WO2009035330A1

WO2009035330A1 - Probiotic and compound derived thereof exhibiting immunomodulatory ability

Info

Publication number: WO2009035330A1
Application number: PCT/NL2008/050600
Authority: WO
Inventors: Sergey Rumenov Konstantinov; Jolanda Lambert; Cindy Mathilda Van Der Meer - Van Kraaij; Willem Meindert De Vos; Michiel Kleerebezem
Original assignee: Stichting Top Institute Food And Nutrition
Priority date: 2007-09-14
Filing date: 2008-09-12
Publication date: 2009-03-19

Abstract

The invention relates to a polypeptide which is able to interact with a DC-SIGN and to a host cell overexpressing this polypetide. Such polypeptide and/or encoding nucleic acid molecule and/or corresponding nucleic acid construct and/or sai d host cell are used as medicament, preferably for preventing or treating inflammatory gastrointestinal tract disease and/or inducing tolerance.

Description

Probiotic and compound derived thereof exhibiting immunomodulatory ability

Field of the invention

The invention relates to a polypeptide and a host cell exhibiting immunomodulatory characteristics and which are used as a food ingredient, or medicament, preferably for preventing or treating inflammatory gastrointestinal tract disease and/or inducing tolerance.

Background of the invention

Dendritic cells (DCs) are highly efficient antigen-presenting cells (APCs) that collect antigen in body tissues and transport them to draining lymph nodes. Antigenic molecules such as peptides or glycosylated peptides are loaded onto major histocompatibility complex (MHC) molecules for presentation to naive T cells, resulting in the induction of cellular and humoral immune responses. DCs take up antigen through phagocytosis, pinocytosis, and endocytosis via different groups of receptor families, such as Fc receptors for antigen-antibody complexes, C-type lectin receptors (CLRs) for glycoproteins, and pattern recognition receptors, such as Toll-like receptors (TLRs), for microbial antigens. Uptake of antigen by CLRs leads to presentation of antigens on MHC class I and II molecules. DCs are well equipped to distinguish between self- and nonself-antigens by the variable expression of cell- surface receptors such as CLRs and TLRs. In the steady state, DCs are not immunologically quiescent but use their antigen-handling capacities to maintain peripheral tolerance. DCs are continuously sampling and presenting self- and harmless environmental proteins to silence immune activation. Uptake of self-components in the intestine and airways are good examples of sites where continuous presentation of self- and foreign antigens occurs without immune activation. In contrast, efficient antigen- specific immune activation occurs upon encounter of DCs with nonself-pathogens. Recognition of pathogens by DCs triggers specific receptors such as TLRs that result in DC maturation and subsequently immune activation.

Lactobacilli and other probiotic bacteria are frequently tested in the management of allergic diseases or gastroenteritis. It is assumed in literature that these probiotics have immunoregulatory properties and promote mucosal tolerance, which is in part mediated by regulatory T cells (Treg cells). On the basis of pathogenic or tissue-specific priming, DCs acquire different T cell- instructive signals and drive the differentiation of naive T cells into either TH 1, TH 2, or regulatory T cells. Previous work (Smits HH, et al. 2005, Journal of Allergy and Clinical Immunology 115:1260-1267) showed that different species of lactobacilli can specifically prime monocyte-derived DCs to drive the development of Treg cells. These Treg cells produced increased levels of IL-IO and inhibited the proliferation of bystander T cells in an IL-10-dependent fashion. Importantly, not all lactobacilli seem to display this capacity and the same study showed a correlation between binding of the lactobacilli to the C-type lectin "DC- specific intercellular adhesion molecule 3 -grabbing non-integrin" (DC-SIGN; Geijtenbeek TB, et al., 2000, Cell, 100:575-585) and Treg induction. This could be illustrated by the observation that antibodies that block DC-SIGN inhibited Treg induction by the lactobacilli, which supports DC-SIGN dependent priming of DCs to induce Treg cells. This study suggests that targeting of DC-SIGN by certain probiotic bacteria could explain their beneficial effect in the treatment of a number of inflammatory diseases, including atopic dermatitis and Crohn's disease.

However, the mechanisms underlying this DC-probiotic bacteria interaction is not yet fully understood and the specific molecule expressed by the probiotic bacteria and involved in this interaction is currently unknown.

Several compounds, among other probiotic are already used to prevent and/or treat inflammatory gastrointestinal disease. However, there is still a need for alternative, even improved compounds exhibiting immunomodulatory characteristics that could be used in such disease.

Description of the invention

The present invention is based on the unravelling of the mechanims underlying this DC-probiotic bacteria interaction and on the identification of a molecule expressed by the probiotic bacteria and involved in this interaction. Polypeptide

In a first aspect, the invention relates to a polypeptide able to interact with DC-SIGN, wherein said polypeptide has an amino acid sequence which has at least 30% identity with the amino acid sequence of SEQ ID NO: 1. The functionality of a polypeptide of the invention (able to interact with a DC-SIGN) is preferably assessed using an in vitro ELISA-based assay employing a DC-SIGN-Fc (DC-SIGN-Fc ELISA assay) and/or a cell-based DC-SIGN binding assay (Raji-based DC-SIGN) both as described in example 1. The detection of a binding in any one of these two assays indicates that said polypeptide is able to interact with a DC-SIGN and that it is therefore functional. Below a short description of both assays is given. DC-SIGN-Fc ELISA assay

Preferably, the DC-SIGN-Fc consists of the extracellular portion of DC-SIGN (amino acid residues 64-404) fused at the C terminus to a human IgGl-Fc fragment (Geijtenbeek et al, 2002, J. Biol. Chem. 277: 11314-11320). Briefly, cells are first washed with a buffer such as phosphate buffered isotonic saline (PBS). Subsequently, approximately 5x10⁵ cfu cells are coated per well, blocked with 1% bovine serum albumin. Binding is assessed by adding a DC-SIGN-Fc, preferably the one as described above and incubated for approximately 30 minutes at 37⁰C. Unbound DC-SIGN-Fc is washed away, and binding is determined using horseradish peroxidase conjugated goat- anti-human-Fc and standard ELISA technique (Geijtenbeek, T.B.H., van Duijnhoven, G.C.F., van Vliet, S.J., Krieger, E., Vriend, G., Figdor, C.G., and van Kooyk, Y. (2002) J. Biol. Chem., 277:11314-11320). Specificity of DC-SIGN-Fc binding is preferably determined by the addition of excess amounts of anti-DC-SIGN monoclonal antibodies (AZN-Dl; approximate concentration 20 μg/ml). As a negative control, no cells are coated in the well (Smits HH, et al. 2005, Journal of Allergy and Clinical Immunology 115:1260-1267).

Cell-based DC-SIGN binding assays (Raji-based DC-SIGN binding assay) This assay determines DC-SIGN dependent binding to transfected cells (Raji-DC- SIGN). Raji-DC-SIGN cells are cultured as previously described (Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS, KewalRamani VN, Littman DR, Figdor CG, van Kooyk Y. (2000) Cell, 100:587-597). Cells (approximatively 10⁹ cfu/mL) are labeled by incubation with Fluorescein Isothiocyanate (FITC; 0.5 mg/mL) for approximately 1 hour at room temperature (RT). After washing (for example 5 times in PBS), cells are resuspended in TSM buffer (20mMTris, 15OmM NaCl, ImM CaC12, and 2 mMMgC12, pH 8.0), which is appropriate for the analyses of their binding to Raji-DC-SIGN cells.

FITC-labeled cells are subsequently incubated with Raji-DC-SIGN cells (ratio Raji cells: cells be tested approximately 1:10) in the presence or absence of excess amounts of anti-DC-SIGN monoclonal antibodies (AZN-Dl; approximate concentration 20 μg/ml) for approximately 45 minutes at approximately 37 ⁰C. Unbound cells are removed by washing the cells ( such as 3 times with TSM). The binding of FITC- labeled cells to Raji-DC-SIGN (detected by fluorescence, preferably mean fluorescence value of approximately 4000 Raji cells) is indicative for DC-SIGN binding and is subsequently examined by FACS. As a negative control, no cells are added. As additional controls, anti-DC SIGN antibodies may also be added.

Optionally, the functionality of a polypeptide of the invention is further confirmed by measuring the induction of Treg and optionally the production of IL- 10 as described m Smits HH, et al. 2005, Journal of Allergy and Clinical Immunology 115:1260-1267, especially at page 1263. A preferred amino acid sequence of a polypeptide of the invention and obtained from the Lactobacillus plantarum strain WCFSl is given in SEQ ID NO:1. A cDNA sequence encoding the amino acid sequence of SEQ ID NO:1 is given in SEQ ID NO:2. The strain WCFSl is a single colony isolate from L. plantarum NCIMB8826, which was originally isolated from human saliva. The strain is present in the National Collection of Industrial and Marine Bacteria, Aberdeen, U.K. (ES). This strain has been sequenced (Kleerebezem M. ,et al. (2003), Proc. Natl. Acad. ScL, vol 100: 1990- 1995).

According to an even more preferred embodiment, a polypeptide used is a homologue of the one represented by SEQ ID NO:1. A homologue of SEQ ID NO:1 has preferably at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%, even more preferably at least 90%, 92%, 95%, 97% , 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1. According to one preferred embodiment, the invention provides a fragment of the polypeptide as defined above, said fragment having the earlier defined identity (30% with SEQ ID NO:1) and functionality (able to interact with DC-SIGN). The identity of a fragment of said polypeptide is preferably defined by comparing its identity with the corresponding fragment of a polypeptide of the invention. Accordingly, in one preferred embodiment, a fragment of a polypeptide of the invention as defined above is fused to any other polypeptide fragment, which is not natively associated with it. In this case, a polypeptide is a so-called hybrid or fusion polypeptide. Preferably, this other polypeptide fragment ensures or at least does not interfere with expression of said hybrid polypeptide at the surface of a cell.

In one preferred embodiment, a polypeptide of the invention comprises an amino acid sequence which is 100% identical to the amino acid sequence of SEQ ID NO:1. Most preferably, the polypeptide has the amino acid sequence of SEQ ID NO:1 or a polypeptide is obtainable by expression of a gene present in the Lactobacilus plantarum strain WCFSl. Accordingly, the polypeptide having the amino acid sequence of SEQ ID NO: 1 or a polypeptide being obtainable by expression of a cDNA present in the Lactobacillus plantarum strain WCFSl as such is also a preferred polypeptide of the invention. Percentage of identity is calculated as the number of identical amino acid residues between aligned sequences divided by the length of the aligned sequences minus the length of all the gaps. Multiple sequence alignment was performed using DNAman 4.0 optimal alignment program using default settings.

The skilled person will understand that a polypeptide of the invention could originate from other hosts than from the herein specified Lactobacillus plantarum strain, eg, from other Lacobacilli species or even from other probiotic species as long as it has the identity and functionality both as earlier herein defined. A preferred host is a bacterium, preferably one of the following: a food-grade bacterium, a commensal bacterium or a (attenuated) pathogenic bacterium. In one embodiment, a host is a food-grade bacterium, particularly a gram-positive bacterium, and more preferably a lactic acid bacterium. A host may also be a probiotic bacterium, which in itself has a beneficial effect when ingested by a subject.

A preferred host is a bacterium that belongs to a genus selected from the group consisting of Lactobacillus, Lactococcus, Leuconostoc, Carnobacterium, Streptococcus, Bifidobacterium, Bacteroides, Eubacterium, Clostridium, Fusobacterium, Propionibacterium, Enter ococcus, Staphylococcus, Peptostreptococcus, and Escherichia. A further preferred host is a bacterium that is a Lactobacillus or Bifidobacterium species selected from the group consisting of L. reuteri, L. fermentum, L. acidophilus, L. crispatus, L. gasseri, L.johnsonii, L. plantarum, L. paracasei, L. murinus, L. jensenii, L. salivarius, L. minutis, L. brevis, L. gallinarum, L. amylovorus, B. bifidum, B. longum, B. infantis, B. breve, B. adolescente, B. animalis, B. gallinarum, B. magnum, and B. thermophilum. Accordingly in a preferred embodiment, a polypeptide of the invention is obtained from a Lactobacillus plantarum strain.

Such polypeptides may be obtained using state of the art molecular biology techniques. Most preferably, a polypeptide used is obtained from a Lactobacillus plantarum strain. It is also encompassed by the invention to isolate several polypeptides of the invention from one single organism. Accordingly, all these polypeptides are also as such part of the invention.

According to another preferred embodiment, a polypeptide of the invention is a variant of any one of the polypeptide sequences defined before. A variant polypeptide may be a non-naturally occurring form of said polypeptide. A polypeptide variant may differ in some engineered way from the polypeptide isolated from its native source. A variant may be made by site-directed mutagenesis starting from the amino acid sequence of SEQ ID NO:1 or from a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:1, which is preferably SEQ ID NO:2. Preferably, a polypeptide variant contains mutations that do not alter the biological function of the encoded polypeptide. According to a preferred embodiment, a polypeptide variant has an enhanced ability to interact with DC-SIGN compared to the corresponding ability of its wild type counterpart as assessed in the assay as earlier defined herein. According to a more preferred embodiment, a polypeptide variant has an enhanced ability to interact with DC-SIGN compared to the polypeptide having SEQ ID NO: 1 as measured in the assay as defined above. According to an even more preferred embodiment, a polypeptide variant has an enhanced ability to interact with DC-SIGN compared to the ability of the corresponding polypeptide of Lactobacilus plantarum strain WCFSl or derivatives as preferably measured using the assay defined above. A polypeptide with an enhanced ability is very useful since it is expected to be more powerful than its wild type counterpart, so that lower dosages might be needed in order to reach a given biological effect.

A polypeptide of the invention is preferably a glycosylated polypeptide. More preferably, a polypeptide is glycosylated on a serine/threonine residue. The polypeptide shown as SEQ ID NO: 1 comprises two repeat regions delimited by amino acids 35-180 and 251-397, the region between these repeats is serine and threonine rich. Even more preferably, a polypeptide of the invention is glycosylated to at least 1 , even more preferably at least 2, at least 3, at least 4, at least 5 or more serine/threonine residues of this serine-threonine rich region. In another preferred embodiment, a polypeptide of the invention is expressed at the surface of a host cell. By virtue of this expression, a polypeptide of the invention will interact with DC-SIGN. Preferred host cells and methods for expressing a polypeptide are later herein defined.

Nucleic acid molecule In a second aspect, the invention relates to a nucleic acid molecule represented by its nucleic acid sequence coding for a preferred polypeptide defined in the previous section as:

- having the ability to interact with a DC-SIGN and - having an amino acid sequence which has at least 30% identity with the amino acid sequence of SEQ ID NO: 1.

According to a preferred embodiment, a nucleic acid sequence representing said nucleic acid molecule is selected from the list consisting of: (a) a nucleic acid sequence having at least 30% identity with the nucleic acid sequence of SEQ ID NO:2 (b) a variant of (a).

Percentage of identity was determined by calculating the ratio of the number of identical nucleotides in the sequence divided by the length of the total nucleotides minus the lengths of any gaps. DNA multiple sequence alignment was performed using DNAman version 4.0 using the Optimal Alignment (Full Alignment) program. The minimal length of a relevant DNA sequence showing 30% or higher identity level should be 40 nucleotides or longer. Preferably, the identity is of at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and even more preferably at least 99%. Most preferably, a nucleic acid sequence is SEQ ID N0:2. According to another preferred embodiment, a nucleic acid sequence of the invention is a variant of the nucleic acid sequence defined above. A nucleic acid sequence variant may be used for preparing a polypeptide variant as defined earlier. A nucleic acid variant may be a fragment of any of the nucleic acid sequences as defined above. A nucleic acid variant may also be a nucleic acid sequence that differs from SEQ ID NO:2 by virtue of the degeneracy of the genetic code. A nucleic acid variant may also be an allelic variant of SEQ ID NO:2. An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosome locus. A preferred nucleic acid variant is a nucleic acid sequence, which contains silent mutation(s). Alternatively or in combination, a nucleic acid variant may also be obtained by introduction of a nucleotide substitution, which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of a polypeptide of the invention. According to a preferred embodiment, a nucleic acid variant encodes a polypeptide still exhibiting its biological function. More preferably, a nucleic acid sequence variant encodes a polypeptide having the ability to interact with a DC-SIGN assessed as earlier herein defined. Even more preferably, a nucleic acid variant encodes a polypeptide with enhanced ability to interact with DC-SIGN as defined earlier. A nucleic acid sequence encoding a polypeptide having the ability to interact with a DC-SIGN may be isolated from any microorganism. All these variants can be obtained using techniques known to the skilled person, such as screening of library by hybridisation (southern blotting procedures) under low to medium to high hybridisation conditions with for the nucleic acid sequence SEQ ID NO:2 or a variant thereof which can be used to design a probe. Low to medium to high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200pg/ml sheared and denatured salmon sperm DNA, and either 25% 35% or 50% formamide for low to medium to high stringencies respectively. Subsequently, the hybridization reaction is washed three times for 30 minutes each using 2XSSC, 0.2%SDS and either 55⁰C, 65⁰C, or 75 ⁰C for low to medium to high stringencies.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of the polypeptide obtainable by expression of the gene present in the Lactobacillus plantarum strain WCSFl containing the nucleic acid sequence coding for the polypeptide of the invention should prevail.

Nucleic acid construct and expression vector

In a third aspect, the invention relates to a nucleic acid construct comprising a nucleic acid sequence defined in the previous section, said nucleic acid sequence encoding a polypeptide able to interact with a DC-SIGN and having an amino acid sequence which has at least 30% identity with the amino acid sequence of SEQ ID NO: 1. Optionally, a nucleic acid sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production of a polypeptide in a suitable expression host. Operably linked is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to a nucleic acid sequence coding for a polypeptide of the invention such that the control sequence directs the production of a polypeptide of the invention.

Expression will be understood to include any step involved in the production of said polypeptide including, but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

Nucleic acid construct is defined as a nucleid acid molecule, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined or juxtaposed in a manner which would not otherwise exist in nature. Control sequence is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and trancriptional and translational stop signals. The invention also relates to an expression vector comprising a nucleic acid construct of the invention. Preferably, an expression vector comprises a nucleic acid sequence of the invention, which is operably linked to one or more control sequences, which direct the production of the encoded polypeptide in a suitable expression host. At a minimum control sequences include a promoter and transcriptional and translational stop signals. An expression vector may be seen as a recombinant expression vector. An expression vector may be any vector (e.g. plasmid, virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleic acid sequence encoding a polypeptide as defined herein. Depending on the identity of the host wherein this expression vector will be introduced and on the origin of a nucleic acid sequence of the invention, the skilled person will know how to choose the most suited expression vector and control sequences.

Host cell In a fourth aspect, the present invention relates to a host cell, which comprises a nucleic acid construct or an expression vector of the invention as defined in the previous paragraph. A host cell expresses a polypeptide of the invention able to interact with a DC-SIGN and having an amino acid sequence which has at least 30% identity with the SEQ ID NO:1. The choice of the host cell will to a large extent depend upon the source of the nucleic acid sequence of the invention. Depending on the identity of the host cell, the skilled person would know how to transform it with the construct or vector of the invention. A nucleic acid sequence may be native for a chosen host cell. Alternatively, a nucleic acid sequence may be heterologous for a chosen host cell. A nucleic acid sequence or polypeptide which has been subjected to any recombinant molecular biology techniques to obtain a variant nucleic acid sequence or polypeptide will be considered as heterologous for the host cell it originated.

A host cell may be any microbial or prokaryotic cell, which is suitable for expression of a polypeptide of the invention. Preferably bacterial cells are used. More preferred are Gram positive bacteria. Even more preferred Gram positive bacteria include bacteria belonging to the genus Bacillus or Lactococcus. Even more preferred bacteria belong to the species Bacillus subtilis or Lactococcus lactis.. Even more preferred are strains from Escherichia coli, . All cells cited under the section "polypeptide" as origin of a polypeptide of the invention are also preferred host cells in the context of this section. In a preferred embodiment, a host cell is a probiotic strain, preferably a Lactobacillus species, even more preferably a Lactobacillus plantarum species. A host cell may be considered as a recombinant host cell. Methods for transforming bacterial cells are known in the art and are for example described in "Genetics and

Biotechnology of Lactic Acid Bacteria", Gasson and de Vos, eds., Chapman and Hall, 1994. In case a host of the invention is constructed through genetic engineering such that the resulting recombinant host comprises only sequences derived from the same species as the host is, albeit in recombined form, the host is said to be obtained through "self-cloning". Hosts obtained through self-cloning have the advantage that there application in food (or pharmaceuticals) is more readily accepted by the public and regulatory authorities as compared hosts comprising foreign (i.e. heterologous) nucleic acid sequences. The present invention thus allows the construction of self-cloned L. plantarum and other lactobacillus hosts for food, pharmaceutical or nutraceutical applications (see also de Vos, 1999, Int. Dairy J. 9: 3-10) and such self-cloned hosts are one further preferred embodiment of the invention.

According to a preferred embodiment, a host cell hence obtained overexpresses, i.e. produces more of a polypeptide of the invention and/or exhibits a higher ability to interact with a DC-SIGN than the parental cell this host cell derives from when both cultured and/or assayed under the same conditions.

"Producing more" is herein defined as producing more of a polypeptide of the invention than what the parental host cell the transformed host cell derives from will produce when both types of cells (parental and transformed cells) are cultured under the same conditions. Preferably, a host cell of the invention produces at least 3%, 6%, 10% or 15% more of a polypeptide of the invention than the parental host cell the transformed host cell derives from will produce when both types of cells (parental and transformed cells) are cultured under the same conditions. Also hosts which produce at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% more of said polypeptide than the parental cell are preferred. According to another preferred embodiment, the production level of said polypeptide of the host cell of the invention is compared to the production level of the WCFSl Lactobacillus plantarum strain, which is taken as control. According to an even more preferred embodiment, when a host cell of the invention is a Lactobacillus plantarum strain, the production level of a polypeptide of the host cell of the invention is compared to the production level of the WCFSl Lactobacillus plantarum strain, which is taken as control.

The assessment of the production level of a polypeptide may be performed at the mRNA level by carrying out a Northern Blot or an array analysis and/or at the polypeptide level by carrying out a Western blot. All these methods are well known to the skilled person.

"Exhibiting a higher ability to interact with a DC-SIGN" is herein defined as exhibiting a higher ability to interact with a DC-SIGN than the one of the parental host cell the transformed host cell derives from using an assay specific for detecting the interaction with a DC-SIGN. Preferably, the assay is the one, which has been already described herein. Preferably, a host cell of the invention exhibits at least 3%, 6%, 10% or 15% higher ability to interact with a DC-SIGN than the parental host cell the transformed host cell derives from will exhibit as assayed using the specific assay as already defined. Also a host cell which exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% more of said activity than the parental cell is preferred. A host cell of the invention which exhibits a ability to interact with a DC- SIGN which is at least twice higher than the ability of the parental host cell the transformed host cell derives from will exhibit as assayed using the specific assay as already defined is also preferred. Also a host cell which exhibits at least 5 times, at least 10 times higher, at least 20 times higher, at least 50 times higher, at least 100 times higher, at least 200 times higher ability to interact with a DC-SIGN than the parental cell is preferred. According to another preferred embodiment, the level of interaction with a DC-SIGN of a host cell of the invention is compared to the corresponding activity of the WCFS 1 Lactobacillus plantarum strain, which is taken as control. According to a more preferred embodiment, when a host cell of the invention is a Lactobacillus plantarum strain, the ability of a host cell of the invention to interact with a DC-SIGN is compared to the corresponding ability of the WCFSl Lactobacillus plantarum, which is taken as control .

The overexpression may have been achieved by conventional methods known in the art, such as by introducing more copies of a nucleic acid sequence encoding a polypeptide of the invention into the host, be it on a carrier or in the chromosome, than naturally present. Alternatively, a nucleic acid sequence of the invention can be overexpressed by fusing it to highly expressed or strong promoter suitable for high level protein expression in the selected organism, or combination of the two approaches. Alternatively or in combination, a polypeptide of the invention having an enhanced activity as defined earlier can be overexpressed in a host cell of the invention. The skilled person will know which strong promoter is the most appropriate depending on the identity of the host cell. Preferably when a host cell is a Lactobacillus plantarum strain, any promoter sequence selected on basis of high activity may be used. The selection of a suited promoter sequence may be realized by arrays or promoter-probe analysis or by in situ GI-tract activity. Such promoters are preferably identified as described in Bron PA, et al, J. Bacteriol. (2004), 186:5721- 5729.

In a preferred embodiment, a polypeptide of the invention is expressed together with at least a nucleic acid molecule in a host cell in order to optimize the maturation of a polypeptide to get an optimal ability to interact with a DC-SIGN. A nucleic acid molecule expressed together with the nucleic acid molecule coding for a polypeptide of the invention is found on a gene cluster named a DC-SIGN ligand gene cluster, which is represented by SEQ ID NO:3. A DC-SIGN ligand gene cluster comprises four open reading frames: a nucleic acid sequence coding for a polypeptide of the invention (also named lp_2145), a nucleic acid sequence coding for a glycosyltransferase (also named lp_2142), and two nucleic acid sequences each coding for an integral membrane protein (also named lp_2143 and lp_2141). A nucleic acid molecule coding for a glycosyltransferase is represented by the SEQ ID NO:4. The corresponding encoded glycosyltransferase is represented by SEQ ID NO:5. The nucleic acid molecules coding for integral membrane proteins are respectively represented by SEQ ID NO:6 and SEQ ID NO:7. The corresponding encoded integral membrane proteins are respectively represented by SEQ ID NO: 8 and SEQ ID NO:9. Each of these polypeptides, homologues thereof (as defined for homologue of SEQ ID NO:1) and corresponding nucleic coding sequences of the homologues are all encompassed by the present invention.

Accordingly in a preferred embodiment, a host cell of the invention expresses a polypeptide of the invention and a polypeptide selected from the group consisting of: a glycosyltransferase, two integral membrane protein or homologues thereof all as defined above. Alternatively, in another preferred embodiment, a host cell of the invention expresses a DC-SIGN ligand cluster gene. Alternatively, a host cell of the invention expresses a polypeptide of the invention and a DC-SIGN ligand cluster gene. In a more preferred embodiment, a host cell of the invention expresses a polypeptide of the invention and a glycosyltransferase or homologue thereof as defined above. To introduce a nucleic acid molecule present in a DC-SIGN ligand cluster gene as defined above into a host cell, the same technology is preferably used as for a nucleic acid molecule encoding a polypeptide of the invention, which is a DC-SIGN ligand. Therefore, the invention is also directed to a nucleic acid construct or an expression vector comprising a nucleic acid molecule encoding a glycosyltransferase, and/or an integral membrane protein or homologues thereof as defined above. A host cell expressing a polypeptide of the invention and a nucleic acid molecule encoding a glycosyltransferase, and/or an integral membrane protein or homologues thereof is also an object of the present invention. Alternatively, a whole DC-SIGN ligand cluster is introduced into a host cell, said cluster comprising all nucleic acid molecules as defined above as well as associated regulating regions. A nucleic acid molecule of a whole DC-SIGN ligand cluster which is further encompassed by the present invention is represented by a sequence having at least 30% identity with SEQ ID NO:3. Preferably, a nucleic acid molecule of a whole DC-SIGN ligand cluster has at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than 95% identity with SEQ ID NO:3. The skilled person will know how to introduce this whole cluster in a bacterial cell. Therefore in a more preferred embodiment, a host cell is provided as defined in the fourth aspect of the invention (see section entitled host cell) said host cell further comprising a nucleic acid construct or an expression vector selected from the group consisting of: a) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding a glycosyltransferase having at least 30% identity with SEQ ID NO: 5, b) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding an integral membrane protein having at least 30% identity with SEQ ID NO: 8, c) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding an integral membrane protein having at least 30% identity with

SEQ ID NO: 9.

In another preferred embodiment, a host cell is provided, which comprises a nucleic acid construct or an expression vector comprising a nucleic acid sequence of the DC- SIGN ligand cluster, having at least 30% identity with SEQ ID NO:3.

Preferably, a nucleic acid molecule encoding a glycosyltransferase as identified and used herein has at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than

95% identity with SEQ ID NO:5. Preferably, a nucleic acid molecule encoding an integral membrane protein as identified and used herein has at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than 95% identity with SEQ ID NO: 8.

Preferably, a nucleic acid molecule encoding an integral membrane protein as identified and used herein has at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than 95 % identity with SEQ ID NO : 9.

Such a host cell is expected to produce an optimal functional polypeptide of the invention as defined in the first aspect of the invention.

The overexpressing host cell may be used to produce substantial amounts of a polypeptide of the invention which can subsequently be used in the below described applications. Accordingly, in a further aspect, the invention relates to a method for producing a polypeptide of the invention as defined above by culturing a host cell of the invention under suitable culture conditions, and optionally isolating it from said host cell. Alternatively, a host cell expressing a polypeptide of the invention may be used as such in any of the below described applications.

Food ingredient, nutraceutical and medicament

Accordingly in a further aspect, a polypeptide of the invention as such and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention as earlier defined herein are for use as a medicament. Said medicament is preferably for preventing or treating an inflammatory gastrointestinal (GI) tract disease and/or for inducing tolerance. Tolerance induction has a major impact on a variety of aberrations of the immune system: for example all types of allergic diseases could be treated and/or prevented by increasing tolerance of the intestinal and/or systemic immune system. Treatment of allergy preferably means allergy symptom reduction. Prevention of allergy preferably means reduction of risk of allergy development.

Furthermore, tolerance induction is also expected to prevent and/or treat any type of autoimmune related defect or disease. Immune reactions against "self (autoimmunity) partially include lack of appropriate tolerance levels. Examples of autoimmune related defects or diseases include rheumatism, arthritis, type-1 diabetes, etc.

In addition, an inflammatory disorder of the intestine could be treated and/or prevented. Preferred inflammatory disorders of the intestine include the Inflammatory Bowel Disease (IBD) and the irritable bowel disorder (IBS).

A further aspect of the invention relates to a composition comprising or consisting of a polypeptide of the invention and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention both as described herein above. A host cell of the invention, preferably a bacterium, is cultured under appropriate conditions, optionally recovered from the culture medium and optionally formulated into a composition suitable for the intended use. Alternatively, a polypeptide of the invention is recovered from the cultured host cell and optionally formulated into a composition suitable for the intended use. Methods for the preparation of such compositions are known per se. A composition for enteral or oral administration may be either a food composition or a pharmaceutical composition whereas a composition for nasal, vaginal or rectal administration will usually be a pharmaceutical composition. A pharmaceutical composition will usually comprise a pharmaceutical carrier in addition to a host cell and/or a polypeptide of the invention. The preferred form depends on the intended mode of administration and (therapeutic) application. A pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver a host cell and/or a polypeptide of the invention to the GI -tract of a subject. E.g. sterile water, or inert solids may be used as a carrier usually complemented with a pharmaceutically acceptable adjuvant, buffering agent, dispersing agent, and the like. A composition will either be in liquid, e.g. a stabilized suspension of the host cell, or in solid forms, e.g. a powder of lyophilized host cells. E.g. for oral administration, a host cell and/or a polypeptide can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. A host cell and/or a polypeptide of the invention can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as e.g. glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. A preferred composition according to the invention is suitable for consumption by a subject, preferably a human or an animal. Such compositions may be in the form of a food supplement or a food or food composition, which besides a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention also contains a suitable food base. A food or food composition is herein understood to include a liquid for human or animal consumption, i.e. a drink or beverage. A food or food composition may be a solid, semi-solid and/or liquid food or food composition, and in particular may be a dairy product, such as a fermented dairy product, including but not limited to a yoghurt, a yoghurt-based drink or buttermilk. Such a food or food composition may be prepared in a manner known per se, e.g. by adding a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention to a suitable food or food base, in a suitable amount. In a further preferred embodiment, a host cell is micro-organism that is used in or for the preparation of a food or food composition, e.g. by fermentation. Examples of such a micro-organism include baker's or brewer's yeast and lactic acid bacteria, such as probiotic lactic acid strains as earlier exemplified herein. In doing so, a host cell of the invention may be used in a manner known per se for the preparation of such fermented foods or food compositions, e.g. in a manner known per se for the preparation of fermented dairy products using lactic acid bacteria. In such methods, a host cell of the invention may be used in addition to a micro-organism usually used, and/or may replace one or more or part of a micro-organism usually used. For example, in the preparation of a fermented dairy product such as yoghurt or yoghurt-based drinks, a food grade lactic acid bacterium of the invention may be added to or used as part of a starter culture or may be suitably added during such a fermentation. A polypeptide of the invention may further be added to these compositions.

Preferably, the above compositions will contain a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention in amounts that allow for convenient (oral) administration of a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention, e.g. as or in one or more doses per day or per week. In particular, a preparation may contain a unit dose of a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention.

In another aspect, the invention relates to a method for (site-specific) production of a polypeptide of the invention at a mucosal surface of a subject as has been exemplified in WO 05/040387. The method comprises the step of administering to the subject a composition comprising a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention. Without wishing to be bound by any theory, it is expected that a polypeptide and/or a host cell of the invention will exert their immunomodulatory action via the presence of a polypeptide of the invention.

Another aspect of the invention relates to the use of a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell according to the invention in the preparation of a medicament for preventing or treating of inflammatory gastrointestinal tract disease in a subject.

Accordingly, in a further aspect, the invention allows the screening for cell expressing a polypeptide of the invention. By detecting the presence of a functional polypeptide of the invention as earlier defined herein, one may identify cells that natively express a polypeptide of the invention, and therefore such strains are expected to interact with a DC-SIGN and have attractive immunomodulatory activity. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a polypeptide or a host cell or a composition of the invention may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. The word "approximately" or "about" when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

"Exhibiting a higher ability to interact with a DC-SIGN" is herein defined as exhibiting a higher ability to interact with a DC-SIGN than the one of the parental host cell the transformed host cell derives from using an assay specific for detecting the interaction with a DC-SIGN. Preferably, the assay is the one, which has been already described herein. Preferably, a host cell of the invention exhibits at least 3%, 6%, 10% or 15% higher ability to interact with a DC-SIGN than the parental host cell the transformed host cell derives from will exhibit as assayed using the specific assay as already defined. Also a host cell which exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% more of said activity than the parental cell is preferred. A host cell of the invention which exhibits a ability to interact with a DC- SIGN which is at least twice higher than the ability of the parental host cell the transformed host cell derives from will exhibit as assayed using the specific assay as already defined is also preferred. Also a host cell which exhibits at least 5 times, at least 10 times higher, at least 20 times higher, at least 50 times higher, at least 100 times higher, at least 200 times higher ability to interact with a DC-SIGN than the parental cell is preferred. According to another preferred embodiment, the level of interaction with a DC-SIGN of a host cell of the invention is compared to the corresponding activity of the WCFS 1 Lactobacillus plantarum strain, which is taken as control. According to a more preferred embodiment, when a host cell of the invention is a Lactobacillus plantarum strain, the ability of a host cell of the invention to interact with a DC-SIGN is compared to the corresponding ability of the WCFSl Lactobacillus plantarum, which is taken as control . The overexpression may have been achieved by conventional methods known in the art, such as by introducing more copies of a nucleic acid sequence encoding a polypeptide of the invention into the host, be it on a carrier or in the chromosome, than naturally present. Alternatively, a nucleic acid sequence of the invention can be overexpressed by fusing it to highly expressed or strong promoter suitable for high level protein expression in the selected organism, or combination of the two approaches. Alternatively or in combination, a polypeptide of the invention having an enhanced activity as defined earlier can be overexpressed in a host cell of the invention. The skilled person will know which strong promoter is the most appropriate depending on the identity of the host cell. Preferably when a host cell is a

Lactobacillus plantarum strain, any promoter sequence selected on basis of high activity may be used. The selection of a suited promoter sequence may be realized by arrays or promoter-probe analysis or by in situ GI-tract activity. Such promoters are preferably identified as described in Bron PA, et al, J. Bacteriol. (2004), 186:5721- 5729.

In a preferred embodiment, a polypeptide of the invention is expressed together with at least a nucleic acid molecule in a host cell in order to optimize the maturation of a polypeptide to get an optimal ability to interact with a DC-SIGN. A nucleic acid molecule expressed together with the nucleic acid molecule coding for a polypeptide of the invention is found on a gene cluster named a DC-SIGN ligand gene cluster, which is represented by SEQ ID NO:3. A DC-SIGN ligand gene cluster comprises four open reading frames: a nucleic acid sequence coding for a polypeptide of the invention (also named lp_2145), a nucleic acid sequence coding for a glycosyltransferase (also named lp_2142), and two nucleic acid sequences each coding for an integral membrane protein (also named lp_2143 and lp_2141). A nucleic acid molecule coding for a glycosyltransferase is represented by the SEQ ID NO:4. The corresponding encoded glycosyltransferase is represented by SEQ ID NO:5. The nucleic acid molecules coding for integral membrane proteins are respectively represented by SEQ ID NO: 6 and SEQ ID NO:7. The corresponding encoded integral membrane proteins are respectively represented by SEQ ID NO: 8 and SEQ ID NO:9. Each of these polypeptides, homologues thereof (as defined for homologue of SEQ ID NO:1) and corresponding nucleic coding sequences of the homologues are all encompassed by the present invention.

Accordingly in a preferred embodiment, a host cell of the invention expresses a polypeptide of the invention and a polypeptide selected from the group consisting of: a glycosyltransferase, two integral membrane protein or homologues thereof all as defined above. Alternatively, in another preferred embodiment, a host cell of the invention expresses a DC-SIGN ligand cluster gene. Alternatively, a host cell of the invention expresses a polypeptide of the invention and a DC-SIGN ligand cluster gene. In a more preferred embodiment, a host cell of the invention expresses a polypeptide of the invention and a glycosyltransferase or homologue thereof as defined above. To introduce a nucleic acid molecule present in a DC-SIGN ligand cluster gene as defined above into a host cell, the same technology is preferably used as for a nucleic acid molecule encoding a polypeptide of the invention, which is a DC-SIGN ligand. Therefore, the invention is also directed to a nucleic acid construct or an expression vector comprising a nucleic acid molecule encoding a glycosyltransferase, and/or an integral membrane protein or homologues thereof as defined above. A host cell expressing a polypeptide of the invention and a nucleic acid molecule encoding a glycosyltransferase, and/or an integral membrane protein or homologues thereof is also an object of the present invention. Alternatively, a whole DC-SIGN ligand cluster is introduced into a host cell, said cluster comprising all nucleic acid molecules as defined above as well as associated regulating regions. A nucleic acid molecule of a whole DC-SIGN ligand cluster which is further encompassed by the present invention is represented by a sequence having at least 30% identity with SEQ ID NO:3. Preferably, a nucleic acid molecule of a whole DC-SIGN ligand cluster has at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than 95% identity with SEQ ID NO:3. The skilled person will know how to introduce this whole cluster in a bacterial cell.

Therefore in a more preferred embodiment, a host cell is provided as defined in the fourth aspect of the invention (see section entitled host cell) said host cell further comprising a nucleic acid construct or an expression vector selected from the group consisting of: a) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding a glycosyltransferase having at least 30% identity with SEQ ID NO: 5, b) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding an integral membrane protein having at least 30% identity with

SEQ ID NO: 8, c) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding an integral membrane protein having at least 30% identity with SEQ ID NO: 9. In another preferred embodiment, a host cell is provided, which comprises a nucleic acid construct or an expression vector comprising a nucleic acid sequence of the DC- SIGN ligand cluster, having at least 30% identity with SEQ ID NO:3. Preferably, a nucleic acid molecule encoding a glycosyltransferase as identified and used herein has at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than 95% identity with SEQ ID NO:5.

Preferably, a nucleic acid molecule encoding an integral membrane protein as identified and used herein has at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than 95% identity with SEQ ID NO: 8. Preferably, a nucleic acid molecule encoding an integral membrane protein as identified and used herein has at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than 95% identity with SEQ ID NO:9.

The overexpressing host cell may be used to produce substantial amounts of a polypeptide of the invention which can subsequently be used in the below described applications. Accordingly, in a further aspect, the invention relates to a method for producing a polypeptide of the invention as defined above by culturing a host cell of the invention under suitable culture conditions, and optionally isolating it from said host cell.

Alternatively, a host cell expressing a polypeptide of the invention may be used as such in any of the below described applications. Food ingredient, nutraceutical and medicament

Accordingly in a further aspect, a polypeptide of the invention as such and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention as earlier defined herein are for use as a medicament. Said medicament is preferably for preventing or treating an inflammatory gastrointestinal (GI) tract disease and/or for inducing tolerance.

Tolerance induction has a major impact on a variety of aberrations of the immune system: for example all types of allergic diseases could be treated and/or prevented by increasing tolerance of the intestinal and/or systemic immune system. Treatment of allergy preferably means allergy symptom reduction. Prevention of allergy preferably means reduction of risk of allergy development.

A further aspect of the invention relates to a composition comprising or consisting of a polypeptide of the invention and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention both as described herein above. A host cell of the invention, preferably a bacterium, is cultured under appropriate conditions, optionally recovered from the culture medium and optionally formulated into a composition suitable for the intended use. Alternatively, a polypeptide of the invention is recovered from the cultured host cell and optionally formulated into a composition suitable for the intended use. Methods for the preparation of such compositions are known per se. A composition for enteral or oral administration may be either a food composition or a pharmaceutical composition whereas a composition for nasal, vaginal or rectal administration will usually be a pharmaceutical composition. A pharmaceutical composition will usually comprise a pharmaceutical carrier in addition to a host cell and/or a polypeptide of the invention. The preferred form depends on the intended mode of administration and (therapeutic) application. A pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver a host cell and/or a polypeptide of the invention to the GI -tract of a subject. E.g. sterile water, or inert solids may be used as a carrier usually complemented with a pharmaceutically acceptable adjuvant, buffering agent, dispersing agent, and the like. A composition will either be in liquid, e.g. a stabilized suspension of the host cell, or in solid forms, e.g. a powder of lyophilized host cells. E.g. for oral administration, a host cell and/or a polypeptide can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. A host cell and/or a polypeptide of the invention can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as e.g. glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.

A preferred composition according to the invention is suitable for consumption by a subject, preferably a human or an animal. Such compositions may be in the form of a food supplement or a food or food composition, which besides a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention also contains a suitable food base. A food or food composition is herein understood to include a liquid for human or animal consumption, i.e. a drink or beverage. A food or food composition may be a solid, semi-solid and/or liquid food or food composition, and in particular may be a dairy product, such as a fermented dairy product, including but not limited to a yoghurt, a yoghurt-based drink or buttermilk. Such a food or food composition may be prepared in a manner known per se, e.g. by adding a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention to a suitable food or food base, in a suitable amount. In a further preferred embodiment, a host cell is micro-organism that is used in or for the preparation of a food or food composition, e.g. by fermentation. Examples of such a micro-organism include baker's or brewer's yeast and lactic acid bacteria, such as probiotic lactic acid strains as earlier exemplified herein. In doing so, a host cell of the invention may be used in a manner known per se for the preparation of such fermented foods or food compositions, e.g. in a manner known per se for the preparation of fermented dairy products using lactic acid bacteria. In such methods, a host cell of the invention may be used in addition to a micro-organism usually used, and/or may replace one or more or part of a micro-organism usually used. For example, in the preparation of a fermented dairy product such as yoghurt or yoghurt-based drinks, a food grade lactic acid bacterium of the invention may be added to or used as part of a starter culture or may be suitably added during such a fermentation. A polypeptide of the invention may further be added to these compositions. Preferably, the above compositions will contain a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention in amounts that allow for convenient (oral) administration of a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention, e.g. as or in one or more doses per day or per week. In particular, a preparation may contain a unit dose of a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell of the invention.

Another aspect of the invention relates to the use of a polypeptide and/or an encoding nucleic acid molecule and/or a nucleic acid construct comprising said nucleic acid molecule and/or a host cell according to the invention in the preparation of a medicament for preventing or treating of inflammatory gastrointestinal tract disease in a subject. Accordingly, in a further aspect, the invention allows the screening for cell expressing a polypeptide of the invention. By detecting the presence of a functional polypeptide of the invention as earlier defined herein, one may identify cells that natively express a polypeptide of the invention, and therefore such strains are expected to interact with a DC-SIGN and have attractive immunomodulatory activity.

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Examples Example 1:

DC-SIGN binding of Lactobacillus ylantarum strains, phenotypic screening.

Previous studies suggested that Lactobacillus plantarum B253 cells were not recognized by DC-SIGN (Smits HH, Engering A, van der Kleij D, de Jong EC, Schipper K, van Capel TM, Zaat BA, Yazdanbakhsh M, Wierenga EA, van Kooyk Y, Kapsenberg ML. (2005) J Allergy Clin Immunol. 115:1260-1267) while two other lactobacilli, e.g. Lactobacillus casei B255 [NIZO food research culture collection, Ede, The Netherlands] and Lactobacillus reuteri DSM20016 [Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany] were recognized by this receptor. However, the strain L. plantarum B253 used in this study was originally obtained from the NIZO food research culture collection in which it is stored as a Lactococcus plantarum and not as a Lactobacillus plantarum. Therefore, it remained to be determined whether Lactobacillus plantarum strains are recognized by DC-SIGN. The experiments described below were performed to establish DC-SIGN recognition of Lactobacillus plantarum cells. In addition, they explore the variation among Lactobacillus plantarum strains in terms of DC-SIGN recognition.

DC-SIGN-FC ELISA assay

Binding of the soluble DC-SIGN mimicking DC-SIGN-Fc to individual L. plantarum cultures was evaluated by ELISA analysis. A total of 24 L. plantarum strains were grown overnight at 30 ⁰C in MRS medium. The 24 strains were selected on basis of the availability of comparative genome hybridization datasets for these strains (see below), and include the Lactobacillus plantarum strain WCFS 1 (also known under number B 1836 [NIZO food research culture collection number]). Cells were washed 3 times with an equal volume of phosphate buffered isotonic saline solution (PBS). Cell densities were determined by enumeration of colony forming units (cfu) on MRS-agar plates using standard techniques (Sambrook, J., F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, US.). All L. plantarum suspensions were coated in ELISA plates at a density of 5 * 10⁵ cfus per well for 1 hour at 4 ⁰C, followed by blocking of the wells with 1% bovine serum albumin for 30 min at 4 ⁰C. DC-SIGN binding was assessed using DC-SIGN-Fc, which consists of the extracellular portion of DC-SIGN (amino acid residues 64-404) fused at the C terminus to a human IgGl-Fc fragment (Fawcett, J., Holness, C. L., Needham, L. A., Turley, H., Gatter, K. C, Mason, D. Y., and Simmons, D. L. (1992) Nature 360, 481-484). DC-SIGN-Fc (A generous gift of the lab of Yvette van Kooyk, Amsterdam, The Netherlands) was added, and the adhesion was performed for 30 min at 37 ⁰C. Unbound DC-SIGN-Fc was washed away, and binding was determined using horseradish peroxidase conjugated goat-anti- human-Fc and standard ELISA technique (Geijtenbeek, T.B.H., van Duijnhoven,

G.C.F., van Vliet, S.J., Krieger, E., Vriend, G., Figdor, C.G., and van Kooyk, Y. (2002) J. Biol. Chem., 277:11314-11320). Specificity of DC-SIGN-Fc binding was determined by the addition of excess amounts of anti-DC-SIGN monoclonal antibodies (AZN-Dl; approximate concentration 20 μg/ml). As a negative control, no bacterial cells were coated in the well.

Of the 24 strains of L. plantarum tested, 21 strains appeared to display significant and specific binding of DC-SIGN-Fc as assessed in the ELISA assay. The 21 positive strains all showed similar quantitative levels of DC-SIGN-Fc binding, which was comparable to that observed in the WCFSl strain for which the genome sequence is available (Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers MW, Stiekema W, Lankhorst RM, Bron PA, Hoffer SM, Groot MN, Kerkhoven R, de Vries M, Ursing B, de Vos WM, Siezen RJ. (2003) Proc Natl Acad Sci USA. 100:1990-1995). The table below shows the ELISA read-out result for 4 strains, three of which appeared to display reduced DC- SIGN-Fc binding (B2766, B2806, B2855) while the fourth strain (B1836; = WCFSl) showed a level of binding that was also seen for the other strains. These results suggest that the majority of L. plantarum strains can be recognized by DC-SIGN, while a few strains have significantly reduced or no DC-SIGN binding capacity. However, the soluble DC-SIGN-Fc based assay should be regarded as a primary screening analysis that requires further confirmation through advanced binding assays. Table 1: DC-SIGN-Fc ELISA assay results of 4 Lactobacillus plantarum strains. Strain B1836 (is the same strain as WCFSl) is included as a representative strain with a positive score, while the three additional strains are the only L. plantarum strains tested here that showed lower DC-SIGN-Fc binding in the ELISA assays performed. The DC- SIGN-Fc + aDC-SIGN column is the specificity control result and present extinctions measured when both DC-SIGN-Fc and excess anti-DC-SIGN are present.

Cell-based DC-SIGN binding assays

Two advanced DC-SIGN recognition assays have been described in the literature. The first assay determines DC-SIGN dependent binding to immature dendritic cells (iDC), while the second assay employs DC-SIGN transfected cells (Raji-DC-SIGN). Immature DCs were generated from monocytes, as previously described (Vieira PL, de Jong EC, Wierenga EA, Kapsenberg ML, Kalinski P. Development of ThI -inducing capacity in myeloid dendritic cells requires environmental instruction. J Immunol 2000; 164:4507- 12.). Raji-DC-SIGN cells were cultured as previously described (Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS, KewalRamani VN, Littman DR, Figdor CG, van Kooyk Y. (2000) Cell, 100:587-597).

Four strains of Lactobacillus plantarum (B1836, B2855, B2766, and B2806; 10⁹ cfu/mL) were labeled by incubation with Fluorescein Isothiocyanate (FITC; 0.5 mg/mL) for 1 hour at room temperature (RT). After washing (5 times in PBS), the bacteria were resuspended in TSM buffer (20mMTris, 15OmM NaCl, ImM CaC12, and 2 mMMgC12, pH 8.0), which is appropriate for the analyses of their binding to iDCs and Raji-DC-SIGN cells.

FITC-labeled L. plantarum cells were incubated with iDC or Raji-DC-SIGN cells (ratio cells: bacteria approximately 1:10) in the presence or absence of excess amounts of anti-Dc-SIGN monoclonal antibodies (AZN-Dl; approximate concentration 20 μg/ml) for 45 minutes at 37 ⁰C. Unbound bacteria were removed by washing the cells 3 times withTSM. The iDC and Raji-DC-SIGN fluorescence was examined by FACS. The numbers presented below are the mean fluorescence level of approximately 4000 iDC or Raji-DC-SIGN cells. As a negative control, no bacteria were added. The results clearly show a distinct difference between the binding capacity of strains B 1836, B2855 and B2766, which is notably higher (2-3 fold) as compared to that observed for strain B2806 (columns DC & Raji-DC-SIGN). Addition of mAb targeted against DC- SIGN in the dendritic cell assay significantly reduces the binding of B1836, B2855, and B2766, indicating that the observed binding is at least partially based on DC-SIGN mediated binding to these cells. In contrast, addition of DC-SIGN specific antibodies in the DC assay with strain B2806 has no significant effect on DC-binding of these bacteria, indicating that the residual binding observed with this strain is DC-SIGN independent. The results obtained after addition of monoclonal antibodies in the Raji- DC-SIGN assay are less clear, although the binding of strains B1836, B2855, and

B2766 is also in this assay significantly reduced. However, the lower level of binding of strain B2806 in this assay also appears to be moderately modulated by the addition DC-SIGN antibodies.) These results indicate that the L. plantarum strains B 1836, B2855, and B2766 bind to DC-SIGN, while strain B2806 does not. These data provide a refined conclusion as compared to the data obtained by DC-SIGN-Fc assay and indicate that from the group of 24 L. plantarum strains analysed here, only a single strain consistently lacks the DC-SIGN binding capacity.

Table 2: Binding of FITC-labeled L. plantarum cells to dendritic and Raji-DC-SIGN cells in the presence and absence of 20 μg/mL anti-DC-SIGN monoclonal antibodies (mAb). Values given are mean fluorescence values of approximately 4000 DC or Raji cells as determined by FACS analysis.

Example 2:

Lactobacillus plantarum genomic biodiversity analysis; DC-SIGN ligand identification.

The DC-SIGN binding capacity of L. plantarum strains appeared to vary between strains and ultimately resulted in the identification of a single strain that clearly does not bind DC-SIGN, while all other strains tested did bind this receptor (23 strains; see example 1). This phenotypic variation can be matched or correlated with genomic variability among the strains analyzed.

To construct a genomic diversity database for the 24 strains used, chromosomal DNA for microarray hybridization experiments was extracted from these 24 strains as previously described by Molenaar et al. (Molenaar D, Bringel F, Schuren FH, de Vos WM, Siezen RJ, Kleerebezem M. (2005) J Bacteriol. 187:6119-6127) and then fragmented by shearing as follows: approximately 50 ng of DNA was mixed with 900 μl of shearing buffer (TE, pH = 8.0 and glycerol 10 %) and loaded in a nebulizer (Invitrogen, The Netherlands). Compressed air was applied for 2 min at 1.7 bar in order to obtain DNA fragments with approximate size of 1 Kb. The DNA integrity and extraction efficiency were verified by electrophoresis in 1 % agarose gel in Ix TAE buffer, stained with ethidium bromide. DNA purity and concentration were evaluated using Nanodrop ND- 1000 Spectrophotometer (NanoDrop Technologies, Germany). The micro arrays used in this study were supplied by Agilent Technologies

(Amstelveen, The Netherlands). Sixtymer oligonucleotide probes were spotted on a standard 1" x 3" glass slide. Two identical arrays were present on each glass slide with H-K features per array, designed on the putative open reading frame (ORF) annotated in the complete sequenced genome of WCFSl strain (Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers MW, Stiekema W, Lankhorst RM, Bron PA, Hoffer SM, Groot MN, Kerkhoven R, de Vries M, Ursing B, de Vos WM, Siezen RJ. (2003) Proc Natl Acad Sci USA. 100:1990-1995). Usually three different probes were present per gene, evenly distributed over the gene sequence.

The sheared chromosomal DNA (1 μg) isolated from 24 L. plantarum strains was labeled by incorporation of Cy3-dUTP or Cy5-dUTP (Amersham Biosciences, UK) using the commercial kit BioPrime® DNA Labelling System (Invitrogen). The L. plantarum WCFSl DNA applied as reference for each hybridization experiment was labeled with Cy3-dUTP dye, and the chromosomal DNA of the 23 tested L. plantarum strains was labeled with Cy5-dUTP. Purification of labeled DNA was performed using CyScribe GFX column (Amersham Biosciences) according to the manufacture's instruction. Then, dye incorporation efficiency was measured using the Nanodrop ND- 1000 Spectrophotometer.

Co -hybridization of the two differently (Cy3 and Cy5) labeled DNA samples was carried out overnight at 60 ⁰C in Ix hybridization buffer following manufacture's instruction and circularly rotated in the stove (Agilent Technologies). After hybridization, the slides were washed 10 min at room temperature in wash solution 1 (6x SSC, 0.005 % triton X- 102), and then transferred to wash solution 2 (O.lx SSC, 0.005 % triton X- 102) for 5 min on ice. The slides were quickly dried using compressed nitrogen and were stored in dark until scanning, which was performed within 1 hour. The hybridization image was acquired using a ScanArray 4000 scanner (Perkin Elmer, USA) setting the resolution at 10 nm. The photo -multiplier tubes value was adjusted to balance signals obtained for both channels (Cy5 and Cy3 signal). Quantification of the signal for each spot was evaluated with ImaGene software, version 4.2 (BioDiscovery, Canada). The quantification data were imported in BASE (http://cran.r- project.org/bin/windows/base/)). The control probes were removed and the background was subtracted from the signal for a preliminary data analysis. Hybridization data were normalized by local fitting of an M-A plot, implementing a LOWESS algorithm in R-2.2.1 program (hUj>^/cπmjy^^ as previously described (Molenaar D, Bringel F, Schuren FH, de Vos WM, Siezen RJ, Kleerebezem M. (2005) J Bacteriol. 187:6119-6127). M value was defined as Iog2 (Chl/Ch2) and A was calculated with the following formula [1Og₂(ChI) + log₂(Ch2)]/2, considering Cy3 (reference DNA of WCFSl strain) as channel 2 (Ch2) and Cy5 signal (tested DNA) as channel 1 , ChI .

Low fluorescence value (M < - 2.0) detected in correspondence of a specific oligonucleotide indicated the absence of the region recognized by this probe. High hybridization signals (M > + 2.0) evidenced for genes probably present in multiple copies in the analyzed strain compared to WCFSl. Intermediate hybridization values (- 1 < M < + 1.0) indicated no differences of gene content between the tested strain and WCFSl. Finally, a small number of probes gave low M values (- 2.0 < M < - 1.0). These low values indicate poor hybridization to the probe target which may indicate variability in the nucleotide sequences rather than the complete absence of the target DNA. The probe specific dataset can be converted to a statistically weighted gene presence or absence dataset using the M-values corresponding to the different probes corresponding to individual genes. Using either the probe specific or gene specific CGH databases of the 24 strains analyzed here can be used for gene-trait matching as described by Pretzer et al. (Pretzer G, Snel J, Molenaar D, Wiersma A, Bron PA, Lambert J, de Vos WM, van der Meer R, Smits MA, Kleerebezem M. (2005) J Bacteriol. 187:6128-6136.). This correlation analysis includes assessment of the significance of the observed correlation, which is based on the assumption that there is a hypergeometric distribution for the probability of the co-occurrence of genes and traits under the null-hypothesis that the observed co-occurrence is caused by random processes alone, as was previously described (Jim, K., Parmar, K., Singh, M., and Tavazoie S. (2004) Genome Res. 14:109-115.). All L. plantarum WCFSl genes were tested for the significance of positive correlation of gene and trait. Although false positives may be expected when testing the null hypotheses for each gene at the same rejection level of, for example, p < 0.05, the number of their occurrence can be reduced by implementing the Bonferroni correction (Jim, K., Parmar, K., Singh, M., and Tavazoie S. (2004) Genome Res. 14:109-115.). However, this correction applies highly conservative constraints and the probability of the rejection of true positives is relatively large. Parallel analysis of the specific characteristics of the predicted proteins encoded by the genes identified to correlate to the trait offers an alternative approach to introduce a correction factor into the gene/trait matching approach. These combined approaches to select the most likely candidate genes that correlate with the DC-SIGN binding phenotype in these 24 L. plantarum strains.

Applying this correlation strategy using the experimental data obtained in DC-SIGN binding assays using the DC and Raji-DC-SIGN cells. The phenotypic input is composed of a relatively simple matrix where 23 of the 24 strains are assigned a positive phenotype-score and a single strain (B2806) is assigned a negative phenotype- score. The gene-trait correlation analysis using the probe-specific CGH-database identified only 3 probes that displayed a perfect correlation with the observed phenotype. Two of these probes correspond with a gene (lp_2145; CDS ID 3588280) encoding a predicted cell-surface protein of unknown function (p<0.05), while the third probe corresponds with the adjacent gene (lp_2143; CDS ID3588270) encoding an integral membrane protein (p<0.05). All other probe-trait correlations displayed lower significance, primarily because the majority of the group of next best correlating probes (p~0.08333) contained a single deviating strain in which either the probe was not giving a significant signal, while this strain scored positive in terms of DC-SIGN binding or vice versa. The majority of the probes falling in this group (p~0.08333) displayed a negative correlation with the trait and corresponded with genes encoding prophage related functions. Nevertheless, besides the prophage related genes there were 4 additional probes positively correlating with the DC-SIGN trait and corresponding to different functions. These probes include the third probe corresponding to the gene lp_2145, and the second and third probe corresponding with the lp_2143 gene (see above). Thereby, all probes belonging to these two genes display a high positive correlation with the variable trait of DC-SIGN binding. Interestingly, the last probe among the group of high positive correlation significance probes (p~0.08333) is one of the probes corresponding to the gene lp_2142, which encodes a putative glycosyltransferase that is closely linked to lp_2145 and lp_2143. Notably, the residual two probes corresponding to the lp_2142 display a positive correlation with the DC- SIGN binding trait that has a far lower significance score (p~0.98533) that is largely due to the observation that these probes do give a signal in strain B2806. These results indicate that the sole locus of the L. plantarum WCFSl genome that positively correlates with high significance with the DC-SIGN binding trait encompasses the directly flanking Ip _ 2145 and lp_2143 and could possibly include the downstream gene lp_2142, suggesting that the proteins encoded within this locus are involved in the production of the L. plantarum DC-SIGN ligand (see Figure 1 for genomic map of the lp_2141 to lp_2145 locus). Since the lp_2145 gene is predicted to encode an exported protein that is anchored to the cell surface through a C-terminal anchor sequence, this gene product seems to be the most likely candidate for the actual DC-SIGN ligand function. The role of the additional candidate genes (lp_2143, and lp_2142) could be in the biogenesis or maturation of the lp_2145 geneproduct. In this respect it is important to realize that DC-SIGN has been reported to recognize glyco- moieties (reviewed in: Zhou T, Chen Y, Hao L, Zhang Y. (2006) Cell MoI Immunol. 3:279-283.) rather than protein moieties, which could suggest that maturation of the lp_2145 gene product involves glycosylation and might depend on the activity of the lp_2142 and lp_2143 gene products. Notably, such a postulated role of the lp_2142 gene in post-translational glycosylation of the lp_2145 gene product is supported by its annotation as a putative glycosyltransferase. Moreover, the serine rich repeats encountered within the amino acid sequence of the lp_2145 gene product provide likely glycosylation target sites in this protein as has also been proposed for serine -rich proteins encoded within the genome sequence of Streptococcus pneumoniae (Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, Heidelberg J, DeBoy RT, Haft DH, Dodson RJ, Durkin AS, Gwinn M, Kolonay JF, Nelson WC, Peterson JD, Umayam LA, White O, Salzberg SL, Lewis MR, Radune D, Holtzapple E, Khouri H, WoIfAM, Utterback TR, Hansen CL, McDonald LA, Feldblyum TV, Angiuoli S, Dickinson T, Hickey EK, Holt IE, Loftus BJ, Yang F, Smith HO, Venter JC, Dougherty BA, Morrison DA, Hollingshead SK, Fraser CM.(2001) Science. 293:498-506.) but have also been found in Staphylococcus epidermidis (McCrea KW, Hartford O, Davis S, Eidhin DN, Lina G, Speziale P, Foster TJ, Hook M. (2000) Microbiology, 146:1535- 1546).

Example 3:

In silico analysis of the Ip 2141 to Ip 2145 locus of Lactobacillus plantarum WCFSl Lp_2141 is predicted to be an in integral membrane protein the function of which is unknown. Several other gram+ organisms have homologues whose function is also uncertain. Scanning using the Prosite suite of motifs does not reveal any motif with a significant high score. (http://ca.expasy.org/tooJs/scanprosite) . The protein shares a PfamB domain with Enterococcus faecalis protein Q83018 (PfamB_162143) but the function of this domain is unknown.

lp_2142 is annotated as being a glycosyltransferase family 2 clan. Pfam entry PS50167 (http://www.sanger.ac.uk/Software/Pfam/) This domain is found in a diverse family of glycosyl transferases that transfer the sugar from UDP-glucose, UDP-N-acetyl- galactosamine, GDP-mannose or CDP-abequose, to a range of substrates including cellulose, dolichol phosphate and teichoic acids, (see

bjn/Pj^yra/gclaccTPFOO^S) There are two paralogues in L.plantarum lp_1524 and lp_2716. Lp_2142 is predicted to be cytoplamic membrane protein and part of a complex which transfers glycosyl residues to proteins which it is associated. It is not possible to state which glycosyl residue is transferred.

Lp_2143 is a small integral membrane protein which has homologues in L.brevis and Oenococcus oeni their functions are unknown.

Lp_2145 is a 433 amino acid 45kDa protein which is predicted to be extracellular using the signalP server

Emanuelsson et al Nature Protocols 2, 953-971 (2007)). The lp_2145 encoded protein is predicted to have a typical N-terminal signal sequence that is probably cleaved after transport by the canonical SEC machinery. Anchoring of the protein to the cell surface is predicted to be achieved through a C-terminal anchor sequence that is predicted to anchor the protein to the cell membrane.

Searching the protein databases with this protein returns very few hits of any significance. L.brevis encodes a protein (LVIS 0893) which has homology to the first and last thirds of lp_2145 but has relatively limited identity with the middle region of lp_2145. Notably the L. brevis homologue of lp_2145 is genetically directly linked to the lp_2143 and lp_2142 homologues identified in that same strain of L. brevis (L. brevis ATCC367, LVIS 0891, LVIS 0892). In this respect, it is remarkable that also in Oenococcus oeni a weak homologue of lp_2145 can be identified (OENOO 59003), which is also genetically directly linked to the homologue of lp_2143 identified in this same strain (O. Oeni ATCC BAA-1163, OENOO 59004). OENOO 59007 corresponds to lp_2141 and the gene designated OENOO 59006 is very similar to lp_2142 Importantly, the middle region of lp_2145 that appears to be missing from the L. brevis and O. oeni homologues, and can arbitrarily be defined from amino acid 174 to 240, is a region with an exceptionally high proportion of serine and threonine residues. Within this 66 amino acid region, 28 serines and 19 threonines are present. The PFAM database describes this as being an area of low complexity which in some proteins have been shown to be functionally important. These serine and threonine residues are the candidate amino acid residues for sugar attachment using lp_2142 the glycosyltransferase. The region from residue 174 to 240 appears to be unique for lp_2145. It has been designated an area of low complexity (domain searching using pfam for example) Low complexity proteins constitute a minor fraction (_™3-7%) in bacterial genomes indicating that they are generally selected against in bacterial evolution. The vast number of proteins identified through genome sequencing indicate that low complexity proteins span a wide range of functions including translation, metabolism, transport and membrane associated, adhesins, cell division proteins and a few proteins with functional roles that can be correlated with the biological characteristics of a given species. For example, the Type III secretion apparatus and other secreted proteins of the enteropathogenic E. coli 0157, the PGRS proteins of M. tuberculosis, colonization factor of V. cholerαe, the cag pathogenicity island protein of H. pylori and the sporulation proteins of Bacillus subtilis. (Nandi et al In Silico Biology 3, 0024 (2003)). It is most likely that this region is glycosylated and that the glycsoylation will involve O-glycoslylation rather than N-glycosylation which requires asparagine residues. There is currently no prediction tool for assigning glycosylation sites in bacterial proteins. Nor does the protein contain the dipeptide repeat region E/V/I S described in the Fapl fimbrial adhesin of Streptococcus parasanguis (Stephenson,Wu,Novak,Tomana,Mintz and Fives-Talyor 2002 Molecular Microbiology 43 147-157). The protein does however contain a novel repeated domain. This was identified using the MEME discovery tool (Bailey and Elkan 1994). This repeat has been described in Boekhorst, WeIs, Kleerebezem and Siezen 2006 Microbiology 152,3175-3183. Using the sequence of lp_2145 in a sequence similarity search by the method of Smith and Waterman (Smith and Waterstone 1981 Journal of Molecular Biology 147,195-197) gives proteins which have some regions of similarity and overlap regions of varying percentages. This data can be found at http://bamics3 ,cmbi,ra.nl/cgi-bin/j^"os/secrctome/S W .pyVORF=lp_2145 . This domain is also seen in pfam (PfamB_59617).

Table 3: structure of the DC-SIGN ligand gene cluster

Example 4:

Construction of L. plantarum WCFSl DC-SIGN interaction mutants.

Gene-trait matching led to the identification of the L. plantarum WCFSl gene lp_2145 as the proposed DC-SIGN ligand. To validate the proposed role oϊlp_2145 in DC- SIGN recognition, gene-replacement mutant derivatives of L. plantarum WCFSl were constructed that lack a functional copy oϊlp_2145, or lp_2142 (proposed glycosyltransferase involved in lp_2145 gene product maturation). The strategy employed is based on previously reported construction of gene replacement mutations in L. plantarum (Lambert JM, Bongers RS, and Kleerebezem M. (2007) Appl Environ Microbiol. 73:1126-1135.)

Mutant constructions Bacterial strains, plasmids and primers. The bacterial strains, plasmids and primers used in this study and their relevant features are listed in Tables 4 and 5. As a model strain for Gram-positive bacteria, L. plantarum WCFS 1 (Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R, Peters SA, Sandbrmk HM, Fiers MW, Stiekema W, Lankhorst RM, Bron PA, Hoffer SM, Groot MN, Kerkhoven R, de Vries M, Ursing B, de Vos WM, Siezen RJ. (2003) Proc Natl Acad Sci USA. 100:1990-1995) was used. L. plantarum was grown at 37°C in MRS broth (Difco, West Molesey, United Kingdom) without aeration. Escherichia coli strains DH5α (Woodcock, D. M., P. J. Crowther, J. Doherty, S. Jefferson, E. DeCruz, M. Noyer-Weidner, S. S. Smith, M. Z. Michael, and M. W. Graham. (1989) Nucleic Acids Res. 17:3469-3478) was used as an intermediate cloning host and was grown at 37°C on TY broth (Killmann, H., C. Herrmann, A. Torun, G. Jung, and V. Braun. (2002) Microbiology 148:3497-3509) with aeration (Sambrook, J., F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: a Laboratory Manuel, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, US.). When appropriate, antibiotics were added to the media. For L. plantarum, 10 μg/ml chloramphenicol and 10 μg/ml or (for replica- plating) 30 μg/ml erythromycin was used. For E. coli, 10 μg/ml chloramphenicol was used.

DNA manipulations. Plasmid DNA was isolated from E. coli on a small scale using the alkaline-lysis method (Birnboim, H. C, and J. DoIy. (1979) Nucleic Acids Res. 7:1513-1523). Large-scale plasmid DNA isolations were performed using Jetstar columns as recommended by the manufacturer (Genomed GmbH, Bad Oberhausen, Germany). For DNA manipulations in E. coli, standard procedures were used (Sambrook, J., F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: a Laboratory Manuel, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, US.).

Transformation of L. plantarum was performed as described previously (Josson, K., T. Scheirlinck, F. Michiels, C. Platteeuw, P. Stanssens, H. Joos, P. Dhaese, M. Zabeau, and J. Mahillon. (1989). Plasmid 21:9-20), with slight modifications. Briefly, a preculture in MRS broth was diluted in MRS broth containing 1% glycine and cells were grown to an OD_OOO of 1. Cells were kept on ice for 10 minutes and pelleted by centrifugation for 10 minutes at 4000 rpm (Megafuge 1.0R, Heraeus, Hanau, Germany). Cells were then resuspended in ice cold 30% PEG-1450 and kept on ice for 10 minutes. Finally, cells were pelleted by centrifugation for 10 minutes at 4000 rpm and concentrated 100-fold into ice cold 30% PEG-1450. Subsequently, 40 μl of cell suspension and up to 5 μl of plasmid DNA solution was electroporated using a GenePulser Xcell electroporator (Biorad, Veenendaal, The Netherlands) in cuvettes with a 2 mm electroporation gap at 1,5 kV, 25 μF capacitance and 400 Ω parallel resistance.

Restriction endonucleases, Taq, Pfic DNA polymerase, and T4 DNA ligase were used as specified by the manufacturers (Promega, Leiden, The Netherlands, and Boehringer, Mannheim, Germany). Primers were obtained from Genset Oligos (Paris, France).

Replacement oflp_2142. For replacement of lp_2142, the mutagenesis vector pNZ5372 (Table 5) was constructed by successive cloning of the PCR-amplified 1.0 kb 5'- and 3 '-chromosomal flanking regions oϊlp_2142 (using Pfic polymerase, L. plantarum WCFSl genomic DNA as a template, and the primer sets 2142kofrlF / 2142kofrlR and 2142kofr2F / 2142kofr2R, respectively [Table 4]) into the Swal and EcIl 3611 restriction site of pNZ5319 (Table 4), respectively (Lambert JM, Bongers RS, and Kleerebezem M. (2007) Appl Environ Microbiol. 73:1126-1135.) (see below). The mutagenesis vector pNZ5372 was checked by restriction analyses and PCR analysis using primersets lp_2142kofrlF / 85 and 87 / lp_2142kofr2R, and sequencing using primers 120 and 85 for double-strand sequencing of the lp_2142 upstream region cloned in pNZ5319, and primers 87 and 20 for double-strand sequencing of the lp_2142 downstream region cloned in pNZ5319 (Baseclear, Roosendaal, the Netherlands).

Subsequently, the mutagenesis vector pNZ5372 was transformed to L. plantarum WCFSl, and putative double cross-over mutants (Ip _ 2142::V '32-cat) were selected based on their Em^s, Cm^R phenotype. A single colony was isolated and analysed by PCR using a primer annealing to uniquely genomic sequences and a primer annealing to the T'32-cat region (2142kogF / 85 and 2142kog2R / 87, respectively). The strain with the confirmed Ip _ 2142::V '32-cat genotype was designated NZ5342 (Table 4).

Replacement oflp_2145. For replacement of lp_2145, the mutagenesis vector pNZ5375 (Table 1) was constructed by successive cloning of the PCR-amplified 1.0 kb 5'- and 3 '-chromosomal flanking regions of Ip _2145 (using Pfic polymerase, L. plantarum WCFSl genomic DNA as a template, and the primer sets 2145kofrlF / 2145kofrlR and 2145kofr2F / 2145kofr2R, respectively [Table 5]) into the Swal and Ecll36II restriction site of pNZ5319 (Table 1), respectively (Lambert JM, Bongers RS, and Kleerebezem M. (2007) Appl Environ Microbiol. 73:1126-1135.) (see below). Mutagenesis vector pNZ5375 was checked by restriction analysis, PCR analysis using primersets lp_2145kofrlF / 85 and 87 / lp_2145kofr2R, and sequencing using primers 120 and 85 for double-strand sequencing of the lp_2145 upstream region cloned in pNZ5319, and primers 87 and 20 for double-strand sequencing of the lp_2145 downstream region cloned in pNZ5319 (Baseclear, Roosendaal, the Netherlands). Subsequently, the mutagenesis vector pNZ5372 was transformed to L. plantarum WCFSl, and putative double cross-over mutants (Ip _ 2745::P₃₂-cαt) were selected based on their Em^s, Cm^R phenotype. A single colony was isolated and analysed by PCR using a primer annealing to uniquely genomic sequences and a primer annealing to the V₃₂-cat region (2145kogF / 85 and 2145kog2R / 87, respectively). The strain with the confirmed Ip _ 2145::¥τ,2-cat genotype was designated NZ5345 (Table 4).

Table 4. Strains and plasmids used in this study.

Table 5. Primers used in this study.

The mutants were made and checked for inactivation.

Claims

1. A polypeptide able to interact with a DC-SIGN, wherein said polypeptide has an amino acid sequence which has at least 30% identity with the amino acid sequence of

SEQ ID NO: 1.

2. A polypeptide according to claim 1, wherein the polypeptide is obtained from a probiotic strain, preferably a Lactobacillus species, more preferably a Lactobacillus plantarum strain.

3. A polypeptide according to claim 1 or 2, wherein the polypeptide is expressed at the surface of a host cell.

4. A polypeptide according to any one of claims 1 to 3, wherein the polypeptide is glycosylated.

5. A nucleic acid molecule coding for the polypeptide as defined in any one of claims l to 4.

6. A nucleic acid molecule according to claim 5, wherein the nucleic acid molecule is represented by its nucleic acid sequence is selected from the list consisting of:

(a) a nucleic acid sequence having at least 30% identity with the nucleic acid sequence of SEQ ID NO:2; and, (b) a variant of (a).

7. A nucleic acid construct comprising the nucleic acid molecule of claim 5 or 6 coding for a polypeptide according to any one of claims 1 to 4.

8. A host cell expressing the polypeptide as defined in any one of claims 1 to 4, comprising the nucleic acid construct of claim 7.

9. A host cell according to claim 8, wherein the host cell produces more of the polypeptide as defined in any one of claims 1 to 4 and/or exhibits a higher ability to interact with a DC-SIGN than the parental cell this host cell derives from when both are cultured and/or assayed under the same conditions.

10. A host cell according to claim 8 or 9, wherein the host cell is a probiotic strain, preferably a Lactobacillus species, more preferably a Lactobacillus plantarum species.

11. A host cell expressing the polypeptide as defined in any one of claims 1 to 4, comprising a nucleic acid construct or an expression vector comprising a nucleic acid sequence of the DC-SIGN ligand cluster, having at least 30% identity with SEQ ID NO:3.

12. A host cell according to any one of claims 8 to 11, wherein the host cell further comprises a nucleic acid construct or an expression vector selected from the group consisting of: a) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding a glycosyltransferase having at least 30% identity with SEQ ID

NO: 5, b) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding an integral membrane protein having at least 30% identity with

SEQ ID NO: 8, and c) a nucleic acid construct or an expression vector comprising a nucleic acid sequence encoding an integral membrane protein having at least 30% identity with SEQ ID NO: 9.

13. A method for producing the polypeptide as defined in any one of claims 1 to 4 by culturing the host cell according to any one of claims 8 to 12 under suitable culture conditions, and optionally isolating it from the host cell.

14. A polypeptide according to any one of claims 1 to 4 and/or a nucleic acid molecule according to claim 5 or 6 and/or a nucleic acid construct according to claim 7 and/or a host cell according to any one of claims 8 to 12 for use as a medicament.

15. Use of the polypeptide according to any one of claims 1 to 4 and/or the nucleic acid molecule according to claim 5 or 6 and/or the nucleic acid construct according to claim 7 and/or the host cell according to any one of claims 8 to 12 for the manufacture of a medicament for preventing or treating inflammatory gastrointestinal tract disease and/or inducing tolerance for preventing or treating allergy.