Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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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. A) Photograph of plate 53 (top) and plate 52 (bottom). B) OD595 readings from plate 53 (top) and plate 52 (bottom).
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, ESPfractions (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, 149D7/129, 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).
Definitions Contacting : The binding, transient or longer lasting, of e. g. a polypeptide to a cell.
Cytokine response: The induction or repression of one or more cytokines.
Degenerated polynucleotide : Different polynucleotides can encode the same poly- peptide 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 (GI) tract.
Food: Where used herein, the term'food'can be any type of food, incl. an edible product. In some embodiments, 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 noncovalent. 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.
Detailed Description of the Invention Preferred embodiments of the present invention is described herein below.
Methods for modulating an immune response in an individual In one embodiment there is provided a method for modulating an immune response and/or the amount and/or composition of mucosa) mucins in an individual, said method comprising the steps of i) providing a microbial cell selected from a Lactobacillus cell and a

Bifidobacterium cell, wherein said cell comprises at least one microbial cell surface polypeptide and a substantially identical intracellular equivalent thereof, wherein 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 iii) modulating an immune response and/or the amount and/or composition of mucosal mucins in an individual.


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, and 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 (MALT) 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.


More preferably, the enzyme is selected from the group consisting of enolase ; glyceraldehyde 3-phosphate dehydrogenase (GAPDH); phosphoglycerate kinase (PGK); and triose phosphate isomerase (TPI). In one presently most preferred embodiment, 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.
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 In another embodiment there is provided a microbial cell comprising at least one microbial cell surface polypeptide and a substantially identical intracellular equivalent thereof, wherein the microbial cell is selected from the group consisting of Lactobacillus species and Bifidobacterium species, and wherein 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 wherein the first and second polynucleotides are not natively associated, and wherein 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.
More preferably, 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.
Polynucleotides, vectors and host cells transformed therewith There is also provided polynucleotides and vectors encoding cell surface polynucleotides, as well as host cells transformed therewith.
In one embodiment 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, wherein the microbial cell is selected from the group consisting of Lactobacillus species and Bifidobacterium species, and wherein 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

wherein the first and second polynucleotides are not natively associated, and wherein 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, nonpathogenic 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 animais, Lactobacillus arizonensis, Lactobacillus aviarius, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus coelohominis, Lactobacillus collinoides, Lactobacillus coryniformis subsp. coryniformis, Lactobacillus coryniformis subsp. torquens, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus cypricasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp delbrueckii, Lactobacillus delbrueckii subsp. lactis, Lactobacillus durianus, Lactobacillus equi, Lactobacillus farciminis, Lactobacillus ferintoshensis, Lactobacillus fermentum, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus helveticus subsp. jugurti, 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. tolerans, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus psittaci, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus sali- varius, Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp. salivarius, 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 animais, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium boum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium choerinum, Bifidobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacterium dentium, Bifidobacterium gallium, Bifidobacterium gallinarum, Bifidobacterium indicum, Bifidobacterium longum, Bifidobacterium longum bv Longum, Bifidobacterium longum bv. Infants, Bifidobacterium longum bv. Suis, Bifidobacterium magnum, Bifidobacterium merycicum, Bifidobacterium minimum, Bifidobacterium pseudocatenulatum, Bifidobacte- rium pseudolongum, Bifidobacterium pseudolongum subsp. globosum, Bifidobacte- rium pseudolongum subsp. pseudolongum, Bifidobacterium psychroaerophilum, Bifidobacterium pullorum, Bifidobacterium ruminantium, Bifidobacterium saeculare, Bifidobacterium scardovii, Bifidobacterium subtile, Bifidobacterium thermoacidophilum, Bifidobacterium thermoacidophilum subsp. suis, Bifidobacterium thermophilum, Bifidobacterium urinais.

Preferred examples of first polynucleotides encoding a cell surface polynucleotide is provided herein below and 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).
Polvnucleotides encoding alvceraldehvde phosphate dehvdrogenase (qapdh) In one preferred embodiment there is provided a polynucleotide selected from the group consisting of i) SEQ ID NO : 1 or a polynucleotide comprising nucleotides 1285 to 2307 of

SEQ ID NO : 11, and ii) a polynucleotide comprising or essentially consisting of the coding se- quence of gap encoding a glyceraldehyde 3-phosphate dehydrogenase of Lactobacillus plantarum 299v, as deposited with DSM under acces- sion number DSM 9843; and iii) a polynucleotide encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO : 2; and iv) a polynucleotide encoding a fragment of a polypeptide encoded by poly- nucleotides (i), (ii) or (iii), wherein said fragment a) has glyceraldehyde 3-phosphate dehydrogenase activity; and/or b) is recognised by an antibody, or a binding fragment thereof, which is capable of recognising SEQ ID NO : 2; and/or 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; and/or b) is recognised by an antibody, or a binding fragment thereof, which is capable of recognising SEQ ID NO : 2; and/or 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, vi) a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in any of (iv) and (v), and the complementary strand of such a polynucleotide.


Polynucleotides encoding phospho qlvcerate kinase (pgk) In one preferred embodiment there is provided a polynucleotide selected from the group consisting of i) SEQ ID NO : 3, or a polynucleotide comprising nucleotides 2428 to 2630 of SEQ ID NO : 11, and ii) a polynucleotide comprising or essentially consisting of the coding se- quence of pgk encoding a phosphoglycerate kinase of Lactobacillus plantarum 299v, as deposited with DSMZ under accession number DSM

9843; and iii) a polynucleotide encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO : 4; and


iv) a polynucleotide encoding a fragment of a polypeptide encoded by poly- nucleotides (i), (ii) or (iii), wherein said fragment a) has phosphoglycerate kinase activity; and/or b) is recognised by an antibody, or a binding fragment thereof, which is capable of recognising SEQ ID NO : 4; and/or 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; 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 phosphoglycerate kinase activity ; and/or b) is recognised by an antibody, or a binding fragment thereof, which is capable of recognising SEQ ID NO : 4; and/or 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, vi) a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in any of (iv) and (v), and the complementary strand of such a polynucleotide.


Polvnucleotides encoding triose phosphate isomerase (tpi) In one preferred embodiment there is provided a 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 ii) a polynucleotide comprising or essentially consisting of the coding se- quence of tpi encoding a triose phosphate isomerase of Lactobacillus plantarum 299v, as deposited with DSMZ under accession number DSM

9843; and iii) a polynucleotide encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO : 6; and iv) a polynucleotide encoding a fragment of a polypeptide encoded by poly- nucleotides (i), (ii) or (iii), wherein said fragment a) has triose phosphate isomerase activity; and/or 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 ; 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 triose phosphate isomerase activity; and/or 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, vi) a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in any of (iv) and (v), and the complementary strand of such a polynucleotide.


Polvnucleotides encoding enolase (eno) In one preferred embodiment there is provided a polynucleotide selected from the group consisting of i) SEQ ID NO : 7, a polynucleotide comprising nucleotides 4497 to 5825 of

SEQ ID NO : 11, and ii) 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 ; and iii) a polynucleotide encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO : 8; and iv) a polynucleotide encoding a fragment of a polypeptide encoded by poly- nucleotides (i), (ii) or (iii), wherein said fragment a) has enolase activity; and/or 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; and/or 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, vi) a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in any of (iv) and (v), and the complementary strand of such a polynucleotide.


Polypeptides The present invention is also directed to polypeptides encoded by the above polynucleotides as well as variants and functional equivalents of such polypeptides.



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