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



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"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.
Accordingly, 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).
It is believed according to one presently preferred hypothesis that at least transient colonisation of the gastro-intestinal (GI) tract is a prerequisite for a probiotic microbial cell to exert its probiotic potential. The attachment of the probiotic microbial cell, optionally in combination with the attachment of 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).


In additionally preferred embodiments of the present invention 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. 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. Such polynucleotides and 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 and TPI are also believed to be located on the surface of Lb plantarum. Ac- cordingly, 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. 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.

Also described herein below is the cloning and characterisation of a gene encoding a regulator protein (GRE) that might regulate the transcription of the operon containing the genes encoding Enolase, GAPDH, PGK and TPI.


The cell surface location of e. g. Enolase, GAPDH, PGK, TPI and GRE of Lactoba- cillus 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.
Moreover, 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. As demonstrated herein, the markers can also serve as important probiotic indicators during production processes and/or con- comitant or subsequent quality control.
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 immunomodulator 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, wherein the microbial cell is selected from the group consisting of Lactobacillus species and Bifidobacterium species, and wherein the microbial cell comprises an altered polynucleotide sequence as compared to a reference microbial cell comprising a reference polynucleotide sequence without said alteration, 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 altered polynucleotide sequence results in an altered, preferably in- creased, 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 reference value indicative of the probiotic potential of a reference microbial cell, and iv) determining the probiotic potential of said candidate microbial cell based on the comparison performed in step iii).


A method for determining the probiotic potential of a starter culture, said 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 determination performed in step ii) with a reference value indicative of the probiotic potential of a reference starter culture, and iv) determining the probiotic potential of said candidate starter culture based on the comparison performed in step iii).
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) comparing the result of the determination performed in step ii) with a reference value indicative of the probiotic potential of a reference end- user product, and iv) determining the probiotic potential of said candidate end-user product based on the comparison performed in step 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 i) providing a microbial cell the probiotic potential of which is to be optimised, ii) cultivating the microbial cell in a growth medium under conditions allowing the microbial cell to undergo at least one cell division, wherein the 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 : a) reducing agents, such as glutathione and/or cysteine, preferably increasing the amount thereof, b) gasses, such oxygen or carbon dioxide, c) yeast extract, or components thereof, d) 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), g) the oxygen content, h) the ionic strength of the growth medium, such as the NaCI content, i) the pH, j) low molecular weight compounds, preferably salts (sulfate, phosphate, ni- trate), and/or metals (e. g. , copper), and/or organic acids, k) cAMP level in the microbial cell, and


I) a cell constituent, or a precursor thereof, preferably a co-factor, a vitamin, a lipid, and the like thereby controlling the production and/or secretion and/or post-translational modification of said cell surface polypeptide.


A method for modulating an immune response and/or the amount and/or composition of mucosal 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.


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 immunomodulating 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.
An antibody against a polypeptide as described herein.
An antagonist capable of inhibiting the activity or expression of a polypeptide as described herein.
An agonist capable of enhancing the activity or expression 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 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 i) contacting a composition comprising the polypeptide with the com- pound 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 compo- nent capable of providing a detectable signal in response to the inter- action of the polypeptide with the compound; and ii) determining whether the compound interacts with and activates or in- hibits an activity of the polypeptide by detecting the presence or ab- sence of a signal generated from the interaction of the compound with the polypeptide.
A method for treating an auto-immune disease in an individual comprising the step of administering to the individual a pharmaceutical effective amount of a polypeptide as described herein, or a host cell as described herein.
A polypeptide and variants and functional equivalents thereof as described herein or a host cell as described herein, for use as a medicament.
Use of a polypeptide and variants and functional equivalents thereof as described herein or a host cell as described herein, for the manufacture of a medicament for treatment of a disease, wherein said treatment benefits from modulation of the immune response.
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.


Use of a 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 nutritional supplement for treatment of a disease which benefit from modulation of the immune response.
A food comprising at least a host cell as describec herein, and/or at least a polypetide and variants and functional equivalents thereof as described herein.
Use of a 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.
In a preferred embodiment, an increased probiotic potential is generated by an increased production and/or an increased secretion and/or an increased or decreased post-translational modification of said microbial cell surface polypeptide, such as an increased production and an increased secretion and an increased or decreased post-translational modification of said microbial cell surface polypeptide, for example an increased production and an increased secretion and an increased post-translational modification of said microbial cell surface polypeptide, such as an increased production and an increased secretion and a decreased post- translational modification of said microbial cell surface polypeptide, for example an increased production and/or an increased or decreased posttranslational modification of said microbial cell surface polypeptide, such as 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, such as an increased secretion and an increased or decreased post-translational modification of said microbial cell surface polypeptide, for example an increased secretion and an increased post-translational modification of said microbial cell surface polypeptide, such as an increased secretion and an decreased post-translational modification of said microbial cell surface polypeptide, for example an increased production of said microbial cell surface polypeptide, such as an increased secretion of said microbial cell surface polypeptide, for example an increased or decreased post-translational modification of said microbial cell surface polypeptide, such as an increased post-translational modification of said microbial cell surface polypeptide, for example a decreased post-translational modification of said microbial cell surface polypeptide.


Description of Drawings Fig. 1 illustrates SDS-PAGE analysis of surface located proteins from L. plantarum 299v.
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 gapdhpgk-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 Lact : obacillus plan arum 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.



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