The invention relates to surface display of proteins on micro-organisms via the targeting and anchoring of heterologous proteins to the outer surface of cells such as yeast, fungi, mammalian and plant cells, and bacteria. The invention provides a proteinaceous substance comprising a reactive group and at least one attaching peptide which comprises a stretch of amino acids having a sequence corresponding to at least a part of the consensus amino acid sequence listed in FIG. 10 (see Original Patent) and comprises a method for attaching a proteinaceous substance to the cell wall of a micro-organism comprising the use of said attaching peptide.

Heterologous surface display of proteins (Stahl and Uhlen, TIBTECH May 1997, 15.+-.185 192) on recombinant micro-organisms via the targeting and anchoring of heterologous proteins to the outer surface of host-cells such as yeast, fungi, mammalian and plant cells, and bacteria has been possible for several years. Display of heterologous proteins at the surface of these cells has taken many forms, varying from the expression of reactive groups such as antigenic determinants, heterologous enzymes, (single-chain) antibodies, polyhistidyl tags, peptides, and other compounds. Heterologous surface display has been applied as a tool for applied and fundamental research in microbiology, molecular biology, vaccinology and biotechnology, and several patent applications have been filed.

Yet another application of bacterial surface display has been the development of live-bacterial-vaccine delivery systems. The cell-surface display of heterologous antigenic determinants has been considered advantageous for the induction of antigen-specific immune responses when using live recombinant cells for immunisation. Another application has been the use of bacterial surface display in generating whole-cell bioadsorbents or biofilters for environmental purposes, microbiocatalysts, and diagnostic tools.

In general, one has used chimeric proteins consisting of an anchoring or targeting part specific and selective for the recombinant organism used and has combined this part with a part comprising a reactive group as described above. A well known anchoring part for example comprise the so-called LPXTG box, that binds covalently to a Staphylococcus bacterial surface, i.e. in the form of a fully integrated membrane protein. In this way, chimeric proteins are composed of at least two (poly)peptides of different genetic origin joined by a normal peptide bond. For example, in patent application WO 94/18830 relating to the isolation of compounds from complex mixtures and the preparation of immobilised ligands (bioadsorbents), a method has been claimed for obtaining such a ligand which comprises anchoring a binding protein in or at the exterior of the cell wall of a recombinant cell. Said binding protein is essentially a chimeric-protein produced by said recombinant cell, and is composed of an N-terminal part, derived from for example an antibody, that is capable of binding to a specific compound joined with a C-terminal anchoring part, derived from an anchoring protein purposely selected for being functional in the specific cell chosen. In patent application WO 97/08553 a method has been claimed for the targeting of proteins selectively to the cell wall of Staphylococcus spp, using as anchoring proteins long stretches of at least 80 90 amino acid long amino acid cell wall-targeting signals derived from the lysostaphin gene or amidase gene of Staphylococcus which encode for proteins that selectively bind to Staphylococcus cell wall components.

Vaccine delivery or immunisation via attenuated bacterial vector strains expressing distinct antigenic determinants against a wide variety of diseases is now commonly being developed. Recently, mucosal (for example nasal or oral) vaccination using such vectors has received a great deal of attention. For example, both systemic and mucosal antibody responses against an antigenic determinant of the hornet venom were detected in mice orally colonised with a genetically engineered human oral commensal Streptococcus gordonii expressing said antigenic determinant on its surface (Medaglini et al., PNAS 1995, 2; 6868 6872). Also, a protective immune response could be elicited by oral delivery of a recombinant bacterial vaccine wherein tetanus toxin fragment C was expressed constitutively in Lactococcus lactis (Robinson et al., Nature Biotechnology 1997, 15; 653 657). Especially mucosal immunisation as a means of inducing IgG and secretory IgA antibodies directed against specific pathogens of mucosal surfaces is considered an effective route of vaccination. Immunogens expressed by bacterial vectors are presented in particulate form to the antigen-presenting cells (for example M-cells) of the immune system and should therefore be less likely to induce tolerance than soluble antigens. In addition, the existence of a common mucosal immune system permits immunisation on one specific mucosal surface to induce secretion of antigen-specific IgA, and other specific immune responses at distant mucosal sites. A drawback to this approach is the potential of the bacterial strain to cause inflammation and disease in itself, potentially leading to fever and bacteraemia. An alternative approach avoids the use of attenuated bacterial strains that may become pathogenic themselves by choosing recombinant commensal bacteria as vaccine carriers, such as Streptococcus spp. and Lactococcus spp.

However, a potential problem with such recombinant organisms is that they may colonise the mucosal surfaces thereby generating a long term exposure to the target antigens expressed and released by these recombinant micro-organisms. Such long term exposure can cause immune tolerance. In addition, the mere fact alone that such organisms are genetically modified and contain recombinant nucleic acid is meeting considerable opposition from the (lay) public as a whole, stemming from a low level of general acceptance for products containing recombinant DNA or RNA. Similar objections exist against the use of (even attenuated) strains of a pathogenic nature or against proteins or parts of proteins derived from pathogenic strains. However, as explained above, present techniques of heterologous surface display of proteins in general entail the use of anchoring or targeting proteins that are specific and selective for a limited set of micro-organisms which in general are of recombinant or pathogenic nature, thereby greatly restricting-their potential applications.

The invention provides substances and methods to anchor or attach said substances to a cell-wall or cell-wall component of a wide range of micro-organisms. A preferred embodiment of the invention provides substances and methods to attach said substances to non-recombinant micro-organisms. Said substances provided by the invention are not limited to (chimeric) proteins alone, but can be fully or only partly of a peptide nature, whereby a peptide part is (covalently) joined to a non-peptide moiety. The invention provides a proteinaceous substance comprising at least one stretch of amino acids derived from a first micro-organism which substance is capable of attaching to a cell-wall of a second micro-organism. Said substance according to the invention is for example produced by a first micro-organism (for example a micro-organism from which the knowledge about the sequence of said stretch of amino acids originates, but another (recombinant) micro-organism can produce said substance as well). After its production said substance is harvested, optionally stored for future use, and then brought in contact with said second micro-organism, where it attaches to its cell-wall. Alternatively, said substance is produced synthetically, by using established peptide synthesis technology. A preferred embodiment of the invention provides a substance wherein said second micro-organism is a non-recombinant micro-organism. With a substance provided by the invention it is now possible to attach or anchor for a example a heterologous or chimeric protein produced by a recombinant micro-organism to an innocuous non-recombinant micro-organism.

A preferred embodiment of the invention provides a proteinaceous substance wherein said stretch of amino acids has a sequence corresponding to a consensus sequence listed in FIG. 10 (SEQ ID NOS. 17), or wherein said stretch of amino acids (herein also called attaching peptide) has a sequence corresponding to a sequence selected from those listed in FIG. 11 (SEQ ID NOS. 20 110), or a homologous sequence derived from another species. The sequences listed in FIG. 11 (SEQ ID NOS. 20 110), and sequences homologuous thereto, are found in a variety of species, both micro-organisms and higher organisms, an example of such a higher organism is C. elegans. Preferably, the attaching peptide is derived from any one of the proteins listed in FIG. 11 (SEQ ID NOS. 20 110), more preferably said attaching peptide comprises an amino acid sequence as shown in FIG. 10 (SEQ ID NOS. 14 17), or a sequence derived thereof. For example, the invention provides a proteinaceous substance wherein said attaching peptide is derived from the major peptidoglycan hydrolase of Lactococcus lactis. (SEQ ID NOS. 14 16 and 20 22).

Yet another preferred embodiment of, the invention provides a proteinaceous substance wherein said second microorganism is selected from any of the group of Gram-positive bacteria and Gram-negative bacteria. Examples are microorganisms, such as Bacillus subtilis (SEQ ID NOS. 75 78, 81 87, 104, 107, 109 110); Clostridium beij erinckii, Lactobacillus plantarum, Lb. buchneri, Listeria inocua, Streptococcus thermophilus, Enterococcus faecalis (SEQ ID NOS. 23 27), E. coli (SEQ ID NOS. 64, 66, 72 73, 79, 106), and others.

The invention provides a proteinaceous substance which is additionally comprising a reactive group. For example, the invention provides a proteinaceous substance comprising a reactive group such as an antigenic determinant, heterologous enzyme, (single-chain) antibody or fragment thereof, polyhistidyl tag, fluorescing protein, luciferase, binding protein or peptide, or another substance such as an antibiotic, hormone, non-peptide antigenic determinant, carbohydrate, fatty acid, aromatic substance and reporter molecule, and an anchoring or targeting protein or part thereof (herein also called attaching peptide) useful in heterologous surface display which is both broadly reactive with cell wall components of a broad range of micro-organisms.

For example, the invention provides a substance wherein said reactive group is a non-protein moiety, for example is selected from the group of antibiotics, hormones, aromatic substances and reporter molecules. Said substance is constructed by binding for example an antibiotic, such as penicillin or tetracycline, but various other antibiotics can be used, or a hormone, such as a steroid hormone, or any other compound to an attaching peptide provided by the invention. Such binding can be achieved by various techniques known in the art, and thereby can label or "flag" the attaching peptide. A preferred example is the binding of an attaching peptide to a reporter molecule such as FITC, or HRPO, whereby tools are generated that can be used in diagnostic assay whereby micro-organisms having peptidoglycan are detected. Similarly, an attaching peptide with an antibiotic bound thereto can be used in vivo by for example parenteral administration into the bloodstream of humans or animals or in vitro to bind to such micro-organisms having peptidoglycan, thereby increasing the concentration of antibiotic around said organism, which then gets killed by the antibiotic action.

The invention provides a substance wherein said reactive group is a protein moiety, for example selected from the group of antigenic determinants, enzymes, (single-chain) antibodies or fragments thereof, polyhistidyl tags, fluorescing proteins, binding proteins or peptides. For example, the invention provides a protein, which comprises as reactive group a protein or (poly)peptide. Also, the invention provides a nucleic acid molecule encoding a protein provided by the invention. Such a nucleic acid molecule (being single- or double stranded DNA, RNA or DNA/RNA) at least comprises nucleic acid sequences specifically encoding a attaching peptide as well as nucleic acid sequences specifically encoding the reactive group polypeptide, but can additionally also comprise other nucleic acid sequences, which for example encode a signal peptide, or comprise for example promoter and/or regulatory nucleic acid sequences. The invention also provides a vector comprising a nucleic acid molecule encoding a protein provided by the invention.

The invention provides a proteinaceous substance comprising a reactive group joined with or bound to at least one attaching peptide which comprises a stretch of amino acids corresponding to the consensus amino acid sequence listed in FIG. 10 (see Original Patent), said substance capable of attaching or anchoring or binding to a cell wall component of a micro-organism.

Corresponding to is defined as having an amino acid sequence homologous to the consensus amino acid sequence listed in FIG. 10 (see Original Patent), or having an amino acid sequence derived of the sequence listed in FIG. 10 which derived sequence comprises a functionally equivalent stretch of amino acids.

Preferably, the attaching peptide is derived from any one of the proteins listed in FIG. 11, or a protein having a repeat sequence related or homologous to the sequence listed in FIG. 10, more preferably said attaching peptide comprises an amino acid sequence as shown in FIG. 10, or a sequence derived thereof. Homology between the various amino acid sequences of related attaching peptides provided by the invention can for instance be determined by performing a homology search between amino acid sequences, such as for example can be found in a protein database, such as the SWISSPROT, PIR and Genbank databases, using a computer programme, such as the BLAST programme, that can determine homology between amino acid sequences. For example, the invention provides a proteinaceous substance wherein said attaching peptide is derived from the major peptidoglycan hydrolase of Lactococcus lactis. The invention provides a proteinaceous substance comprising a reactive compound wherein at least two stretches of amino acids, corresponding to an attaching peptide sequence, are located adjacent to each other, possibly separated by one or more amino acid residues. Said stretches or repeats can be separated by a short distance, for example 3 6 to 10 15 amino acids apart, or by a medium distance 15 100 amino acids apart, or by longer distances (>100 amino acid residues apart). Examples of such distances can be found in FIG. 11, but longer distances are also possible. The distances between said stretches or repeats can also be used for an (additional) reactive group, whereby a reactive group is inserted between repeats, thereby allowing an even better anchoring to a cell wall component. A preferred embodiment provided by the invention is a proteinaceous substance comprising a reactive group and at least one attaching peptide which comprises a stretch of amino acids having a sequence corresponding to the consensus amino acid sequence listed in FIG. 10, wherein said substance is capable of attaching to a cell wall component of a micro-organism, such as can be found among from any of the group of yeast, moulds, Gram-positive bacteria and Gram-negative bacteria. Examples are micro-organisms, such as Bacillus subtilis, Clostridium beijerinckii, Lactobacillus plantarum, Lb. buchneri, Listeria inocua, Streptococcus thermophilus, Enterococcus faecalis, E. coli, and others. A preferred embodiment provided by the invention is a proteinaceous substance which is capable of attaching to a cell wall component of a conventional (non-recombinant) micro-organism. In this embodiment, the invention provides for example non-recombinant organisms which displaying heterologous proteins, these may colonise the mucosal surfaces without causing problems such as immune tolerance, since they do not generate a long term exposure to the target antigens expressed. In addition, the mere fact alone that such organisms provided by the invention are not genetically modified and do not contain recombinant nucleic acid will alleviate the opposition from the (lay) public as a whole against recombinant micro-organisms, which is stemming from a low level of general acceptance for products containing recombinant DNA or RNA. Similar objections that exist against the use of (even attenuated) strains of a pathogenic nature or against proteins or parts of proteins derived from pathogenic strains are now also overcome by the invention, in that is now possible to attach a proteinaceous substance to a non-recombinant, non-pathogenic micro-organism, such as L. lactis which is generally considered as safe. The invention provides a proteinaceous substance comprising a reactive group such as an antigenic determinant, (heterologous) enzyme, (single-chain) antibody or fragment thereof, polyhistidyl tag, fluorescing protein, luciferase, binding protein or peptide, or another compound such as an antibiotic, hormone, non-peptide antigenic determinant, carbohydrate, fatty acid, aromatic compound and reporter molecule, and an anchoring or targeting protein or part thereof (herein also called attaching peptide) useful in heterologous surface display which is both broadly reactive with cell wall components of a broad range of micro-organisms. Said attaching peptide is preferably derived from a micro-organism which is generally recognised as safe (G.R.A.S.), thereby greatly enhancing the potential of applications of the heterologous surface display technique. Lactococcus lactis is a non-pathogenic, non-invasive, and non-colonising Gram-positive bacterium which is not adapted for growth in body or even the gut; it does not belong to the commensal species of lactic acid bacteria. L. lactis has a history of safe use of several thousand years. The major cell wall hydrolase AcmA of the Gram-positive bacterium Lactococcus lactis subsp. cremoris MG1363 is an N-acetylmuramidase which is required for cell separation and is responsible for cell lysis during stationary phase. The protein consists of three separate domains (FIG. 9, Buist et al., J. Bacteriol. (1995) 177:1554 1563) of which the first 57 amino acids of the N-terminal domain encompasses the signal peptide needed for secretion. This domain is followed by the active site domain running from the Ala at position 58 to Ser-218. The active site domain was overproduced in and purified from Escherichia coli as a thioredoxin fusion protein. The AcmA part was released by proteolytic cleavage with enterokinase and shown to be active in vitro. Three homologous repeated regions (or stretches of amino acids) of 35 55, more often 40 50 amino acid residues are present in the C-terminus of for example AcmA which are separated by non-homologous sequences (FIG. 10). The repeat sequences of AcmA (cA) can be deleted and additional repeat sequences could be added without impairing cell wall hydrolysing activity in vitro. The AcmA deletion derivatives lacking one or two repeat sequences and the protein containing at least one additional repeat were able to bind to lactococcal cells when added from the outside. The derivative lacking all three repeats did not bind to the cells nor did the purified active site domain. The invention provides an attaching peptide that comprise at least one repeat sequence as shown in FIG. 10 or a sequence that is similar to the sequence of FIG. 10, similar being defined as comprising at least a part of a consensus sequence as shown in FIG. 11. Also, attaching peptides are provided by the invention which are comprising amino acid sequences that are derived from a sequence as shown in FIG. 11. Derived herein meaning among others by comparison with heterologous sequences whereby a consensus sequence is obtained, or derived via conventional amino acid substitutions whereby amino acids are being substituted by like amino acids, or derived via substitutions whereby functional amino acids are being replaced by functionally alike or better amino acids identified by methods such as PEPSCAN techniques or replacement mapping. The invention provides a proteinaceous substance comprising a reactive group and at least one attaching peptide which comprises a stretch of amino acids having a sequence corresponding to at least a part of the consensus amino acid sequence provided in FIG. 10. Repeats similar to those in AcmA were for example shown to be present in various cell wall hydrolases and other (secreted) proteins of Gram-positive and Gram-negative bacteria and other micro-organisms and constitute a general cell wall binding domain in these proteins. An attaching peptide comprising at least one AcmA repeat or an amino acid sequence similar to the AcmA repeat provided by the invention represents a general and broadly reactive tool to bind or attach reactive groups such as antigenic determinants, enzymes, antibodies, proteins or peptides to cell walls of micro-organisms. Said repeat comprises a peptide composed of a stretch of amino acids having a sequence corresponding to at least a part of the consensus amino acid sequence provided in FIG. 10. Furthermore, we also demonstrated that an attaching peptide provided by the invention bound or attached to cells of other, e.g. non-recombinant micro-organisms, such as Bacillus subtilis, Clostridium beijerinckii, Lactobacillus plantarum, Lb. buchneri, Listeria inocua, Streptococcus thermophilus, Enterococcus faecalis, E. coli, and others. Binding of the attaching peptide and reactive group joined therewith, as provided by the invention is stable at pH values ranging from 2 10. Moreover, the attaching peptide provided by the invention is, when attached to the cell wall, protected against proteolytic degradation. One embodiment of the invention is a protein wherein the attaching peptide is derived from any of the proteins listed in FIG. 11. An example of such an attaching peptide is provided in the experimental part of this description wherein an attaching peptide having a sequence as shown in FIG. 10, or a sequence similar thereto, is used. Furthermore, the invention provides a protein, which comprises as reactive group a protein or (poly)peptide. Also, the invention provides a nucleic acid molecule encoding a protein provided by the invention. Such a nucleic acid molecule (being single- or double stranded DNA, RNA or DNA/RNA) at least comprises nucleic acid sequences specifically encoding a attaching peptide as well as nucleic acid sequences specifically encoding the reactive group polypeptide, but can additionally also comprise other nucleic acid sequences, which for example encode a signal peptide, or comprise for example promoter and/or regulatory nucleic-acid sequences. The invention also provides a vector comprising a nucleic acid molecule encoding a protein provided by the invention. Such a vector can for example be a plasmid, phage, or virus, and can now be constructed using a nucleic acid provided by the invention and routine skills of the art. Examples of such a vector can be found in the experimental part of the description, other examples can e.g. be a baculovirus vector, or comparable vector viruses through which a protein provided by the invention can be expressed or produced in (insect) cells. The invention also provides a host cell or expression system comprising a nucleic acid molecule according to the invention or a vector according to the invention. Such a host cell expressing a protein is in it self provided by the invention as a micro-organism to which a protein provided by the invention is attached. Such a host cell or expression system can for example be a Gram-positive- or Gram-negative bacterium, or a yeast cell or insect cell or plant- or mammalian cell, or even a cell-free expression system such as a reticulocyte lysate, and can now be constructed or obtained using a nucleic acid or vector provided by the invention and routine skills of the art. Examples of such a host cell or expression system can be found in the experimental part of the description, other examples can be obtained using a nucleic acid or vector provided by the invention and routine skills of the art.

The invention provides a method for attaching a substance to the cell wall of a micro-organism comprising the use of an attaching peptide which comprises a stretch of amino acids having a sequence corresponding to at least a part of the consensus amino acid sequence provided in FIG. 10. An example of the method provided by the invention is anchoring of recombinant poly(peptides), being (chimeric) proteins fused to the cell wall anchoring repeats of AcmA of Lactococcus lactis MG1363, to the cell wall of (Gram-positive) bacteria. The recombinant proteins are obtained by the expression of DNA sequences encoding these recombinant (poly)peptides in a suitable production strain (e.g. E. coli or L. lactis) and subsequent purification of the expression products. The recombinant proteins are than mixed, either in vitro or in vivo, with a non-recombinant target bacterium to obtain binding to the cell wall. Another example of the method provided by the invention is anchoring of recombinant poly(peptides), being (chimeric) proteins fused to the cell wall anchoring repeats of AcmA of Lactococcus lactis, to the cell wall of said recombinant Lactococcus lactis which produces the protein itself. In a preferred embodiment of the method provided by the invention the binding of (purified) proteins to bacterial cells upon addition from the outside, the method is an excellent tool to anchor recombinant proteins or other substances to non-recombinant bacterial cells.

A preferred method according to the invention comprises the use of an attaching peptide which is derived from the major peptidoglycan hydrolase of Lactococcus lactis. Another method according to the invention is provided wherein said substance is a (poly)peptide or a protein, for example being part of a protein provided by the invention. Post-translational modifications occurring to such a (poly)peptide or protein are inherent to the host cell or expression system used, a post-translationally modified protein as provided by the invention is therefore also provided. However, yet another method according to the invention is provided wherein said compound is selected from the group composed of antibiotics, hormones, antigenic determinants, carbohydrate chains, fatty acids, aromatic compounds and reporter molecules. Said substance is constructed by binding for example an antibiotic, such as penicillin or tetracycline, but various other antibiotics can be used, or a hormone, such as a steroid hormone, or any other compound to an attaching peptide provided by the invention. Such binding can be achieved by various techniques known in the art, and thereby can label or "flag" the attaching peptide. A preferred example is the binding of an attaching peptide to a reporter molecule such as FITC, or HRPO, whereby tools are generated that can be used in diagnostic assay whereby micro-organisms having peptidoglycan are detected. Similarly, an attaching peptide with an antibiotic bound thereto can be used in vivo by for example parenteral administration into the bloodstream of humans or animals or in vitro to bind to such micro-organisms having peptidoglycan, thereby increasing the concentration of antibiotic around said organism, which than can get killed by the antibiotic action. Said micro-organism is preferably selected from any of the group of yeast, moulds, Gram-positive bacteria and Gram-negative bacteria. For example, the experimental part of this description describes mixing of .beta.-lactamase::cA fusion protein with lactococcal cells which resulted in binding to the cells whereas this was not the case when mature .beta.-lactamase not joined with an attachement protein was added. Also, fusion of .beta.-lactamase of E. coli and .alpha.-amylase of Bacillus licheniformis to the attaching peptide provided by the invention and subsequent production of these fusion proteins resulted in active, secreted proteins which were located (attached) in L. lactis cell walls. Binding of AcmA and the .beta.-lactamase::cA fusion protein was also demonstrated to isolated lactococcal cell walls and SDS-washed cell walls (the major part of this fraction is peptidoglycan).

Anchoring of recombinant proteins to non-recombinant micro-organisms such as lactococci (or other bacteria) or fungi, is especially attractive if the use of recombinant bacteria is not desired, e.g. in food processes or as pharmaceuticals for medical use such as in vaccines or in anti-bacterial therapy. The invention provides for example vaccine delivery or immunisation via micro-organisms provided by the invention which are labelled with distinct antigenic determinants, which may be directed against a wide variety of diseases. A protective immune response can for example be elicited by oral delivery of a bacterial vaccine provided by the invention wherein tetanus toxin fragment C is attached via a protein provided by the invention to a non-recombinant Lactococcus lactis. Such immunogens expressed by micro-organisms provided by the invention are presented in particulate form to the antigen-presenting cells (for example M-cells) of the immune system and are therefore less likely to induce tolerance than soluble antigens. In addition, the existence of a common mucosal immune system permits immunisation on one specific mucosal surface to induce secretion of antigen-specific IgA, and other specific immune responses at distant mucosal sites. The invention solves the drawback of earlier bacterial vaccines whereby the potential to flourish on mucosal surfaces of the (attenuated or recombinant) bacterial strain used can cause problems such as inflammation and disease in itself, potentially leading to fever and bacteraemia, or to the induction of immune tolerance. Also, the invention avoids the potential risks that are involved when using recombinant DNA containing bacterial vectors for vaccination. In yet another possible vaccine and vaccine use provided by the invention, certain (killed) micro-organisms with adjuvant properties (such as the mycobacteria used in BCG) are labelled or loaded with a protein or substance composed of an antigenic determinant and an attaching peptide. These micro-organisms than function as adjuvant, thereby greatly enhancing the immune response directed against the specific antigenic determinant. Yet another use provided by the invention comprises anchoring proteins from the outside to a micro-organism which provides a means to present proteins or peptides which normally can not be overexpressed (and/or secreted) by said micro-organism. For example, the subunit B of cholera toxin (CTB) can be overproduced in E. coli but expression in L. lactis has been unsuccessful until now. The adjuvant activity of CTB in experimental recombinant vaccines is well documented and the ability of CTB or part thereof to bind to GM1 ganglioside on eucaryotic cell surfaces is of interest with respect to the use of L. lactis (or other Gram-positives) in vaccines which specifically require targeting to mucosal surfaces. Yet another medical use provided by the invention is the addition of (purified) antigen::cA fusion proteins in vivo by parenteral administration into the bloodstream of humans or animals to combat bacterial infections. In this case the antigen::cA fusion protein is used as a "flag" for the immune system. The antigenic determinant of a protein provided by the invention being a subunit of a vaccine regularly used for the immunisation of humans (preferably children) or animals, e.g. a subunit of the Rubella, Pertusis, Poliomyelitis, tetanus or measles vaccine. After delivery in the bloodstream, the "flag" will bind through the AcmA repeats to the pathogenic bacterium present in the blood. A "flag" protein provided by the invention will then activate a memory response, i.e. the response to the antigenic determinant present in the protein. The antibodies thus produced recognise the "flag"--labelled bacteria, which will then be neutralised by the immune system. In this way the protein is used to stimulate a pre-existing (memory) immune response, non-related to the bacterial infection, to clear bacterial infections from the system. Yet another use (which alternatively may be considered medical use or food use) provided by the invention is the use wherein a protein provided by the invention has the ability to bind to cells, such as mucosal cells, e.g. of the gut. The reactive group of such a protein is in such a case for example partly or wholly derived from a fimbriae protein or another gut attachment protein, as for example present in various E. coli strains. Micro-organisms to which such a protein is attached will specifically home or bind to certain areas of the gut, a property which for example is beneficial for certain bacterial strains (i.e. lactococcal or lactobacillar strains) used as a probiotic. In another food or use of food provided by the invention, the protein or substance provided by the invention is a composed of a food additive (such as an enzyme or flavour compound) which affects quality, flavour, shelf-life, food value or texture, joined with an attaching peptide, and subsequently attached or anchored to a micro-organism which is than mixed with the foodstuff. The anchoring of such proteins to a bacterial carrier offers the additional advantage that the additive can be targeted to a solid (bacteria-containing) matrix (e.g. curd) in a process for the preparation of food, e.g. cheese or tofu. Yet another use of a proteinaceous substance or micro-organism provided by the invention is the use of bacterial surface display in generating whole-cell bioadsorbents or biofilters for environmental purposes, microbiocatalysts, and diagnostic tools

Claim 1 of 11 Claims

1. A method for attaching a substance to the cell wall of a microorganism, said method comprising: attaching the substance to a cell wall of the microorganism with an attaching peptide comprising at least one AcmA repeat comprising SEQ ID NO:17, wherein the AcmA repeat is capable of attaching the substance to the cell wall of the microorganism.

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