surface polypeptide, or determining another characteristic of said candidate microbial cell, wherein said other characteristic is related to or correlates with the production and/or secretion and/or post-translational modification of said microbial cell surface polypeptide, iii) comparing the result of the determination performed in step ii) with
  • a method for determining the probiotic potential of a starter culture comprising a plurality of microbial cells, preferably selected from the group consisting of Lactobacillus species and Bifidobacterium species, such as, but not limited to, a plurality of microbial cells as described herein, said cells each comprising a microbial cell surface polypeptide and a substantially identical intracellular equivalent capable of converting a substrate in a metabolic pathway of the microbial cell, said method comprising the steps of i) providing a sample from a candidate starter culture for which the probiotic potential is to be determined, ii) performing on said sample a qualitative and/or quantitative determination of the production and/or secretion and/or post-translational modification of said microbial cell surface polypeptide, or determining another characteristic on said sample, wherein said other characteristic is related to or correlates with the production and/or secretion and/or post-translational modification of said microbial cell surface polypeptide, iii) comparing the result of the
  • a method for determining the probiotic potential of an end-user product preferably selected from the group consisting of Lactobacillus species and Bifidobacterium species, said end-user product comprising a plurality of microbial cells, such as, but not limited to, a plurality of microbial cells as described herein, said cells each comprising a microbial cell surface polypeptide and a substantially identical intracellular equivalent capable of converting a substrate in a metabolic pathway of the microbial cell
  • said method comprising the steps of i) providing a sample from a candidate end-user product for which the probiotic potential is to be determined, ii) performing on said sample a qualitative and/or quantitative determination of the production and/or secretion and/or post-translational modification of said microbial cell surface polypeptide, or determining another characteristic on said sample, wherein said other characteristic is related to or correlates with the production and/or secretion and/or post-translational modification of said microbial cell surface polypeptide, iii)
  • a method for identifying a microbial cell with altered probiotic potential comprising the steps of i) providing a plurality of cells of a Lactobacillus species or a plurality of cells of a Bifidobacterium species ii) subjecting said plurality of cells to a selection and/or mutagenesis procedure, and iii) identifying a microbial cell with altered probiotic potential as compared to the cells provided in step i), by identifying a cell with an altered production and/or secretion and/or post-translational modification of cell surface polypeptide, said cell surface polypeptide having a substantially identical intracellular equivalent, wherein the activity of the intracellular equivalent is capable of converting a substrate in a metabolic pathway of the cell.
  • a microbial cell having an altered probiotic potential obtainable by the above method for identifying.
  • a method for improving the probiotic potential of a microbial cell preferably selected from the group consisting of Lactobacillus species and Bifidobacterium species, said cell comprising a cell surface polypeptide having a substantially identical intracellular equivalent, wherein the activity of the intracellular equivalent is capable of converting a substrate in a metabolic pathway of the cell, said method comprising the steps of
  • probiotic potential of the microbial cell is improved by controlling, during the cultivation of the microbial cell, the presence or amount of one or more of the following components:
  • reducing agents such as glutathione and/or cysteine, preferably increasing the amount thereof
  • gasses such oxygen or carbon dioxide
  • c) yeast extract or components thereof
  • organic acids e) the carbon source, preferably carbohydrates, f) the nitrogen source, preferably proteins, peptides (like casaminoacids), amino acids, including any composition of naturally occurring amino acids, and precursors and/or derivatives thereof, as well as inorganic salts (like ammonium sulfate, acetamide, nitrates or nitrites),
  • the oxygen content h) the ionic strength of the growth medium, such as the NaCl content, i) the pH, j) low molecular weight compounds, preferably salts (sulfate, phosphate, nitrate), and/or metals (e.g., copper), and/or organic acids, k) cAMP level in the microbial cell, and I) a cell constituent, or a
  • a method for modulating an immune response and/or the amount and/or composition of mucosal mucins in an individual comprising the steps of
  • microbial cell selected from a Lactobacillus cell and a
  • said cell comprises at least one microbial cell surface polypeptide and a substantially identical intracellular equivalent thereof
  • the activity of the intracellular equivalent is capable of converting a substrate in a metabolic pathway of the cell
  • MALT mucosa-associated lymphoid tissue
  • An isolated polynucleotide comprising a nucleic acid sequence which is at least 90% identical to at least one of SEQ ID NO:1 ; SEQ ID NO:3; SEQ ID NO:5; and SEQ ID NO:7, wherein the percentage of identical nucleotides is determined by aligning the sequence and the compare sequences using the BLASTN algorithm version 2.04 set at default parameters described herein above, identifying the number of identical nucleotides over aligned portions of the sequence and the compare sequences, dividing the number of identical nucleotides by the total number of nucleic acids of the compare sequence, and multiplying by 100 to determine the percentage identical nucleotides.
  • a vector comprising a polynucleotide as described herein.
  • a host cell comprising a polynucleotide as described herein.
  • a method for producing a microbial cell surface polypeptide capable of modulating an immune response, or a fragment thereof comprising the step of culturing a host cell as described herein under conditions suitable for the production of said immu- nomodulating polypeptide, or fragment thereof.
  • a method for producing a microbial cell surface polypeptide capable of modulating the amount and/or composition of mucosal mucins, or a fragment thereof comprising the step of culturing a host cell as described herein under conditions suitable for the production of said immunomodulating polypeptide, or fragment thereof.
  • a method for producing an epithelial adhesive polypeptide, or a fragment thereof comprising the step of culturing the host cell as described herein under conditions suitable for the production of said epithelial adhesive polypeptide, or fragment thereof.
  • a polypeptide comprising an amino acid sequence which is at least 90% identical to at least one of SEQ ID NO:2; SEQ ID NO: 4; SEQ ID NO:6; and SEQ ID NO:8, including variants and functional equivalents thereof.
  • a method for the treatment of an individual comprising the step of administering to the individual a therapeutically effective amount of a polypeptide as described herein.
  • a method for the treatment of an individual comprising the step of administering to the individual a therapeutically effective amount of a host cell as described herein.
  • a method for identifying compounds which interact with and inhibit or activate an activity of a polypeptide as described herein comprising the steps of
  • composition comprising the polypeptide with the compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound;
  • a method for treating an auto-immune disease in an individual comprising the step of administering to the individual a pharmaceutically effective amount of a polypeptide as described herein, or a host cell as described herein.
  • a pharmaceutical composition comprising a therapeutically effective amount of at least one polypeptide and variants and functional equivalents thereof as described herein or a host cell as described herein, and at least one excipient.
  • a nutritional supplement comprising at least a host cell as described herein and/or at least a polypeptide and variants and functional equivalents thereof as described herein.
  • a food comprising at least a host cell as describee herein, and/or at least a polypeptide and variants and functional equivalents thereof as described herein.
  • polypeptide and variants and functional equivalents thereof as described herein and/or at least a host cell as described herein for the manufacture of a food for treatment of a disease which benefit from modulation of the immune response.
  • an increased probiotic potential is generated by an increased production and/or an increased secretion and/or an increased or decrea- sed post-translational modification of said microbial cell surface polypeptide,
  • an increased production and an increased post-translational modification of said microbial cell surface polypeptide for example an increased production and a decreased post-translational modification of said microbial cell surface polypeptide
  • Fig. 1 illustrates SDS-PAGE analysis of surface located proteins from L. plantarum
  • Fig. 2 illustrates data obtained from mass spectrometric analysis of the tryptic digest from band at MW 38.5 kDa (Fig. 1).
  • Fig. 3 illustrates a nucleotide sequence of L. plantarum 299v encoding the regulator and the genes encoding gapdh-pgk-tpi-eno.
  • Fig. 4 illustrates the amino acid sequence of L. plantarum Gapdh.
  • Fig. 5 illustrates the amino acid sequence of L. plantarum Pgk.
  • Fig. 6 illustrates the amino acid sequence of L. plantarum Tpi.
  • Fig. 7 illustrates the amino acid sequence of L. plantarum Eno.
  • Fig. 8 illustrates the amino acid sequence of the regulator of expression of gapdh- pgk-tpi-eno in L. plantarum.
  • Fig. 9 illustrates the difference between extracellular/surface-located GAPDH activity in MRS broth (black bars) and in the modified sMRS medium (white bars). Each result is the mean of two parallel cultures of Lactobacillus plantarum 299v.
  • Fig. 10 illustrates the development of extracellular/surface-located GAPDH in cultures of Lactobacillus plantarum strains 299v (diamonds) and WCFS1 (triangles) during incubation for three days at 30°C.
  • Fig. 11 shows GAPDH activity in culture supernatant, in ESP (the fraction eluted from harvested cells by washing with PBS), and in the suspension of washed cells.
  • Fig. 12 shows the distribution of GAPDH (white bars) and LDH activity (black bars) between the extracellular (culture supernatant, ESP, and washed cells) and intracellular (cell lysate) fractions from a stationary phase culture of Lactobacillus plantarum 299v.
  • Fig. 13 shows a western blot illustrating the cross reaction between anti-GAPDH and GAPDH-GST fusion protein or GAPDH wild type protein.
  • Fig. 14 illustrates the extracellular/surface-located GAPDH and LDH activities in cultures of 23 different Lactococcus strains.
  • Fig. 15 shows western blots of ESP-fractions from 23 different Lactococcus strains. anti-GAPDH and anti-ENO, respectively, were used as primary antibodies.
  • Fig. 16 illustrates the binding of GAPDH to fibronectin.
  • Fig. 17 illustrates the binding of enolase to fibronectin.
  • Fig. 18 illustrates the binding of GAPDH to plasminogen.
  • Fig. 19 illustrates the binding of enolase to plasminogen.
  • Fig. 20 illustrates the binding of GAPDH to mucin.
  • Fig. 21 illustrates the binding of enolase to mucin.
  • Fig. 22 illustrates the IL-10 stimulation assay.
  • Fig. 23 illustrates screening of 192 mutants of 299v.
  • Fig. 24 illustrates LDH and GAPDH activities found in culture supernatants and ESP-fractions of L. plantarum strains 299v, WCFS1 , 149-D7, 147-D7/129 and UP102.
  • Fig. 25 shows immunoblots (western blots) of proteins from lysed cells, ESP- fractions (surface attached proteins), and culture supernatants of Lb. plantarum strains 299v, WCFS1, and 149-D7. A Coomassie stained SDS-PAGE gel is also shown.
  • Fig. 26 shows immunoblots of proteins from culture supernatants and ESP-fractions (surface attached proteins) of Lb. plantarum strains 299v, WCFS1 , 149-D7, 149- D7/129, and UP102. Anti-GAPDH, anti-ENO, and anti-PGK, respectively, are used as primary antibodies.
  • Fig. 27 illustrates plasmid constructs used to complement L. plantarum WCFS1.
  • Fig. 28 shows a silver stained 2D-PAGE loaded with ESP from L. plantarum 299v.
  • Fig. 29 illustrates part of the result from analysis of the GAPDH tryptic digest.
  • Fig. 30 illustrates Nano-ESI analysis of the ions at m/z 612.87.
  • Fig. 31 illustrates Nano-ESI analysis of the ions at m/z 827.38.
  • Fig. 32 illustrates the inactivation of hom2-thrB in L. plantarum 299v.
  • Fig. 33 illustrates Southern blot analysis of L. plantarum 299v and L. plantarum PSM2012 (hybridised with hom2-thrB DNA).
  • Cytokine response The induction or repression of one or more cytokines.
  • Degenerated polynucleotide Different polynucleotides can encode the same polypeptide as the genetic code is degenerated.
  • Enzyme Polypeptide comprising an activity allowing the polypeptide to convert a substrate into a product resulting from the enzymatic reaction.
  • Epithelial cell Cell of the gastro-intestinal (Gl) tract.
  • the term 'food' can be any type of food, incl. an edible product.
  • the edible product is a food for special medical purposes, or a functional food or a novel food.
  • Microbial cell surface polypeptide Polypeptide located on the cell surface or attached thereto or associated therewith. The attachment can be covalent or non- covalent. The polypeptide can be exposed to the extracellular medium or act in the cell membrane to present other polypeptides to the extracellular medium.
  • Modulating Changing the expression or production of one or more compounds. Modulating can be inducing or repressive leading to increased expression/production and reduced expression/production, respectively.
  • Probiotic marker Surface located polypeptide acting as a determinant for the probiotic potential of a cell.
  • Probiotic potential the potential for immunomodulation and/or mucin production and/or the adhesion to intestinal epithelium.
  • a method for modulating an immune response and/or the amount and/or composition of mucosal mucins in an individual comprising the steps of
  • microbial cell selected from a Lactobacillus cell and a
  • said cell comprises at least one microbial cell surface polypeptide and a substantially identical intracellular equivalent thereof
  • the activity of the intracellular equivalent is capable of converting a substrate in a metabolic pathway of the cell, ii) contacting an epithelial cell or a cell of the mucosa-associated lymphoid tissue (MALT) of the individual with at least one microbial cell surface polypeptide, and
  • the modulation of the immune response preferably comprises a cytokine response, such as a modulation of the synthesis and/or secretion of at least one cytokine selected from the group consisting of IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
  • IL-10 IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 and IL-19
  • TNF-alpha TNF-beta, LT-beta
  • CD40 ligand Fas ligand
  • CD27 ligand CD30 ligand
  • 4-1 BBL TGF-beta
  • interferons including IFN-alpha, IFN-beta, and IFN-gamma.
  • the modulation of the immune response can further comprise one or more of i) an increased or decreased IgA production, ii) an increased or decreased IgE production, iii) a stimulation or repression of macrophage function, iv) a stimulation or repression of natural killer cell activity, and v) an activation or repression of the MALT system.
  • the epithelial cell is preferably selected from the group consisting of epithelial cells from an animal or human individual.
  • the cell of the mucosa-associated lymphoid tissue is selected from the group consisting of M-cells, antigen presenting cells (APCs), dendritic cells (DCs), T-lymphocytes, including Th1 , Th2, and CTL cells, IgA-committed B cells, macrophages, and natural killer (NK) cells.
  • the substantially identical intracellular equivalent of the cell surface polypeptide is preferably selected from the group consisting of Lactobacillus enzymes acting in a metabolic pathway and Bifidobacterium enzymes acting in a metabolic pathway.
  • the metabolic pathway is preferably the glycolytic pathway or the pathway for uptake of carbohydrates (phosphotransferase uptake system).
  • the enzyme acting in a metabolic pathway in Lactobacillus and/or Bifidobacterium is preferably selected from the group consisting of hexokinase; glucose 6-phosphate isomerase; phosphofructokinase; aldolase; triose phosphate isomerase (TPI); glyceraldehyde 3-phosphate dehydrogenase (GAPDH); phosphoglycerate kinase (PGK); phosphoglycerate mutase; enolase; and pyruvate kinase.
  • the enzyme is selected from the group consisting of enolase; glyceraldehyde 3-phosphate dehydrogenase (GAPDH); phosphoglycerate kinase
  • the enzyme is selected from the group consisting of enolase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
  • the microbial cell surface polypeptide can be covalently or non-covalently bound to the surface of a microbial cell, such as a Lactobacillus cell or a Bifidobacterium cell.
  • a microbial cell such as a Lactobacillus cell or a Bifidobacterium cell.
  • the microbial cell can natively produce the .cell surface polypeptide, or the microbial cell is can be a cell not natively producing the cell surface polypeptide.
  • the cell surface polypeptide can modified as compared to the polypeptide or its substantially identical equivalent/homolog when it is located intracellularly.
  • the modification can be a covalent modification, such as a covalent modification selected from the group consisting of ribosylation, phosphorylation, methylation acetylation, alkylation, glycosylation, sulfation, amidation, proteolytic processing.
  • Microbial cells capable of producing a microbial cell surface polypeptide
  • a microbial cell comprising at least one microbial cell surface polypeptide and a substantially identical intracellular equivalent thereof
  • the microbial cell is selected from the group consisting of Lactobacillus species and Bifidobacterium species, and
  • the activity of the intracellular equivalent is capable of converting a substrate in a Lactobacillus metabolic pathway and/or a Bifidobacterium metabolic pathway, and wherein the at least one microbial cell surface polypeptide is encoded by a first polynucleotide operably linked to a second polynucleotide capable of directing the expression of said first polynucleotide, and
  • first and second polynucleotides are not natively associated, and
  • the production and/or secretion and/or modification of the at least one microbial cell surface polypeptide is altered as compared to the production thereof when the first polynucleotide is operably linked to its native expression signal.
  • First and second polynucleotides not natively associated shall comprise the introduction of a heterologous expression signal operably linked to the gene encoding the cell surface polynucleotide as well as a mutagenised expression signal which differs from the native expression signal by at least one nucleotide deletion, addition or substitution.
  • An altered expression of the cell surface polypeptide can be determined by e.g. enzymatic assays and/or immunological assays.
  • the intracellular equivalent of the microbial cell surface polypeptide is preferably selected from the group consisting of Lactobacillus enzymes and Bifidobacterium enzymes acting in a metabolic pathway.
  • the metabolic pathway is preferably selected from the glycolytic pathway and the phosphotransferase system, and the enzyme is preferably selected from the group consisting of hexokinase; glucose 6-phosphate isomerase; phosphofructokinase; aldolase; triose phosphate isomerase (TPI); glyceraldehyde 3-phosphate dehydrogenase (GAPDH); phosphoglycerate kinase (PGK); phosphoglycerate mutase; enolase; and pyruvate kinase.
  • the enzyme is selected from the group consisting of enolase; glyceraldehyde 3-phosphate dehydrogenase (GAPDH); phosphoglycerate kinase (PGK); and triose phosphate isomerase (TPI).
  • the enzyme in a presently most preferred embodiment is selected from the group consisting of enolase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
  • the microbial cell surface polypeptide can be covalently or non-covalently bound to the surface of the microbial cell, and the microbial cell can natively produce the cell surface polypeptide or be a cell which does not natively produce the cell surface polypeptide.
  • the cell surface polypeptide can be modified as compared to the substantially identical intracellularly equivalent/homolog thereof, and the modification can be a covalent or non-covalent modification.
  • the covalent modification is preferably selected from the group consisting of ribosylation, phosphorylation, methylation acetylation, alkylation, glycosylation, sulfation, amidation, and proteolytic processing.
  • the host cell is the microbial cell described herein above, i.e. a microbial cell comprising at least one microbial cell surface polypeptide and a substantially identical intracellular equivalent thereof,
  • the microbial cell is selected from the group consisting of Lactobacillus species and Bifidobacterium species, and
  • the activity of the intracellular equivalent is capable of converting a substrate in a Lactobacillus metabolic pathway and/or a Bifidobacterium metabolic pathway, and
  • the at least one microbial cell surface polypeptide is encoded by a first polynucleotide operably linked to a second polynucleotide capable of directing the expression of said first polynucleotide, and wherein the first and second polynucleotides are not natively associated, and
  • the production and/or secretion and/or modification of the at least one microbial cell surface polypeptide is altered as compared to the production thereof when the first polynucleotide is operably linked to its native expression signal.
  • the host cell transformed with the below polynucleotides can also be a cell where the expression of the gene encoding the cell surface polynucleotide is directed by a native expression signal, and wherein the secretion and/or modification of the cell surface polynucleotide is altered as a result of mutagenesis, or altered expression of one or more chaperones or one or more components of the secretion machinery or one or more enzymes involved in performing post-translational modifications of polypeptides.
  • An increased secretion and/or modification can be determined by suitable enzymatic assays and/or immunological assays.
  • Preferred host cells are selected from the group consisting of Gram-positive, non- pathogenic bacteria, such as from the group consisting of the genus of Lactobacillus and the genus of Bifidobacterium.
  • Presently preferred host cells comprise Lactobacillus acetotolerans, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus arizonensis, Lacto- bacillus aviarius, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus coelohominis, Lactobacillus collinoides, Lactobacillus coryniformis subsp.
  • lactis Lactobacillus durianus, Lactobacillus equi, Lactobacillus farciminis, Lactobacillus ferintoshensis, Lactobacillus fer- mentum, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus helveticus subsp.
  • Lactobacillus heterohiochii Lactobacillus hilgardii, Lacto- bacillus homohiochii, Lactobacillus intestinalis, Lactobacillus japonicus, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacillus kimchii, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillus letivazi, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus maltaromi- cus, Lactobacillus manihotivorans, Lactobacillus mindensis, Lactobacillus mucosae,
  • Lactobacillus murinus Lactobacillus nagelii, Lactobacillus oris, Lactobacillus panis, Lactobacillus pantheri, Lactobacillus parabuchneri, Lactobacillus paracasei subsp. paracasei, Lactobacillus paracasei subsp. pseudoplantarum,, Lactobacillus paracasei subsp.
  • Lactobacillus parakefiri Lactobacillus paralimentarius
  • Lactoba- cillus paraplantarum Lactobacillus pentosus
  • Lactobacillus perolens Lactobacillus plantarum
  • Lactobacillus pontis Lactobacillus psittaci
  • Lactobacillus reuteri Lactobacillus rhamnosus
  • Lactobacillus ruminis Lactobacillus sakei
  • Lactobacillus salivarius Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp.
  • Lactobacillus sanfranciscensis Lactobacillus sharpeae, Lactobacillus suebicus, Lactobacillus thermophilus, Lactobacillus thermotolerans, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vitulinus, Lactobacillus vermiforme, Lactobacillus zeae
  • Additional preferred host cells comprise Bifidobacterium adolescentis, Bifidobacte- rium aerophilum, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bourn, B dobacterium breve, Bifidobacterium catenulatum, Bifidobacterium choerinum, B dobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacterium dentium, B dobacterium gallicum, Bifidobacterium gallinarum, Bifidobacterium indicum, B dobacterium longum, Bifidobacterium longum bv Longum, Bifidobacterium longum bv. Infantis,
  • Bifidobacterium scardovii Bifidobacterium subtile, Bifidobacterium thermoacidophi- lum, Bifidobacterium thermoacidophilum subsp. suis, Bifidobacterium thermophilum, Bifidobacterium urinalis.
  • first polynucleotides encoding a cell surface polynucleotide includes SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7 and fragments thereof encoding a polypeptide capable of acting as a cell surface polypeptide and capable of binding an epithelial cell and/or a cell of the mucosa-associated lymphoid tissue (MALT).
  • a polynucleotide comprising or essentially consisting of the coding sequence of gap encoding a glyceraldehyde 3-phosphate dehydrogenase of Lactobacillus plantarum 299v, as deposited with DSMZ under accession number DSM 9843;
  • a polynucleotide encoding a fragment of a polypeptide encoded by polynucleotides (i), (ii) or (iii), wherein said fragment
  • a) has glyceraldehyde 3-phosphate dehydrogenase activity
  • c) is competing with a polypeptide comprising or essentially consisting of the amino acid sequence as shown in SEQ ID NO:2 for binding to at least one predetermined binding partner; and v) a polynucleotide, the complementary strand of which hybridises, under stringent conditions, with a polynucleotide as defined in any of (i), (ii) (iii), and (iv), and encodes a polypeptide that
  • a) has glyceraldehyde 3-phosphate dehydrogenase activity
  • c) is competing with a polypeptide comprising or essentially consisting of the amino acid sequence as shown in SEQ ID NO:2 for binding to at least one predetermined binding partner,
  • a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in any of (iv) and (v),
  • SEQ ID NO:3 SEQ ID NO:3, or a polynucleotide comprising nucleotides 2428 to 2630 of SEQ ID NO:11 , and
  • a polynucleotide comprising or essentially consisting of the coding sequence of pgk encoding a phosphoglycerate kinase of Lactobacillus plantarum 299v, as deposited with DSMZ under accession number DSM
  • a) has phosphoglycerate kinase activity
  • c) is competing with a polypeptide comprising or essentially consisting of the amino acid sequence as shown in SEQ ID NO:4 for binding to at least one predetermined binding partner;
  • v a polynucleotide, the complementary strand of which hybridises, under stringent conditions, with a polynucleotide as defined in any of (i), (ii) (iii), and (iv), and encodes a polypeptide that
  • a) has phosphoglycerate kinase activity
  • c) is competing with a polypeptide comprising or essentially consisting of the amino acid sequence as shown in SEQ ID NO:4 for binding to at least one predetermined binding partner,
  • a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in any of (iv) and (v),
  • polynucleotide selected from the group consisting of i) SEQ ID NO:5, or a polynucleotide comprising nucleotides 3657 to 4415 of SEQ ID NO:11 , and
  • a polynucleotide comprising or essentially consisting of the coding sequence of tpi encoding a triose phosphate isomerase of Lactobacillus plantarum 299v, as deposited with DSMZ under accession number DSM 9843;
  • a polynucleotide encoding a fragment of a polypeptide encoded by polynucleotides (i), (ii) or (iii), wherein said fragment
  • a) has triose phosphate isomerase activity
  • c) is competing with a polypeptide comprising or essentially consisting of the amino acid sequence as shown in SEQ ID NO:6 for binding to at least one predetermined binding partner;
  • v a polynucleotide, the complementary strand of which hybridises, under stringent conditions, with a polynucleotide as defined in any of (i), (ii) (iii), and (iv), and encodes a polypeptide that
  • a) has triose phosphate isomerase activity
  • b) is recognised by an antibody, or a binding fragment thereof, which is capable of recognising SEQ ID NO:6; and/or c) is competing with a polypeptide comprising or essentially consisting of the amino acid sequence as shown in SEQ ID NO:6 for binding to at least one predetermined binding partner,
  • a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in any of (iv) and (v),
  • SEQ ID NO:7 a polynucleotide comprising nucleotides 4497 to 5825 of
  • a polynucleotide comprising or essentially consisting of the coding se- quence of eno encoding an enolase of Lactobacillus plantarum 299v, as deposited with DSMZ under accession number DSM 9843;
  • a polynucleotide encoding a fragment of a polypeptide encoded by polynucleotides (i), (ii) or (iii), wherein said fragment
  • a) has enolase activity
  • b) is recognised by an antibody, or a binding fragment thereof, which is capable of recognising SEQ ID NO:8; and/or c) is competing with a polypeptide comprising or essentially consisting of the amino acid sequence as shown in SEQ ID NO:8 for binding to at least one predetermined binding partner; and
  • v a polynucleotide, the complementary strand of which hybridises, under stringent conditions, with a polynucleotide as defined in any of (i), (ii) (iii), and (iv), and encodes a polypeptide that
  • a) has enolase activity
  • c) is competing with a polypeptide comprising or essentially consisting of the amino acid sequence as shown in SEQ ID NO:8 for binding to at least one predetermined binding partner,
  • a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in any of (iv) and (v),
  • the present invention is also directed to polypeptides encoded by the above polynucleotides as well as variants and functional equivalents of such polypeptides.
  • polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, including fragments, variants and functional equivalents thereof as described below in more detail.
  • variants are determined on the basis of their degree of identity or their degree of homology with any predetermined sequence of consecutive amino acid sequences of a fragment of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8.
  • variants preferably have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85 % sequence identity, for example at least 90 % sequence identity, such as at least 91 % sequence identity, for example at least 91% sequence identity, such as at least 92 % sequence identity, for example at least 93 % sequence identity, such as at least 94 % sequence identity, for example at least 95 % sequence identity, such as at least 96 % sequence identity, for example at least 97% sequence identity, such as at least 98 % sequence identity, for example 99% sequence identity with the predetermined SEQ ID NO:2,
  • SEQ ID NO:4 SEQ ID NO:6, AND SEQ ID NO:8 sequence of consecutive amino acid residues.
  • Sequence identity is determined in one embodiment by using the algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.
  • predetermined sequence is a defined sequence used as a basis for a sequence comparison; a predetermined sequence may be a subset of a larger sequence.
  • Optimal alignment of sequences for aligning a comparison window may be con- ducted by the local homology algorithm of Smith and Waterman (1981 ) Adv. Appl.
  • Math. 2: 482 by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerised implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wis- consin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
  • sequence identity means that two amino acid sequences are identical over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which identical amino acid residues occur 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.
  • a degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences.
  • a degree of homology or similarity of amino acid sequences is a function of the number of amino acids, i.e. structurally related, at positions shared by the amino acid sequences.
  • substantially identical means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75 percent sequence identity, such as at least 80 percent sequence identity, for example at least 85 percent sequence identity, such as e.g.
  • amino acid residues such as 10 amino acid residues, for example 11 amino acid residues, such as 12 amino acid residues, for example 13 amino acid residues, such as 14 amino acid residues, for example 15 amino acid residues, such as 20 amino acid residues, for example 30 amino acid residues, such as 40 amino acid residues, for example 50 amino acid residues, such as 60 amino acid residues, for example 70 amino acid residues, such as 80 amino acid residues, for example 90 amino acid residues, such as 100 amino acid residues, for example 110 amino acid residues, such as 120 amino acid residues, for example 130 amino acid residues, such as 140 amino acid residues, for example 150 amino acid residues, such as 175 amino acid residues, for example 200 amino acid residues, such as 225 amino acid residues, for example 250 amino acid residues, such as 275
  • Conservative amino acid substitutions refer in one embodiment to the interchange- ability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine, a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine.
  • variants are also determined based on a predetermined number of conservative amino acid substitutions as defined herein below.
  • Conservative amino acid substitution as used herein relates to the substitution of one amino acid (within a predetermined group of amino acids) for another amino acid (within the same group), wherein the amino acids exhibit similar or substantially similar characteristics.
  • Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr,
  • Amino acids having non-polar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met)
  • Amino acids having aliphatic side chains (Gly, Ala Val, Leu, lie)
  • Amino acids having acidic side chains (Asp, Glu)
  • a variant or a fragment thereof according to the invention may comprise at least one substitution, such as a plurality of substitutions introduced independ- ently of one another. It is clear from the above outline that the same variant or fragment thereof may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.
  • the addition or deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids.
  • additions or deletions of more than 50 amino acids such as additions from 50 to 100 amino acids, addition of 100 to 150 amino acids, addition of 150-250 amino acids, are also comprised within the present invention.
  • the deletion and/or the addition may - independently of one another - be a deletion and/or an addition within a sequence and/or at the end of a sequence.
  • polypeptide fragments according to the present invention may in one embodiment comprise a sequence of consecutive SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 amino acid residues of less than 250 amino acid residues, such as less than 240 amino acid residues, for example less than 225 amino acid residues, such as less than 200 amino acid residues, for example less than 180 amino acid residues, such as less than 160 amino acid residues, for example less than 150 amino acid residues, such as less than 140 amino acid residues, for example less than 130 amino acid residues, such as less than 120 amino acid residues, for example less than 110 amino acid residues, such as less than 100 amino acid residues, for example less than 90 amino acid residues, such as less than 85 amino acid residues, for example less than 80 amino acid residues, such as less than 75 amino acid residues, for example less than 70 amino acid residues, such as less than 65 amino acid residues, for example less than 60 amino acid residues, such
  • Frctional equivalency as used in the present invention is according to one pre- ferred embodiment established by means of reference to the corresponding functionality of a predetermined fragment of the sequence.
  • Functional equivalents or variants or fragments of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 as described herein will be understood to exhibit amino acid sequences gradually differing from preferred, predetermined sequences, as the number and scope of insertions, deletions and substitutions including conservative substitutions, increases. This difference is measured as a reduction in homology between a preferred, predetermined sequence and the SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 fragment or SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 variant or functional equivalent.
  • SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID N0:8 fragments comprising or consisting of consecutive SEQ ID N0:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 amino acid residues as well as variants and functional equivalents thereof are included within the scope of this invention, regardless of the degree of homology they show to a predetermined sequence. The reason for this is that some regions of the SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 fragments are most likely readily mutatable, or capable of being completely deleted, without any significant effect on e.g. the binding activity of the resulting fragment.
  • a functional variant obtained by substitution may well exhibit some form or degree of native binding activity, and yet be less homologous, if residues containing functionally similar amino acid side chains are substituted.
  • Functionally similar in this respect refers to dominant characteristics of the side chains such as hydrophobic, basic, neutral or acidic, or the presence or absence of steric bulk. Accordingly, in one embodiment of the invention, the degree of identity is not a principal measure of a fragment being a variant or functional equivalent of a preferred predetermined fragment according to the present invention.
  • the homology between amino acid sequences may be calculated using well known algorithms such as any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.
  • Fragments sharing homology with fragments comprising or consisting of consecutive SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 amino acid residues are to be considered as falling within the scope of the present invention when they are preferably at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 percent homologous, for example at least 99 percent homologous with a predetermined SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 fragment.
  • the homology percentages indicated above are identity percentages. Additional factors that may be taken into consideration when determining functional equivalence according to the meaning used herein are i) the ability of antisera to detect a SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 fragment according to the present invention, or ii) the ability of a functionally equivalent SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 fragment to compete with a predetermined SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 fragment in an assay.
  • One method for determining a sequence of immunogenically active amino acids within a known amino acid sequence has been described by Geysen in US 5,595,915 and is incorporated herein by reference.
  • SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 fragments it may also be desirable to introduce non-conservative substitutions in any one or more positions of such a fragment.
  • a non-conservative substitution leading to the formation of a functionally equivalent fragment would for example i) differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, lie, Leu, Phe or Met) substituted for a residue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gin or a charged amino acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such
  • Variants obtained by substitution of amino acids may in one preferred embodiment be made based upon the hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like.
  • Exemplary amino acid substitutions which take various of the foregoing char- acteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the present invention relates to functional variants comprising substituted amino acids having hydrophilic values or hydropathic indices that are within +/-4.9, for example within +/-4.7, such as within +/-4.5, for example within +A-4.3, such as within +/-4.1 , for example within +/-3.9, such as within +/-3.7, for example within +/- 3.5, such as within +/-3.3, for example within +/- 3.1 , such as within +/- 2.9, for example within +/- 2.7, such as within +/-2.5, for example within +/-
  • +/- 2.3 such as within +/- 2.1 , for example within +/- 2.0, such as within +/- 1.8, for example within +/- 1.6, such as within +/- 1.5, for example within +/- 1.4, such as within +/- 1.3 for example within +/- 1.2, such as within +/- 1.1 , for example within +/- 1.0, such as within +/- 0.9, for example within +/- 0.8, such as within +/- 0.7, for example within +/- 0.6, such as within +/- 0.5, for example within +/- 0.4, such as within +/- 0.3, for example within +/- 0.25, such as within +/- 0.2 of the value of the amino acid it has substituted.
  • hydrophilic and hydropathic amino acid indices in conferring interactive biologic function on a protein is well understood in the art (Kyte & Doolit- tle, 1982 and Hopp, U.S. Pat. No. 4,554,101 , each incorporated herein by reference).
  • amino acid hydropathic index values as used herein are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4 ); threonine (-0.7 ); serine (-0.8 ); tryptophan (-0.9); tyrosine (-1.3); praline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5) (Kyte & Doolittle, 1982).
  • amino acid hydrophilicity values are: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1 ); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); praline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4) (U.S. 4,554,101).
  • sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be employed to modify the amino terminus of, e.g., a di-arginine peptide backbone, to mimic a tetra peptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • variants and functional equivalents of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 also includes derivatives of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 or fragments thereof, for example fragments substituted with one or more chemical moieties.
  • Peptides with N-terminal alkylations and C-terminal esterifications are also encompassed within the present invention.
  • Functional equivalents also comprise glycosyl- ated and covalent or aggregative conjugates formed with the same or other polypeptide selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8, or a fragment thereof, including dimers and unrelated chemical moieties.
  • Such functional equivalents are prepared by linkage of functionalities to groups which are found in fragment including at any one or both of the N- and C-termini, by means known in the art.
  • Functional equivalents may thus comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:4, SEQ ID NO:
  • SEQ ID NO:8 or fragments thereof conjugated to aliphatic or acyl esters or amides of the carboxyl terminus, alkylamines or residues containing carboxyl side chains, e.g., conjugates to alkylamines at aspartic acid residues; O-acyl derivatives of hydroxyl group-containing residues and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g. conjugates with fMet-Leu-Phe or immunogenic proteins.
  • Derivatives of the acyl groups are selected from the group of alkyl-moieties (including C3 to C10 normal alkyl), thereby forming alkanoyl species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species.
  • the reactive groups preferably are difunctional compounds known per se for use in cross-linking proteins to insoluble matrices through reactive side groups. Covalent or aggregative functional equivalents and derivatives thereof are useful as reagents in immunoassays or for affinity purification procedures.
  • a SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 or fragments thereof according to the present invention may be insolubilized by covalent bonding to cyanogen bromide-activated Sepharose by methods known per se or adsorbed to polyolefin surfaces, either with or without glutaraldehyde cross-linking, for use in an assay or purification of anti-SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 antibodies or cell surface receptors.
  • Fragments may also be labelled with a detectable group, e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in e.g. diagnostic assays.
  • a detectable group e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in e.g. diagnostic assays.
  • SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 or fragments thereof according to the invention may be synthesised both in vitro and in vivo.
  • the SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 fragments of the invention are synthesised by automated synthesis. Any of the commercially available solid-phase techniques may be employed, such as the Merrifield solid phase synthesis method, in which amino acids are sequentially added to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963).
  • Insertions shall be understood to include amino- terminal and/or carboxyl-terminal fusions, e.g. with a hydrophobic or immunogenic protein or a carrier such as any polypeptide or scaffold structure capable as serving as a carrier.
  • Oligomers including dimers including homodimers and heterodimers of any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 or fragments thereof fragments according to the invention are also provided and fall under the scope of the invention.
  • Functional equivalents and variants of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8, or fragments thereof can be produced as homodimers or heterodimers with other amino acid sequences or with native SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 sequences.
  • Heterodimers include dimers containing immunoreactive SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 variants and fragments as well as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8 variants and fragments that need not have or exert any biological activity.
  • a method for producing a microbial cell surface polypeptide, or a fragment thereof capable of modulating an immune re- sponse in an individual or modulating the amount and/or composition of mucosal mucins comprising the step of culturing a host cell as described herein under conditions suitable for the production of said polypeptide, or fragment thereof.
  • the cell surface polypeptide is preferably selected from any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8, including functional equivalents and variants and fragments thereof.
  • a method for producing an epithelial adhe- sive polypeptide or a MALT cell adhesive polypeptide, or a fragment thereof comprising the step of culturing a host cell as described herein under conditions suitable for the production of said epithelial adhesive polypeptide, or fragment thereof.
  • the adhesive polypeptide is preferably selected from any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8, including functional equivalents and vari- ants and fragments thereof.
  • Antibodies there is also provided a polyclonal antibody or a monoclonal antibody specific for any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8, including functional equivalents and variants and fragments thereof.
  • the invention also provided antagonists and agonists for any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8, including functional equivalents and variants and fragments thereof.
  • compositions and methods for treatment of an individual are provided.
  • The is provided a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide selected from any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, AND SEQ ID NO:8, includ- ing functional equivalents and variants and fragments thereof.
  • the polypeptide can be provided in purified or isolated form or the polypeptide can be provided as part of a Lactobacillus cell and/or Bifidobacterium cell in a composition comprising such cells.
  • the invention in preferred embodiments relates to pharmaceutical compositions which comprise the above-mentioned polypeptides as well as variants or fragments of these molecules as defined herein above for the treatment of disorders of the immune system.
  • compositions according to the invention can be formulated according to known methods such as by the admixture of one or more pharmaceutically or veterinary acceptable excipients or carriers. Examples of such excipients, carriers and methods of formulation may be found e.g. in Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, PA). To form a pharmaceutically or veterinary acceptable composition suitable for effective administration, such compositions will contain an effective amount of a polypeptide, nucleic acid, antibody or compound modulator.
  • compositions of the invention are administered to an individual (mammal-human or animal) or used in amounts sufficient to treat or diagnose apoptosis-related disorders.
  • the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • the term functional derivative includes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or de- crease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such infor- mation can then be used to determine useful doses and routes for administration in humans and other animals.
  • a therapeutically effective dose refers to that amount of compound, peptide, antibody or nucleic acid which ameliorate or prevent a dysfunctional apoptotic condition. The exact dosage is chosen by the individual physician in view of the patient to be treated.
  • compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Administration of pharmaceutical compositions is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the tissue), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
  • compositions containing compounds identified according to this invention as the active ingredient for use in the modulation of a protein which mediates apoptosis can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
  • the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • An effective but non-toxic amount of the compound, nucleic acid, or peptide desired can be employed as an apoptosis modulating agent.
  • the daily dosage of the products may be varied over a wide range such as e.g. from about 1 to 10,000 mg per adult human/per day.
  • the compo- sitions are preferably provided in the form of scored or unscored tablets containing
  • An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day.
  • the range is more par- ticularly from about 0.001 mg/kg to preferably less than 100 mg/kg of body weight per day.
  • the dosage level will vary depending upon the potency of the particular compound. Certain compounds will be more potent than others. In addition, the dosage level will vary depending upon the bioavailability of the compound. The more bioavailable and potent the compound, the less compound will need to be administered through any delivery route, including but not limited to oral delivery.
  • dosages of living modulators are adjusted when combined to achieve desired effects.
  • dosages of these various agents may be independently optimised and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
  • Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors.
  • combination therapies comprising the step of administering the vaccine compositions according to the invention in combination with a chemotherapeutic agent and/or an immunotherapeutic agent and/or a cancer vaccine.
  • Conditions capable of being treated include, but is not limited to auto-immune dis- eases.
  • Autoimmune diseases may be loosely grouped into those primarily restricted to specific organs or tissues and those that affect the entire body.
  • organ-specific disorders include multiple sclerosis (myelin coating on nerve processes), type I diabetes mellitus (pancreas), Hashimotos thy- roiditis (thyroid gland), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at neuromuscular junction), rheumatoid arthritis (joint lining), uveitis (eye), psoriasis (skin), Guillain-Barre Syndrome (nerve cells) and Grave's disease (thyroid).
  • Systemic autoimmune diseases include systemic lupus erythematosus and dermatomyositis.
  • intestinal inflammations such as Coeliac disease, proctitis, eosinophilia gastroenteritis, mastocytosis, inflammatory bowel disease, Crohn's disease and ulcerative colitis, as well as food-related allergies.
  • a microbial cell surface polypeptide the intracellular equivalent of which is selected from glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, triose phosphate isomerase, and enolase, including variants and functional equivalents thereof, and
  • iii) optimising the production and/or secretion and/or modification in the microbial cell of a polypeptide selected from the group consisting of glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, triose phosphate isomerase, and enolase, including variants and functional equivalents thereof, and
  • Example 1 Isolation of major surface located proteins from Lactobacillus plantarum strain 299v Lactobacillus plantarum strain 299v was pre-cultivated on Man-Rogosa-Sharpe
  • MRS Magnetic Reagar
  • L. plantarum 299v agar plate for 48 hours at 37° C.
  • Glass tubes containing 15 mL MRS medium was subsequently inoculated with pre- cultured cells of L. plantarum 299v and left overnight at 37° C without aeration.
  • the L. plantarum 299v culture (OD 60 o of 6.5) was harvested by centrifugation (4000 x g/4°C) and washed once in PBS buffer (136.9 mM sodium chloride, 2.68 mM potassium chloride, 8.1 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate). The pellet was resuspended in PBS to a final optical density at 600 nm of 65. The suspension was incubated statically at 37° C for 3 hours, and proteins released from the outer cell surface of L. plantarum 299v into the PBS buffer was analysed by SDS-PAGE.
  • Samples were analysed by SDS-PAGE using 12 % Invitrogen NuPage TM BIS-TRIS gels (Invitrogen, San Diego, Calif.), gelmatrix: acrylamide/bis-acrylamide, size: 8x8 cm 2 , 1 mm gel thickness.
  • the running buffer used was a 2-(N- morpholino)ethanesulfonic acid (MES) SDS buffer.
  • Samples for SDS-PAGE analysis were prepared by mixing 22.5 ⁇ l sample, 12.5 ⁇ l NuPage TM lithium dodecyl sulphate (LDS) sample buffer (Invitrogen) and 5 ⁇ l NuPage TM sample reducing agent (Invitrogen). The mixture was incubated at 56°C for 20 min prior to analysis.
  • LDS lithium dodecyl sulphate
  • Example 2 Analysis of released surface proteins by mass spectrometry Bands visualised by Coomassie blue staining were excised from the gel and in-gel digested with trypsin. The excised gel bands were transferred to 1.5 mL eppendorf microcentrifuge tubes and incubated with 200 ⁇ l ultra pure water. After 10 min of incubation the gel pieces were transferred to a clean glass plate, cut into small pieces (approximately 1 mm 3 ) and rinsed with 200 ⁇ l ultra pure water in eppendorf microcentrifuge tubes. The gel pieces were rinsed, shrunk by adding 30 ⁇ l 100% acetonitrile, and subsequently dried in a vacuum centrifuge.
  • the column was washed with 50 ⁇ L formic acid and the bound peptides were eluted directly into a nanospray needle (Protana, Odense, Denmark) with 2 ⁇ L of a 50% MeOH-1 % formic acid solution.
  • the digestion mixtures were analysed by nanoelectrospray mass spectrometry (nano ESI MS) using a Q-Tof mass spectrometer (Micromass, Manchester, United Kingdom). Selected peptides were sequenced by nanoelectrospray tandem mass spectrometry (nano ESI MS/MS). The resulting peptide sequences were used to search for short nearly exact matches in the non-redundant Blast protein-protein (www.ncbi.nlm.nih/gov/BLAST/) sequence database, enabling identification of the proteins.
  • Table 1 Peptide sequences and protein identifications/assignments from proteins derived from one-dimensional SDS-PAGE separation of cell surface associated proteins. The bands were in-gel digested with trypsin and selected peptides sequenced by ESI MS/MS analysis. Proteins were identified using the non-redundant Blast protein-protein (www.ncbi.nlm.nih/oov/BLAST/) sequence database.
  • Example 4 Cloning of the gene encoding glyceraldehyde 3 phosphate dehydrogenase (GAPDH) from Lactobacillus plantarum 299v
  • GPD-Nterm (INGFGRIG (SEQ ID NO:21)) (5' ATHAAYGGNTTYGGNMGNATHGGN
  • GPD-mid REV (TGAAKAVGK (SEQ ID NO:23)) (5' YTTNCCNACNGCYTTNGC
  • Total DNA from L. plantarum 299v was used as template.
  • a standard PCR reaction condition with the Taq DNA polymerase (Invitrogen, Carlsbad, Calif.), expect that the concentration of each primer was 5 ⁇ M, was used to amplify the gapdh gene.
  • a PCR product of the expected size was purified from an agarose gel using the GFXTM PCR DNA and gel band purification kit (Amersham Biosciences Corp., Pis- cataway, NJ) and inserted into the pCR ® 2.1-TOPO vector (Invitrogen, Carlsbad, Calif.). The DNA sequence of the insert was determined using an ALFexpress DNA sequencer and universal M13 forward and reverse primers.
  • the remaining part of the gapdh gene and the adjacent DNA regions were amplified by consecutive rounds of inverse PCR (Ochman et al.; 1988).
  • total DNA of L. plantarum 299v was digested with either EcoRI or Hindlll and religated in a large volume.
  • PCR amplifications were carried out using DNA primers based on DNA sequences that were obtained during the successive rounds of inverse PCR.
  • the polynucleotide sequence and the polypeptide sequence of gapdh are shown in Figs. 3 and 4.
  • the gapdh gene of 299v encodes a 340 aa protein.
  • a blastp similarity search showed that gapdh protein of L.
  • plantarum is 96% (low complexity filter on) identical to the gapdh gene from L. plantarum WCFS1 (Ace. No. CAD63377) and 81% (low complexity filter on) identical to a hypothetical protein from Lb. gasseri (Ace. No. ZP_00047412.1).
  • Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is an enzyme of the glycolytic pathway, in which it catalyzes the oxidative conversion of D- glyceraldehyde 3-phosphate and phosphate to 3-phospho-D-glyceroyl phosphate using NAD + as an acceptor.
  • Example 5 Cloning of the gene encoding phosphoglycerate kinase (PGK) from Lactobacillus plantarum 299v During the course of cloning and sequencing of the gapdh gene we identified the 5' end of the gene encoding phosphoglycerate kinase (PGK; EC. 2. 7. 2. 3.). In the glycolytic pathway, PGK catalyses the phosphotransferase reaction between 3- phospho-D-glyceroyl phosphate and ADP to produce ATP and 3-phospho-D- glycerate. The pgk gene is located immediately downstream of the gapdh gene of L. plantarum 299v.
  • the remaining part of the pgk gene was cloned by consecutive rounds of inverse PCR as described above.
  • the polynucleotide sequence and the polypeptide sequence of pgk are shown in Figs. 3 and 5.
  • the pgk gene encodes a 400 aa protein.
  • a blastp similarity search showed that the pgk protein from L. plan- tarum is 99% (low complexity filter on) identical to the pgk gene from L. plantarum
  • WCFS1 (Ace. No. CAD63378) and 74% (low complexity filter on) identical to the pgk gene from Lb. delbrueckii (Ace. No. CAD56495).
  • TPI triosephosphate isomerase
  • TPI triosephosphate isomerase
  • plantarum is 99% (low complexity filter on) identical to the tpi gene from Lactobacillus plantarum WCFS1 (Ace No. CAD63379) and 71% (low complexity filter on) identical to the tpi gene from L. delbrueckii (Ace. No. 032757).
  • TPI catalyzes the isomerisation of D-glyceraldehyde 3-phosphate to glycerone phosphate and vice versa in the glycolytic pathway.
  • Example 7 Cloning of the gene encoding enolase (eno) (phosphoenolpyru- vate hydratase) from Lactobacillus plantarum 299v
  • Eno-Deg1 corresponds to the peptide sequence VEVELYTES (SEQ ID NO:27), which, is found in the amino terminal of the enolase whereas Eno-Deg2 corresponds to the peptide sequence GDDLFVTN (SEQ ID NO:28) located approximately 300 amino acids downstream of the start codon of the enolase.
  • the following PCR profile was used to amplify an internal 0.9 kb enolase fragment: 94 °C 2 min
  • Total DNA from L. plantarum 299v was used as template.
  • a PCR product of approximately 0.9 kb was purified from an agarose gel using the GFXTM PCR DNA and gel band purification kit (Amersham Biosciences Corp., Pis- cataway, NJ) and inserted into the pCR ® 2.1-TOPO vector.
  • the DNA sequence of the insert was determined using the ALFexpress DNA sequencer and universal M13 forward and reverse primers. The remaining part of the enolase gene and the adjacent DNA regions were amplified by consecutive rounds of inverse PCR as described above.
  • triosephosphate isomerase gene upstream of the 5' end of the enolase gene, which consequently re- vealed that the four genes are placed in the order gapdh-pgk-tpi-eno and suggests that the genes are clustered in an operon.
  • Enolase of 299v encodes a 442 aa protein.
  • a blastp similarity search showed that enolase protein of L. plantarum 299v is 98% (low complexity filter on) identical to the phosphopyruvate hydratase from L. plantarum WCFS1 (Ace No. CAD63380) and 77% identical to a hypothetical protein from Lb. gasseri (Ace. No. ZP_00047409).
  • Example 8 Cloning of the gene encoding a putative regulator of the gapdh- pgk-tpi-eno operon from Lactobacillus plantarum 299v
  • the regulatory gene is located upstream of the gapdh gene of L. plantarum 299v.
  • the remaining part of the regulator gene was cloned by consecutive rounds of inverse PCR as described above.
  • the polynucleotide sequence and the polypeptide sequence of the regulator are shown in Figs. 3 and 8.
  • the glycolytic regulator encodes a 343 aa protein.
  • a blastp similarity search showed that the glycolytic regulator from L. plantarum 299v is 93% (low complexity filter on) identical to the central glycolytic regulator from L. plantarum WCFS1 (Ace. No. CAD63376) and 45% (low complexity filter on) identical to a hypothetical transcriptional regulator from Listeria innocua (Ace. No. NP_471884).
  • Gapdh (SEQ ID NO:1 ):
  • Tpi SEQ ID NO:5: Start position: 3657 (Valine)
  • GAPDH Detection of GAPDH activity in untreated culture samples As described in Example 3, GAPDH appeared as a major band when proteins released from the cells in PBS were analyzed by SDS-PAGE. To test whether the surface-located GAPDH protein was enzymatically active, we performed activity assays on untreated culture samples. NAD and NADH, which take part in the GAPDH reaction, are not taken up by intact cells. Therefore, the intracellular GAPDH will not be detected without prior lysis or permeabilization of the cells. We refer to the activity measured in untreated culture samples as "extracellular/surface located".
  • GAPDH assay was performed with a modification of the procedure described by Gil- Navarro et al. (1997). 16 ⁇ L sample was mixed in a 1 cm light path cuvette with reaction mixture to a final volume of 0.8 mL. The reaction mixture contained 1 mM
  • a 340 was measured at intervals throughout a total incubation time of 5-180 min, depending on the activity of the sample. For each sample, the slope of A 340 versus time was calculated, and the slope of the control reaction without glyceralde- hyde 3-phosphate was subtracted. Further correction was made for A 340 decrease in reaction mixture with buffer added instead of sample. To obtain the activity in units/mL, the corrected slope was multiplied by the reaction volume and divided by the sample volume and the millimolar extinction coefficient of NADH, 6.3 (mM-cm) "1 . 1 unit of GAPDH will catalyse production of 1 ⁇ mole 1 ,3-diphosphoglyceric acid per minute.
  • L. plantarum 299v was grown without shaking or aeration at 30°C in MRS broth (De Man et al.; 1960) prepared from dehydrated medium (OXOID Ltd., Basingstoke, Hampshire, England).
  • the extracellular/surface-located GAPDH activity was found to be low during active growth of the cultures, but increased in stationary phase to above 0.05 u/mL. Thus, the extracellular/surface-located GAPDH appears to be growth phase dependent.
  • sMRS surface-located GAPDH activity
  • this medium shown in Table 9-1
  • the dehydrated MRS broth contains all the final components, which are dissolved in water and autoclaved together.
  • sMRS some components are sterilised separately to avoid breakdown or precipitation of important nutrient factors:
  • a basal medium consisting of peptones and meat extract are autoclaved with Tween 80, salts, acetate and citrate.
  • Yeast extract is filter-sterilised (0.22 ⁇ m pore size filter).
  • Fig. 9 shows the results from parallel cultures in sMRS and in a low-yielding batch of MRS.
  • GAPDH assays were performed in untreated culture samples taken in late growth phase (OD 60 o 7.5-8.6) and again 22 hours later in stationary phase (OD 60 o 12-12.5). Even after such prolonged incubation, the GAPDH activity in the MRS cultures was below 0.03 u/mL.
  • GAPDH activity followed the pattern observed earlier for some other batches of MRS, i.e. increasing activity in stationary phase.
  • sMRS we consistently obtained high activities in untreated samples from stationary phase cultures. Therefore, sMRS was used in all subsequent experiments on the surface-located GAPDH, unless otherwise stated.
  • the ratio between the culture surface and the volume of the cultivation medium was found to influence the amount of GAPDH activity located extracellulariy and/or on the cell surface. This was demonstrated by growing L. plantarum 299v in two screw- capped 15 mL tubes containing 5 mL sMRS medium. Incubating one tube horizontally and the other in an upright position resulted in a remarkable difference in extra- cellular/surface-located GAPDH activity, as shown in Table 9-2. The difference is probably connected to the exchange of oxygen, carbon dioxide and/or volatile metabolites between headspace and culture liquid. If nothing else is stated, the Lactobacillus strains were cultivated in closed tubes or flasks in upright position and with a medium to headspace ratio of at least 0.25.
  • LDH L- and D-lactate dehydrogenase
  • washed cells were lysed by ultrasound treatment with glass beads: 500 ⁇ L of cell suspension in PBS was mixed with an equal volume of glass beads (Sigma G 9143, 212-300 ⁇ m) and subjected to ultrasound at maximum effect in an ultrasound bath (Elma Transsonic Digital S) with ice for a total of 15 min. Cells and glass beads were mixed at 1-2 minute intervals by inversion of the tubes. The resulting lysate represent the intracellular fraction plus the proteins still attached to the cell surface after washing. The latter can be measured in the suspension of washed cells, and in the case of GAPDH and LDH, the activities were low (see below). For simplicity, we will refer to the GAPDH and LDH activities measured in the lysate as "intracellular".
  • LDH and GAPDH assays were performed on the lysate, culture supernatant, ESP fraction, and washed cells suspension of a stationary phase L. plantarum 299v culture. The results are shown in Fig. 12.
  • the culture supernatant and the suspen- sion of washed cells contained only negligible amounts of LDH and GAPDH activities. In the lysate, both activities were high (3 u/mL of LDH and 1 u/mL of GAPDH).
  • the ESP fraction contained a high activity of GAPDH (0.37 u/mL) and, surprisingly, also a significant amount of LDH activity (0.09 u/mL). This could indicate that LDH, like GAPDH, was presented on the cell surface in the stationary phase culture.
  • the Lactobacillus strains were inoculated in 150 or 200 ⁇ L sMRS in 300 ⁇ L wells in sterile microtiter plates (96 well polystyrene plates with round-bottom wells, Nunc a/s, Roskilde, Denmark).
  • the microtiter plates were incubated at 30°C with Anaerocult A or Anaerocult IS (Merck, Darmstadt, Germany) in anaerobic jars or sealed polyethylene bags or in an atmosphere of 10% H 2 , 10% C0 2 , 80% N 2 in a MK3 Anaerobic Work Station (DW Scientific, Shipley, West Yorkshire, UK).
  • the culture in each well was mixed with a pipette and a sample of 5 ⁇ L was transferred to the corresponding well in another microtiter plate where it was mixed with 120 or 150 ⁇ L reaction mixture.
  • the reaction mixture contained 1 mM NAD and 2 mM glyceraldehyde 3-phosphate in 0.1 mM dithiothreitol, 5 mM EDTA, 50 mM sodium phosphate, and 40 mM triethanolamine, adjusted to pH 8.6 with HCI. After incubation at ambient temperature for 30-120 min, the plates were photographed on a UV trans-illuminator. Wells with GAPDH activity were identified by their yellow fluorescence (450 nm). The microtiter plate-based GAPDH assay was used for screening purposes as described in example 14 and 15.
  • Example 10 Expression and purification of recombinant PGK, GAPDH and ENO and generation of antibodies.
  • the coding regions of the PGK, GAPDH and ENO encoding genes were amplified from the genome of Lactobacillus plantarum 299v by PCR.
  • the PCR was performed on the three individual genes using total DNA from L. plantarum 299v and three sets of primers containing engineered BamHI (GGATCC) and Xhol (C7 " CG>4G) recognition sites:
  • the resulting PCR products were 1225 bp comprising the pgk gene, 1351 bp comprising the eno gene and 1022 bp comprising the gapdh gene and contained the translation start site (ATG) and the stop codon (TAA) of the each gene.
  • the DNA fragments were BamHI/Xhol-digested and cloned into the same sites of the pGEX- 4T-3 (Pharmacia) expression vector.
  • the ligation mixtures were transformed into E. coli DH10 (Invitrogen, Carlsbad, CA, USA) according to standard procedures.
  • the recombinant PGK, GAPDH and ENO are produced as a fusion protein with the 26 kDa glutathione-S-transferase (GST) polypeptide.
  • GST glutathione-S-transferase
  • the procedure for induction and expression were optimised as follows. Overnight E. coli cultures were diluted 50 times in 100 mL fresh LB medium containing 100 ⁇ g/mL ampicillin and were incubated for 2 h at 37°C in large 1000 mL flasks at 200 RPM. The temperature was lowered to 25°C and after 0.5 h IPTG was added to a final concentration of 0.1 mM. Expression was allowed overnight at 25°C and 200 RPM. Harvested cells were washed in 5 mL
  • Fig. 13 shows the cross reaction against the GAPDH fusion protein and the wild type GAPDH protein from L. plantarum 299v.
  • Example 11 Extracellular/surface-located activity of Lactobacillus spp.
  • Lactobacillus species L. plantarum, rhamnosus, gasseri, casei and paracasei Each strain was inoculated with a small amount of material ( ⁇ 50 ⁇ L) from a frozen cryo- culture into 5 mL of sMRS medium and incubated in a 15 mL screw-capped tube at 30°C for at least 45 hours. Strains that had not developed a dense culture in 2 days were incubated for one additional day. OD 60 o was measured and GAPDH and LDH activity was determined in untreated culture samples. For detection of GAPDH and ENO by Western Blotting, ESP-fractions were prepared from the cultures and frozen for later analysis.
  • Fig. 14 shows the extracellular/surface-located activities as a mean of the result from the two tests.
  • L. plantarum ATCC8014 differs by a higher LDH activity.
  • L. plantarum C had a lower GAPDH activity (0.15 u/mL) and an LDH activity below
  • strain WCFS1 diverges by showing low activities of both enzymes.
  • L. rhamnosus strains ATCC7469, E, R, GG, and T varied considerably with respect to extracellular/surface-located GAPDH activity. Only two of the strains, E and T, showed activities that were comparable with those of L. plantarum 299v. LDH was low in all tested strains of the L. rhamnosus species. Only the L gasseri AA and Z showed high GAPDH/LDH ratios indicating that GAPDH is not directed to the surface by means of lysis. The other L. gasseri tested showed low GAPDH/LDH activity ratios indicating that the extracellular GAPDH activity is due to lysis.
  • Example 12 Immobilized binding assay.
  • GAPDH and ENO proteins were eluted from the surface of L. plantarum 299v using the procedure described in example 9. Subsequently, the eluted surface proteins (ESP) were concentrated 20 times using 4 mL spin columns with cut-off at MW 10 kDA (Millipore, MA, USA). Maxisorb microtiter wells (Nunc, Roskilde, DK) were coated with 20 ⁇ g/mL human plasma fibronectin (Sigma-Aldrich, St.
  • GAPDH and ENO The specific affinity of GAPDH and ENO towards plasminogen is significant and the binding curves display saturation at lower GAPDH and ENO concentrations than that towards fibronectin (Fig. 18 and Fig. 19).
  • GAPDH and ENO binds more strongly to plasminogen than to fibronectin.
  • ENO shows a higher affinity to plasminogen than that of GAPDH.
  • the adhesion properties of GAPDH and ENO to mucin were also investigated. These results show that GAPDH specifically binds to immobilised mucin (Fig. 20). However, the affinity of ENO to mucin is low and not significant (Fig. 21).
  • Dendritic cells play an essential immunoregulatory role in the Th1 , Th2, and Th3 cell balance and are present throughout the gastrointestinal tract. Thus, dendritic cells may be targets for modulation by gut microbes, including ingested probiotics. It has been shown that incubation of dendritic cells with killed Lactobacillus induces a strain dependent cytokine production. In this example the eluted surface proteins from the surface of L. plantarum 299v were tested for immunomodulating potential. Bone marrow cells were isolated from the femora and tibiae from two female
  • RPMI 1640 Sigma-Aldrich, St. Louis, MO, USA
  • 4 mM L-glutamine 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 50 ⁇ M 2-Mercaptoethanol, 10% (v/v) heat-inactivated FBS (Atlanta Biologicals, Nor- cross, GA, USA), and 15 ng/mL murine GM-CSF.
  • GM-CSF was added as 5-10% (v/v) culture supernatant harvested from a GM-CSF-producing cell line (GM-CSF transfected Ag8.653 myeloma cell line) and GM-CSF was quantified using a specific ELISA kit (BD PharMingen, San Diego, CA).
  • Nonadherent cells were gently pipetted from the petri dishes containing 8-day old dendritic cell-enriched cultures. The collected cells were centrifuged for 5 min at 300 x g and resuspended in medium supplemented with only 10 ng/mL GM-CSF. Cells were seeded in 48-well tissue culture plates at 1.4 x 10 6 /500 ⁇ L/well. The 20x concentrated ESP from L. plantarum 299v was then added at 100 ⁇ L/well in a series of 2 fold dilutions. The PBS alone was used as negative control. Furthermore, wells containing 100 ⁇ l supernatants of the L. plantarum 299v cultures were included.
  • IL-10 were analyzed using commercially available ELISA kits (BD PharMingen) according to manufacturer's instructions.
  • the ESP from L. plantarum 299v induces IL-10 production in dendritic cells in a concentration dependent matter.
  • the lack of IL-10 induction from PBS alone (Fig. 22) or from the culture supernatants (data not shown) of L. plantarum 299v shows that a component in the ESP is responsible for the induction of IL-10.
  • Example 14 High throughput screening of L. plantarum 299v mutant strains for low extracellular amounts of GAPDH
  • a library of random mutants of Lactobacillus plantarum 299v was generated by a modified version of the method of Bennett and O'Sullivan, 2000. Strain 299v was grown in MRS broth (Oxoid, Basingstoke, Hampshire, England) at 30°C for 24h. The optical density of the culture (at 600 nm) was 8.0. The culture was harvested (5000 RPM, 20°C, 10 min) and resuspended in 10 mL of 100 mM K 2 HP0 4 /KH 2 P0 4 buffer, pH 7.5. 100 ⁇ L of the cell suspension was withdrawn, and the remaining cell sus- pension was mixed with 1 ,2 mL ethyl methanesulfonate (EMS) (Sigma Co., St.
  • EMS ethyl methanesulfonate
  • kill rates are:
  • Microwell plates were incu- bated 21-27h at 30°C in a gas mixture containing 10% H 2 , 10% C0 2 , and 80% N 2 in a MK3 Anaerobic work station from DW scientific (Shipley, West Yorkshire, UK).
  • the assay for extracellular GAPDH activity was modified for microwell plates (example 9). Cultures were mixed using a multi channel pipette to resuspend precipi- tated cells, and 5 ⁇ L of the cultures were transferred to new microwell plates. The assay reactions were initiated by the addition of 150 ⁇ L reaction mixture. Plates were incubated 45 min at room temperature and photographed on an UV transil- luminator to record fluorescence of NADH at 450 nm. During incubation, the optical densities at 595 nm (OD 59 ) were determined using a microwell plate reader.
  • Photographs from plates were visually inspected and cultures resulting in lower fluorescence, indicating lower levels of extracellular GAPDH, were selected. For selected isolates, OD 5 g 5 readings were examined to estimate, whether low fluores- cence was a result of poor growth or low levels of extracellular GAPDH. If low fluorescence was estimated to be due to low levels of extracellular GAPDH, then isolates were selected for further analysis.
  • example 14 demonstrates generation of random mutants by EMS mutagenesis of L. plantarum 299v. Furthermore, 15000 mutant strains could be investigated for the presence of extracellular/surface-located GAPDH by use of a high-through-put screening method. Of the 15000 screened clones the high-through- put screening produced nine final candidates with apparent low amounts of extracellular/surface-located GAPDH.
  • Example 15 GAPDH and LDH activities in culture supernatants and ESP- fraction of selected isolates Overnight cultures of L plantarum 299v, L. plantarum WCFS1 , and nine selected mutants of L. plantarum 299v, were assayed for GAPDH and LDH activity in the ESP-fraction and in culture supernatants as described in example 9. The result is shown in the table below.
  • Isolate 8-C8 was deselected. This mutant showed very slow growth and had no LDH activity in a cell lysate prepared as in example 9 (not shown). It was assumed to be an LDH mutant. Several of the other clones showed a high extracellular LDH activity indicating a high degree of lysis.
  • Isolate 149-D7 was selected for further work because this strain showed normal growth, and reproducible low GAPDH activity in culture supernatants and in ESP- fractions. Lactobacillus plantarum strain 149-D7 was deposited at the DSMZ (Deut- sche Sammlung von Mikroorganismen und Zellkulturen GmbH), and has been registered under number DSM 16241.
  • New activity assays were made to confirm the low GAPDH activity in culture supernatants and ESP-fractions of strain 149-D7, and to study the activity levels for other strains.
  • Fig. 24 shows a comparison of GAPDH and LDH activities in the culture supernatants and ESP-fractions of L. plantarum strains 299v, WCFS1 , and 149-D7.
  • the GAPDH activity in the ESP fraction of 149-D7 is significantly lower than in equivalent fraction from the wild type 299v, indicating that the mutation in 149-D7 has affected genes involved in surface display of GAPDH.
  • strains 149-D7/129 and UP102 were isolated from a library of WCFS1 containing DNA fragments from L. plantarum 299v during screening for clones that displayed higher levels of extracellular/surface-associated GAPDH than the host strain WCFS1 (see example 17). Lactobacillus plantarum strain UP-102 was deposited at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), and has been registered under number DSM 16240. The plasmid in strain UP102, pUP102 was transformed into strain 149-D7 to obtain the strain 149-D7/129.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • strain 149-D7/129 had higher GAPDH activities than 149-D7 in both culture supernatants and ESP-fractions. Although the activities did not reach the levels of strain 299v, the presence of pUP102 partially complement the mutation in 149-D7 with respect to surface display of GAPDH. A high proportion of the extracellular activity was found in the ESP fraction, indicating that the complemented 149-D7 mutant has retained the 299v wild type phenotype of binding GAPDH to the surface.
  • Example 16 Immuno-detection of GAPDH, ENO and PGK from selected mu- tants of 299v.
  • Proteins in culture supernatants and ESP-fractions (surface proteins) from the L. plantarum strains 299v, WCFS1 , 149-D7, 149-D7/129 and UP102 were separated by SDS-PAGE and blotted onto nitrocellulose membranes as described above.
  • Strain UP102 described in example 18 had higher levels of all three pro- teins in both culture supernatants and at cell surfaces compared to the host strain WCFS1.
  • the levels of GAPDH, ENO and PGK were also higher for strain 149- D7/129 than for strain 149-D7. This confirms the results from activity assays for GAPDH in culture supernatants and in ESP-fractions from these strains (example 16).
  • levels of ENO and PGK correlate to the levels of GAPDH in these strains.
  • Example 17 Screening for genes involved in surface display of GAPDH
  • L. plantarum 299v is able to display the normally intracellular located enzyme glyc- eraldehyde-3-phosphate dehydrogenase (GAPDH) on the cell surface.
  • GAPDH glyc- eraldehyde-3-phosphate dehydrogenase
  • Genomic DNA was isolated from L. plantarum 299v and partially digested with Sau3AI. The partially digested DNA fragments were separated on agarose gel and fragments with a minimum size of 5 kb were isolated. These fragments were ligated with the vector pTRKL2 (O'Sullivan and Klaenhammer), which had been digested with BamHI. The ligation mixture was transformed into E. coli and transformants selected on LB agar plates containing erythromycin (200 ⁇ g/mL). Pools of transfor- mant colonies were washed off the selective agar plates and plasmid DNA was isolated from these transformant pools. The obtained plasmid DNA pools were used for transformation of L.
  • pTRKL2 O'Sullivan and Klaenhammer
  • Plasmid DNA was isolated from these clones and restriction enzyme analysis using EcoRI indicated that the three plasmids contained the same insert.
  • pUPO102 has been further characterised.
  • the plasmid contains an insert of
  • 6.2 kb (Fig. 27) and sequence analysis indicate the presence of the 3' end of a regulatory gene, the entire rpoB gene including the promoter region and the 5' end of the rpoC gene.
  • Deletions in the rpoB sequence were made by digestion with Fspl (pUP0164), Nrul (pUP0165) or Bglll (pUP0163) (Fig. 27).
  • the resulting plasmids were transformed into L. plantarum WCFS1 and the transformants analysed for surface-associated GAPDH activity. None of these transformants displayed extracellular/surface-associated GAPDH activity (Fig. 27).
  • the rpoB gene encodes the ⁇ subunit present in the core enzyme ( 2 ⁇ ') of the RNA polymerase complex.
  • the ⁇ subunit is implicated in the binding of nucleotides needed for RNA polymerisation.
  • Example 18 Analysis of eluted surface proteins by 2D-PAGE
  • Plasmid pTN1 was recently developed and successfully used for gene inactivation in
  • L. gasseri (Nue and Henrich, 2003).
  • the pTN1 vector replicates at 35°C whereas replication is efficiently shut down at 42°C, allowing the use of the vector for single copy integrations in L. gasseri.
  • the present example describes the use of pTN1 for construction of a threonine auxotroph mutant in L. plantarum 299v.
  • Inactivation of genes needed for threonine biosynthesis serves as an example of inactivation of specific genes.
  • a similar approach can be used for inactivation of genes that are expected to be essential for probiotic activity.
  • the complete genome sequence of L. plantarum WCFS1 revealed the presence of a threonine biosynthetic pathway.
  • a 500 bp PCR fragment covering an internal region of the hom2 gene was obtained using the primers hom2-thrB-1 (5' GAGGA- TATTGCGGAAGCTC 3' (SEQ ID NO:50)) and hom2-thrB-2 (5' GCGCCGGTCAAT- CATTCATGGCATGGGTAATG 3' (SEQ ID NO:51 )) and genomic L plantarum 299v DNA as template.
  • a 500 bp PCR fragment covering an internal region of the thrB gene was obtained using the primers hom2-thrB-3 (5' CATGAATGATT-
  • the two primers hom2-thrB 2 and hom2-thrB 3 contain 5 ' ends that are complementary to each other.
  • the two synthesised PCR fragments containing overlapping regions were allowed to anneal to each other before extension and amplification using the outer primers hom2-thrB 1 and hom2-thrB 4.
  • the extended PCR product was purified using the GFXTM PCR DNA and gel band purification kit (Amersham Biosciences) and inserted into the pCR2.1 ® -TOPO vector (Invitrogen) resulting in plasmid pPSM1081.
  • the polylinker region of pCR2.1 ® - TOPO contains two EcoRI restriction sites that flank the hom2-thrB insert in pPSM1081.
  • Plasmid pPSM1081 was digested with EcoRI, the 1000 bp fragment was purified and inserted into plasmid pTN1 (integration vector), which was pre- digested with EcoRI and treated with bacterial alkaline phosphatase.
  • the replicon present in pTN1 is unable to replicate in E. coli whereas replication in
  • L. lactis is efficient.
  • the ligation mixture was therefore transformed into L. lactis MG1363 and selected on M17 agar (Oxoid) with 5 g/L glucose and 5 ⁇ g/mL of erythromycin.
  • the resulting plasmid was named pPSM652.
  • Plasmid pPSM652 was isolated from L. lactis and electroporated into L. plantarum
  • strain PSM2009 was grown overnight in selective medium at the permissive temperature (30°C).
  • the overnight culture was diluted 1000 fold in fresh MRS medium and incubated overnight at 41 °C.
  • the overnight culture was diluted 10 "4 and plated on MRS agar plates with antibiotic at the non- permissive temperature (41 °C) for 48 hours to obtain single copy integrations of pPSM652 into the chromosome of L. plantarum 299v.
  • Fig.. 32 shows the strategy for pPSM652 integration.
  • plasmid pPSM652 Erythromycin resistant clones were isolated and integration of plasmid pPSM652 was verified using the following primers hom2-thrB-5 (5' CGCGACCCTGCTTGATCCGTCC 3') (SEQ ID NO:54) and pTN1-frw (5' GGAA- CAGAACATTTTTTTGTTAAGA 3').
  • the primer sequence of hom2-thrB-5 is not included in the fragment that was inserted in pPSM652, but located in a position on the chromosome of 299v, which is further upstream of the sequence in pPSM652. Consequently, only erythromycin resistant clones that contain an integrated plasmid pPSM652 will give rise to a PCR product.
  • PSM2011 clone containing pPSM652 on the chromosome was named PSM2011.
  • Excision of plasmid pPSM652 in PSM2011 by a second single-crossover event is allowed by growth in non-selective MRS broth at the permissive temperature (30°C).
  • PSM2011 was incubated in MRS broth at 30°C overnight and diluted to 10 ⁇ 3 in fresh medium. The overnight incubation and dilution was repeated three times and cells were spread on MRS agar plates and incubated at 30°C for two days.
  • By replica- plating to MRS agar plates containing erythromycin clones that were unable to grow in the presence of erythromycin were identified. These clones were expected to have excised the integrated pPSM652 plasmid.
  • the single-crossover event will result either in a mutant strain or a wild-type strain depending on how the recombination takes place.
  • the two types of events can be distinguished using the primers hom2-thrB-5 and hom2-thrB-4. Wild type clones will result in a 1908 bp PCR fragment, whereas mutant clones will result in a 1509 bp PCR fragment.
  • a mutant clone was isolated and named PSM2012.
  • the presence of an internal 399 bp deletion in the hom2-thrB genes of strain PSM2012 was verified by Southern blot analysis. Genomic DNA was prepared from strains PSM2012 and L plantarum 299v. Isolated genomic DNA from both strains was digested with either Hindi or Accl and separated on a 1% agarose gel. The agarose gel was treated for hybridisation as described previously (Arnau et al.;
  • PSM2012 digested with Accl and hybridised with the same probe resulted in bands of approximately 0.7 kb and 1.8 kb, respectively.
  • the wild type strain (299v) was expected to give rise to fragments of 1038 and >730 bp, respectively
  • the deletion strain was expected to give rise to fragments of 639 and >730 bp, respectively, when digested with Accl.
  • the sizes of the fragments revealed by Southern hybridisation correspond to the expected sizes of the smallest fragment for both the wild type and the deletion strain and further indicate the presence of an Accl site 1.8 kb upstream of the Accl site located in the probe sequence.
  • the Southern blot analysis revealed two bands of approximately 0.9 kb and 1.5 kb, respectively, when L. plantarum 299v genomic DNA was digested with Hindi and hybridised with the hom2-thrB probe.
  • genomic DNA isolated from PSM2012 digested with Hindi and hybridised with the same probe resulted in a band of approximately 1.3 kb.
  • the wild type strain (299v) was expected to give rise to fragments of 826 and >747 bp, respectively
  • the deletion strain (PSM2012) was expected to give rise to fragments of 1204 bp and >747 bp, respectively, when digested with Hindi.
  • the smallest fragments revealed by Southern hybridisation when wild type DNA was digested with Hindi correspond to the expected 826 bp fragment and the largest band indicate the presence of an Hindi site approximately 1.3 kb upstream of the Hindi site located in the probe sequence.
  • a band of 1206 bp was expected predicted from the genome sequence and a band of 1.3 kb was expected from the hybridisation pattern observed for the wild type strain.
  • the observed band of approximately 1.3 kb represents a double band.
  • the Southern blot analysis of chromosomal DNA from PSM2012 resulted in the hybridisation pattern expected for a deletion strain.
  • L. plantarum strains 299v and PSM2012 were inoculated in defined medium with out threonine and as a control in the same medium supplemented with threonine (Lbp-
  • V24-G10 The medium components are listed in the box below.
  • the wild type strain (299v) was able to grow in both media.
  • the deletion strain (PSM2012) was unable to grow in the threonine deficient medium, but able to grow in the same medium supplemented with threonine indicating a block in the threonine biosynthetic pathway.
  • this example shows that specific gene inactivation can be achieved in L. plantarum by use of plasmid pT 1.
  • the chemically defined medium Lbp-V24-G10 for L plantarum contains: Carbohydrate: 10 g/L D-Glucose;
  • Buffers 3.6 g/L sodium acetate, 3 g/L potassium dihydrogen phosphate, 3 g/L dipoiassium hydrogen phosphate; Fatty acid ester: 1 mL/L Tween 80;
  • Amino acids 1.2 g/L L-alanine, 0.8 g/L L-arginine, 0.4 g/L L-asparagine, 0.2 g/L L-cysteine, 1.2 g/L glutamic acid, 0.4 g/L glutamine, 0.8 g/L glycine, 0.2 g/L L-histidine, 0.4 g/L L- isoleucine, 0.4 g/L L-leucine, 1.0 g/L L-lysine-HCI, 0.4 g/L L-methionine, 0.8 g/L L- phenylalanine, 1.2 g/L L-proline, 1.2 g/L L-serine, 0.8 g/L L-threonine, 0.1 g/L L-tryptophane, 0.2 g/L L-tyrosine, and 0.4 g/L L-valine;
  • Nucleotide bases and vitamins 0.05 g/L adenine, 0.05 g/L guanine, 0.05 g/L xanthine, 0.05 g/L uracil, 0.2 mg/L potassium p-aminobenzoate, 0.05 mg/L biotin, 0.05 mg/L cyanocobala- min, 1 mg/L riboflavin, 1 mg/L nicotinic acid, 1 mg/L niacinamid, 0.05 mg/L folic acid, 2 mg/L pyridoxal-HCI, 2 mg/L pyridoxin-HCI, 1 mg/L thiamin-HCI, 0.1 mg/L lipoic acid, 5 mg/L inos- ine, 3.7 mg/L thymidine, and 5 mg/L potassium orotate;
  • the present example illustrates various methods for analysing the immunomodulatory effects of pure polypeptides with or without the parallel use of probiotic strains, which are e.g. wild type optimised for selected probiotics properties, null-mutants, secretion deficient mutants, or modification deficient mutants.
  • GENE PRODUCTS The identified genes encoding Enolase, GAPDH, PGK and TPI (in the following termed GENE PRODUCTS) will each be inserted into expression vectors for lactic acid bacteria but also into expression vectors for other bacteria such as E. coli (as in example 10). The resulting vectors will be introduced into appropriate strains, which then will be grown under controlled conditions in fermentors (Bredmose et al.; 2001). A pure preparation of GENE PRODUCTS can be obtained using the above techniques followed by standard purification techniques.
  • the secretion, localisation on the cell surface, and/or possibly chemical modifications could be imperative for the GENE PRODUCTS to be capable of exerting immunomodulation, or changing the amount and/or composition of the mucins in animals or humans.
  • This analysis will be carried out according to the description below where the application of a probiotic strain includes the use of a null-mutant with respect to the gene or genes encoding the relevant GENE PRODUCT(S), or a mutant that is deficient in the ability to secrete the GENE PRODUCT(S), such as the 299v mutant
  • GENE PRODUCTS Pure preparations of one or more GENE PRODUCTS can be used alone or in combination with probiotic strains in the developed in vitro assays (example 13) aiming at testing and establishing the immunomodulatory properties of GENE PRODUCTS alone or in combination with probiotic strains and derivatives thereof, such as e.g. the mutagenised strain 149-D7.
  • the probiotic strains could be wild type, naturally improved or improved using recombinant gene technology techniques as described in the following example.
  • Immunomodulatory effects means that the production increases or decreases of one or more of either the cytokines IL1 , IL2 etc.
  • the animal models could include a colitis model where the intestines of animals are treated with dextran sulfate sodium (Okayasu et al.; 1990) to induce colitis symptoms. Following induction, the animals are nourished with feed containing the compound ⁇ ) and or the strains to be tested. Also, a control with no compound(s) or strains is included. The animals are killed after an appropriate time of treatment and their intestines will be examined. In addition, an analysis of the levels of selected markers such as cytokines could be carried out. Moreover, an analysis of the mucin production and composition before and after the treatment could be performed. Subsequently, human trials will be carried out if the examination and/or the levels of markers show that the compounds and/or the bacterial strains demonstrate the expected beneficial effects.
  • the human trials will be carried out in patients with e.g. autoimmune diseases, including, but not limited to, Inflammatory Bowel Disease or rheumatoid arthritis.
  • autoimmune diseases including, but not limited to, Inflammatory Bowel Disease or rheumatoid arthritis.
  • the compound(s) with or without probiotic strain(s) and/or supporting compounds could be encapsulated using an appropriated substance that releases the contents at desired locations in the intestine. Examination of symptoms and analysis of relevant marker such as TNF-alpha and other cytokines will be performed. Also, an analysis of the mucin production and composition before and after the treatment is relevant.
  • Novel drugs will result from the above program.
  • the drugs can either be used alone or in combination with existing drugs to treat or prevent several diseases including autoimmune diseases, cancers and microbial infections.
  • Example 22 Selected applications of the present invention
  • the present invention in preferred embodiments is directed to e.g. methods for developing or constructing probiotic strains with impaired or improved probiotic properties, methods for setting up a quality control in the manufacturing process of probiotic starter cultures and end-user products, and methods for screening for new probiotic strains, as described in more detail herein below.
  • Improved probiotic strains can be developed when the GENE PRODUCTS alone or on the surface of a probiotic microorganism have been demonstrated to exert immunomodulatory effects or alterations in the mucin production in in vitro assays and/or in animals and/or in humans. Two approaches can be used namely i) tradi- tional mutagenesis followed by screening procedures and ii) the use of recombinant gene technology to enhance or reduce the levels of the GENE PRODUCTS.
  • the first approach uses EMS (ethyl-methane-sulfonate), as described in example 14, or UV irradiation for the mutagenesis of a known probiotic strain such as L. plantarum 299v.
  • EMS ethyl-methane-sulfonate
  • UV irradiation for the mutagenesis of a known probiotic strain such as L. plantarum 299v.
  • a large number, preferably but not restricted to more than 10 4 , of mutagenised bacteria will subsequently be analysed using a high throughput screening (HTS) technology, as described in example 14.
  • the HTS is based on growth of the lactic acid bacterium mutants in microtiter wells followed by the monitoring of the levels of one or more GENE PRODUCTS.
  • mutants could overproduce one or more GENE PRODUCTS and/or have a lower production of one or more other GENE PRODUCTS and/or a have a higher or lower production of any other metabolic products produced by the bacterium.
  • mutants that do not contain one or more GENE PRODUCTS on the surface will be useful for analysing the importance of the GENE PRODUCTS on the surface of Lactobacillus and/or the role of a possible modification of the GENE PRODUCTS.
  • Enzyme activity assays or specific antibodies could be used for the quantification of the production levels of the GENE PRODUCTS or any other metabolic product produced by the bacterium.
  • the preferred mutants will be analysed in in vitro assays (as in example 13) and animal models as described in the former example.
  • the GENE PRODUCTS or any other supporting compounds could be included together with the mutants in the analysis. Mutants that show the expected effects alone or in any combination with GENE PRODUCTS or any other supporting compounds will be used in human trials also as described in the former example.
  • one or more genes encoding the GENE PRODUCTS will be inserted into an appropriate expression vector such as pVS2 (von Wright et al.; 1987) containing expression signals that ensure the desired production levels of the GENE PRODUCTS.
  • Expression signals include promoters, Shine Dalgarno se- quences (RBS-sequences), secretion signals and the modulation of the distances between these units themselves and the distances to the start codon of the gene(s).
  • one or more genes encoding the GENE PRODUCTS together with the appropriate expression signals could be inserted into the chromosome of the bacterium using the described techniques (Madsen et al.; 1996).
  • null-mutants con- taining a deletion in one or more genes encoding the GENE PRODUCTS can be constructed using for instance gene replacement techniques (Madsen et al.; 1996), as described in example 20. Also, it will be possible to construct strains that are deficient in the secretion of one or more of the GENE PRODUCTS. This will be done using an approach that allows the generation of randomly located and tagged inser- tions into the genome of Lactobacillus followed by screenings according to the description above. The construction of null-mutants requires the use of growth media containing compounds that replace the metabolic products produced in the reactions catalysed by the GENE PRODUCTS in the wild type.
  • null- mutants or the secretion deficient mutants in the analysis will provide evidence whether secretion, surface localisation and/or possibly chemical modifications are imperative for the GENE PRODUCTS to be capable of exerting immunomodulations or changing the levels and the composition of the mucins in animals or humans.
  • the resulting recombinant strains will be analysed for the expected over-production and/or lowered production of the GENE PRODUCTS and possibly also other metabolic compounds produced by the bacterium. Analyses of increased or decreased secretion of GENE PRODUCTS as well as analyses of the modification of the GENE PRODUCTS can also be performed.
  • the analysis could be carried out using the same methods as described above for the HTS technique.
  • the recombinant strains will be analysed in in vitro assays and in animal models as described above for the preferred mutant. Also as described for the preferred mutants, the recombinant strains could also be tested in humans.
  • Quality control (QC) in the manufacturing process of probiotic starter cultures and probiotic end-user products can be established when the GENE PRODUCTS have been demonstrated to exert immunomodulatory effects or alterations in the mucin production in in vitro assays and/or in animals and/or in humans.
  • Starter culture companies can perform QC on probiotic cultures in the laboratory and in the manufacturing process using methods that take advantage of the GENE PRODUCTS as probiotic markers. Analysing for appropriate levels of the GENE PRODUCTS in the probiotic starter can be performed during inoculation, propaga- tion and the manufacturing of the cultures.
  • the analysis can include monitoring of the levels of one or more GENE PRODUCTS, the presence of the genes encoding one or more GENE PRODUCTS and/or the levels of mRNA related to the genes encoding one or more GENE PRODUCTS.
  • the companies that produce end-user probiotic products can perform the same QC by using the same techniques.
  • these techniques can be used for process optimisations in the production of probiotic starter cultures and/or end-user probiotic products, c.f. the different GAPDH activities at different growth stages and conditions as shown e.g. in Example 9.
  • these techniques are useful for the identification of and screening for new probiotic strains that could be found anywhere in the environment such as in the Gl- tract of humans or animals, in dairy products and in cereals. The screening could be performed using the HTS technology described above.
  • al- pha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol. Microbiol. 40:1273-1287.

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Abstract

La présente invention concerne des méthodes de modulation (i) d'une réponse immunitaire et/ou (ii) de la quantité et/ou de la composition de mucines des muqueuses, consistant à mettre en contact une partie formant cellule d'un tissu lymphoïde associé à des muqueuses (MALT), ou une cellule épithéliale avec un polypeptide de surface d'une cellule microbienne. La modulation de la réponse immunitaire fait intervenir de préférence une ou plusieurs cytokines. Le polypeptide de surface de cellule microbienne est de préférence un polypeptide obtenu à partir de Lactobacillus ou de Bifidobacterium. Curieusement, on a constaté que les enzymes intracellulaires agissant dans les voies métaboliques de Lactobacillus et de Bifidobacterium, ou bien des polypeptides sensiblement identiques à de tels enzymes intracellulaires, étaient transportés à la surface de la cellule où ils pouvaient être au moins partiellement exposés au milieu extra-cellulaire. Par voie de conséquence, les polypeptides de surface cellulaire préférés ont des équivalents intracellulaires (c'est-à-dire associés au cytoplasme) agissant dans les voies métaboliques, telles que la glycolyse, dans les espèces probiotiques de Lactobacillus et/ou de Bifidobacterium. Les polypetides associés à la surface et leurs équivalents intracellulaires partagent une suite importante de résidus d'acides aminés consécutifs, mais sont situées dans des parties différentes de la cellule.
PCT/DK2004/000138 2003-02-27 2004-02-27 Composes prtobiotiques immunomodulateurs WO2004076615A2 (fr)

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WO2006063592A1 (fr) * 2004-12-14 2006-06-22 Alk Abelló A/S Préparations pharmaceutiques comprenant une cellule bactérienne présentant un composé protéique hétérologue
WO2007040446A1 (fr) * 2005-10-06 2007-04-12 Probi Ab Utilisation de lactobacillus pour le traitement des maladies autoimmunes
WO2008075685A1 (fr) * 2006-12-21 2008-06-26 Calpis Co., Ltd. Promoteur de la production d'iga
WO2008117266A2 (fr) 2007-03-28 2008-10-02 Alimentary Health Limited Souches de bifidobacterium probiotique
WO2008117267A2 (fr) 2007-03-28 2008-10-02 Alimentary Health Limited Souches de bifidobacterium probiotiques
FR2922450A1 (fr) * 2007-10-19 2009-04-24 Lallemand Sas Soc Par Actions Utilisation de bacteries lactique pour la prevention et/ou le traitement de pathologies cutanees
WO2009080862A1 (fr) * 2007-12-24 2009-07-02 Consejo Superior De Investigaciones Científicas Micro-organismes pour améliorer l'état de santé d'individus présentant des troubles associés à l'ingestion de gluten
US20120183513A1 (en) * 2009-02-18 2012-07-19 Josef Neu Lactobacillus Supplement for Alleviating Type I Diabetes
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WO2013099883A1 (fr) * 2011-12-28 2013-07-04 株式会社山田養蜂場本社 Nouvelle bactérie lactique ayant une activité favorisant la production d'iga, et son utilisation
WO2015121458A3 (fr) * 2014-02-14 2015-10-08 Vesale Pharma Sa Utilisations de compositions comprenant du bifidobacterium animalis ssp. lactis lmg p-28149
US9339055B2 (en) 2005-03-04 2016-05-17 Asahi Group Holdings, Ltd. T cell apoptosis inducer and method therefore
WO2018183685A1 (fr) * 2017-03-29 2018-10-04 President And Fellows Of Harvard College Méthodes de régulation de l'expression génique dans une cellule
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CN112501049A (zh) * 2020-10-28 2021-03-16 石河子大学 产转糖基活性β-半乳糖苷酶的开菲尔乳杆菌及制备的β-半乳糖苷酶生产低聚半乳糖的方法
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WO2009050677A3 (fr) * 2007-10-19 2010-01-21 Danstar Ferment Ag Utilisation de bacteries lactiques pour la prevention et/ou le traitement de pathologies cutanees
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