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WO1999051631A1 - Une region proteique responsable de la liaison avec des types de cellules epitheliales et une sequence d'adn codant pour cette region - Google Patents

Une region proteique responsable de la liaison avec des types de cellules epitheliales et une sequence d'adn codant pour cette region Download PDF

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Publication number
WO1999051631A1
WO1999051631A1 PCT/FI1999/000290 FI9900290W WO9951631A1 WO 1999051631 A1 WO1999051631 A1 WO 1999051631A1 FI 9900290 W FI9900290 W FI 9900290W WO 9951631 A1 WO9951631 A1 WO 9951631A1
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Prior art keywords
thr
ala
seq
gly
binding
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PCT/FI1999/000290
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Inventor
Timo Korhonen
Airi Palva
Ilkka Palva
Ulla HYNÖNEN
Benita WESTERLUND-WIKSTRÖM
Original Assignee
Timo Korhonen
Airi Palva
Ilkka Palva
Hynoenen Ulla
Westerlund Wikstroem Benita
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Application filed by Timo Korhonen, Airi Palva, Ilkka Palva, Hynoenen Ulla, Westerlund Wikstroem Benita filed Critical Timo Korhonen
Priority to AU33343/99A priority Critical patent/AU3334399A/en
Publication of WO1999051631A1 publication Critical patent/WO1999051631A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis

Definitions

  • the present invention relates to DNA molecules encoding polypeptides responsible of binding to human and/or animal epithelial cell types, like intestinal, urogenital and/or endothelial cells. Further this invention relates to vectors containing the DNA molecules and hosts transformed with the DNA molecules of this invention.
  • the present invention relates also to a method of constructing new hosts or new proteins capable of binding to human and/or animal epithelial cell types.
  • This invention also relates to genes encoding preselected proteins modified to bind to human and/or animal epithelial cell types.
  • this invention relates to a host cell, to a protein and to a method for carrying preselected factors/properties to human and/or animal epithelial cells or cell surfaces by using a Lactobacillus brevis strain or L. brevis SlpA protein.
  • Bacterial adhesion to human epithelial and subepithelial tissues is a decisive initial event in successful colonization of tissue sites by invading bacteria.
  • Several molecular ligand- receptor interactions have been characterized for bacterial species that cause infectious diseases in man or animals.
  • Adhesion of pathogenic bacteria to the tissue at the infection site helps the bacteria to resist mechanical defences of our body, such as peristalsis in the intestine or flow of urine in the urinary tract.
  • Adhesion is a key determinant in the host, tissue and cell-type tropism of bacterial infections. Attachment to tissues is also important for those bacteria that establish themselves as members of the normal bacterial flora in the human body.
  • Lactobacillus are major members of the indigenous bacterial flora in the gastrointestinal and the genital tract of man and animals. Lactobacilli are thought to be beneficial to their host organism and have a long history of use in the gastrointestinal and the urogenital tract to prevent or cure various minor illnesses. Probiotic effects of lactobacilli include exclusion of invasive pathogens from intestinal and vaginal surfaces, production of antimicrobial substances, stimulation of immune systems, as well as other physiological effects. As lactobacilli are members of the normal bacterial flora and food- grade organisms, their possible use as carriers of vaccine antigens in the intestine has aroused interest.
  • lactobacilli bring about the probiotic effects have remained uncharacterized, but it is generally agreed that efficient adhesion to epithelial surfaces is important for the colonization of the intestine as well as for the effects associated with these bacteria. Isolates of lactobacilli have been shown to adhere to the intestinal epithelium of their hosts (Coconnier et al, Appl. Environ. Microbiol 58:2034 - 2039
  • S-layers are paracrystalline surface protein arrays that are commonly expressed by species of Eubacteria and Archaebacteria (reviewed in Messner and Sleytr, Adv. Microb. Physiol. 33: 213 -275 (1992) and Sleytr and Sara, Trends Biotechnol. 15:20-26 (1997). Most S- layers are composed of a single protein species, the S-layer protein, greatly varying in size in different bacterial genera. The S-layer subunits are very hydrophobic and crystallize to form a two-dimensional layer on the bacterial surface. The genes encoding S-layers are efficiently transcribed, and the S-layer protein is the dominant protein species representing 10-20% of the total cellular protein of the bacterial cell.
  • Differing functions have been attributed to the S-layers of different bacterial species. These functions include maintenance of the cell shape, protection of cells from hostile environment, anchorage of extracellular enzymes to the bacterial cell wall and mediation of bacterial attachment to animal tissues (Chu et al, J. Biol. Chem.266 : 15258- 15265 (1991), Schneitz et al, J. Appl. Microbiol. 74: 290- 294 (1993) and Toba et al, Appl. Environ. Microbiol. 61 :2467-2471 (1995)).
  • the S- layer of the fish pathogen Aeromonas salmonicida binds to extracellular matrix proteins and increases bacterial virulence by promoting bacterial spread to cause systemic infection in the fish (Chu et al, J. Biol Chem.266: 15258-15265 (1991)).
  • Most S-layer proteins aggregate in physiological buffers and their functional analysis have been restricted to solid phase assays (Sleytr and Sara, Trends Biotechnol. 15:20-26 (1997)), which has remained a severe limitation in the functional analysis of these important surface proteins.
  • the predicted lactobacillar S-layer proteins are 40 to 50 kDa in molecular size and show similarity in amino acid compositions.
  • Lactobacilli are important bacterial colonizers of our intestinal surfaces. Despite their high number and potential symbiotic effects in our body, our knowledge of the colonization mechanisms that the lactobacilli use to attach and multiply our intestine have remained uncharacterized. This has been in part due to the restricted methodology to genetically manipulate these bacteria and also due to the lack of suitable methods to study the binding mechanisms of these bacteria to intestinal or other mucosal surfaces.
  • WO 97/14802 suggests the use of Lactobacillus fermentum 104R 29 kD adherence factor for promoting the activity of microorganism cells to bind to a receptor recognized on mucus.
  • the finding of a factor capable of binding to mucus does not solve the problem of specifically carrying preselected factors to human or animal epithelial cells. Mucus on the surface of intestinal tract and any factors bound to the mucus are easily rinsed out from the intestine.
  • a DNA molecule encoding a protein region responsible of binding of the protein to intestinal, urogenital, endothelial and/or other epithelial cell types has for the first time been identified and characterized. Although there has been some preliminary notions about the possible binding capability of the S-layer proteins of lactic acid bacteria (LAB) to human or animal cells, the binding property has not been confirmed to be due to the S-layer protein.
  • the DNA molecule encoding a protein region responsible of binding was unexpectedly found from a gene encoding the S-layer protein of a species of lactic acid bacteria, Lactobacillus brevis.
  • a homologous DNA molecule encoding similar advantageous binding properties may be synthetic or semisynthetic or originate from the same or another group of microorganisms.
  • This invention results in various advantages.
  • This invention makes it for the first time possible to modify or improve the binding capacity of various prokaryotic or eukaryotic cells to human or animal epithelial cell types, like intestinal, urogenital and/or endothelial cell types by using lactobacillar surface structures.
  • the nucleotide sequences of this invention it is possible with the nucleotide sequences of this invention to improve the binding properties of a host cell having probiotic effects to human and/or animal epithelial cell types.
  • nucleotide sequences of this invention to modify or improve the binding properties of a preselected protein to human and/or animal epithelial cell types.
  • the probiotic properties of various strains of Lactobacillus brevis species can be improved by genetic modification. Totally new properties can be transferred to the strains of Lactobacillus brevis species or other hosts of this invention, which may carry these properties to human or animal gastrointestinal or urogenital tract.
  • Various host cells having or being modified to have the binding capability can be used to colonize the human or animal gastrointestinal or urogenital tract and to exclude pathogens by binding to cell receptors, which would otherwise be bound by pathogens or by growing to cell surface areas which would otherwise be colonized by pathogens.
  • Figure 1 Effect of S-layer removal on the adherence of L. brevis ATCC 8287 to human Intestine 407 cells.
  • Panel a shows adherence of untreated bacteria to the intestinal cells
  • panel b shows the adhesiveness of bacteria treated with guanidine hydrochloride to remove S-layer from the bacterial surface.
  • FIG. 1 Quantitative analysis of the effect of S-layer removal on the adherence of L.brevis to Intestine 407 cells.
  • Panel A shows the number of adherent bacteria per epithelial cell; the adhesion test with the treated and the untreated bacteria was performed at four different bacterial cell densities indicated below.
  • Panel B shows the polyacrylamide gel electro- phoresis in sodium dodecyl sulfate (SDS-PAGE) of the proteins released from the bacterial surface by the Laemmli sample buffer used in SDS-PAGE.
  • the S-layer peptide is indicated by the arrow.
  • the S-layer peptide is the dominant peptide species released from the cells but not the only one.
  • FIG. 3 Schematic representation of the SlpA fragments expressed as fusion to flagellin.
  • the bars indicate the fragments expressed in ⁇ iC, the numbers refer to the N- and C-terminal amino acids in the SlpA peptide.
  • Binding of the chimeric flagella to Intestine 407 (Int 407) cells is indicated on the right.
  • FIG. 4 Binding of the SlpA96-200/ FliC chimeric flagella to the human Intestine 407 (Panel a) and urinary bladder T24 cells (Panel c). The binding was visualized by indirect immunofluorescence. Panels b and d show the corresponding fields by light microscopy. Panel e shows the binding of the ⁇ FliC flagella to Intestine 407 cells, and Panel g the control staining without flagella; the corresponding light microscopic fields are shown in Panels f and h. Arrows indicate binding of the chimeric flagella, arrowhead indicates lack of binding.
  • Figure 5 The nucleotide sequence of Lactobacillus brevis SlpA gene and the corresponding amino acid sequence.
  • the first and last nucleotide residue of the coding sequence and of the various fragments is marked with an arrow.
  • the first and last amino acid residue of the entire peptide and of the fragments is marked with a circle.
  • a DNA molecule encoding a protein region responsible of binding to human and/or animal intestinal, urogenital, endothelial and/or other epithelial cell types has for the first time been identified and characterized.
  • the S-layer protein SlpA of Lactobacillus brevis has adhesive properties to epithelial cell types.
  • the primary structure of this protein and the corresponding gene has been described in WO 94/00581 and in Vidgren et al ., J. Bacteriol. 174: 7419-7427 (1992). According to Palva, A in H. Bahl et al./, FEMS
  • the S-layer peptides needed for the binding were contained in fragments of 270, 215, 275, 150 and 105 amino acid residues respectively, the shortest fragment representing residues 96 through 200 in the S-layer protein.
  • any fragment being a partial amino acid sequence of these sequences or of the entire Lactobacillus brevis S-layer protein and possessing similar binding capacity as the above mentioned fragments is a polypeptide of this invention and any DNA molecule encoding these polypeptides is a DNA molecule of this invention.
  • Chimeric flagella harbouring inserts that represented the N-terminal part of the S-layer protein bound to intestinal as well as urinary bladder cells, whereas the C-terminal part of the S-layer protein did not confer binding on the chimeric flagella.
  • the C-terminal parts of the protein may have an effect in enhancing the efficiency of binding.
  • the S-layer expressing bacterium Lactobacillus brevis ATCC 8287 efficiently adhered to the human small intestinal cell line and to the human urinary bladder cell line. Bacterial adhesiveness to both cell lines was completely abolished after removal of the S- layer protein (SlpA) from the bacterial surface by guanidine hydrochloride extraction.
  • SlpA S- layer protein
  • This invention is directed to a DNA molecule encoding a polypeptide capable of binding to human and/or animal epithelial cell types, like intestinal, urogenital and/or endothelial cells.
  • the cells may originate from human or animal origin, like from porcine or poultry origin or from pet animals.
  • the cells may be normal or e.g. tumour cells.
  • the DNA molecule may be a DNA molecule having the full length or the partial sequence i.e. the coding sequence 8 contained in the nucleotide sequence of any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ LD NO. 3, SEQ ID NO. 4 , SEQ ID NO 5 or SEQ LD NO. 6, representing the various fragments of the Lactobacillus brevis sip A gene, excluding, however, the full length SEQ LD NO. 6.
  • a partial nucleotide sequence is meant a nucleotide sequence lacking at least one nucleotide residue as compared to the nucleotide sequences of SEQ LD 1 to SEQ LD 6.
  • SEQ LD NO. 1 represents the 315 nucleotide residues from 286 to 600
  • SEQ LD NO. 2 represents the 450 nucleotide residues from 286 to 735
  • SEQ LD NO. 3 represents the 825 nucleotide residues from 286 to 1110
  • SEQ LD NO. 4 represents the 645 nucleotide residues from 91 to 735
  • SEQ ID NO 5 represents the 810 nucleotide residues from 91 to 900
  • SEQ LD NO. 6 represents the entire coding sequence of Lactobacillus brevis slpA gene from 1 to 1395 nucleotide residues.
  • the first and last nucleotide residue of the nucleotide sequences of SEQ ID NO. 1 to SEQ LD NO. 6 is marked with an arrow in Figure c
  • the DNA molecule of this invention may be a DNA molecule encoding a polypeptide having the full length or the partial amino acid sequence i.e. the amino acid sequence contained in any one of SEQ LD NO. 7, SEQ LD NO. 8 , SEQ LD NO. 9, SEQ LD NO.10 ,
  • SEQ LD NO. 7 represents the shortest amino acid sequence of 105 amino acid residues between 96 and 200
  • SEQ LD NO. 8 represents 150 amino acid residues between 96 and 245
  • SEQ LD NO. 9 represents 275 amino acid residues between 96 and 370
  • SEQ ID NO.10 represents 215 amino acid residues between 31 and 245
  • SEQ ID NO 11 represents 270 amino acid residues between 31 and 300.
  • SEQ LD NO 12 represents the amino acid sequence of the entire Lactobacillus brevis SlpA protein between 1 and 465.
  • the first and last amino acid residue of the amino acid sequences of SEQ ID NO. 7 to SEQ LD NO. 12 is marked with a circle in Figure 5.
  • a partial amino acid sequence is meant an amino acid sequence lacking at least one amino acid compared to the amino acid sequences SEQ ID NO. 7 to SEQ ID NO. 12.
  • the present invention furthermore relates to DNA molecules, the sequences of which differ from the sequences of the above-identified molecules due to degeneracy of the genetic code, and which code for a polypeptide capable of binding to human and/or animal epithelial cell types, like intestinal, urogenital and/or endothelial cells.
  • the present invention relates also to DNA molecules, the sequences of which hybridize to any one of the DNA molecules above encoding a polypeptide capable of binding to human and/or animal epithelial cell types, like intestinal, urogenital and/or endothelial cells.
  • hybridization in this context means hybridization under conventional hybridization conditions, preferably under stringent conditions such as described by, e.g. Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Typical stringent hybridization conditions are exemplified in example 7, but equal hybridizations can be carried out in slightly different conditions as is known to a person skilled in the art.
  • example 7 the slpA gene of L. brevis has been hybridized to the chromosomal DNA of other Lactobacillus strains.
  • the hybridization method is very useful and reliable method to find new DNA molecules of this invention.
  • All the L. brevis strains tested gave positive hybridisation signal except two strains, which were shown to lack the S-layer protein.
  • Other S-protein expressing lactobacilli belonging to other lactobacilli species gave negative result.
  • L. buchneri which is closely related to L. brevis gave also positive signal.
  • L. buchneri carries S-layer and has the capability of binding to human and/or animal epithelial cell types, like intestinal, urogenital and/or endothelial cells.
  • nucleic acid molecules that hybridize to the nucleic acid molecules of the present 10 invention can in principle be derived from any organism possessing such nucleic acid molecules. Preferably, they are derived from lactic acid bacteria or bifidobacteria. Nucleic acid molecules hybridizing to the nucleic acid molecules of the present invention can be isolated, e.g., from genomic libraries or cDNA libraries of various organisms.
  • nucleic acid molecules can be identified and isolated by using the nucleic acid molecules of the present invention or fragments of these molecules or the reverse complements of these molecules, e.g. by hybridization according to standard techniques (see Sambrook et al.( 1989)).
  • hybridization probe e.g. nucleic acid molecules can be used that have exactly or substantially the same nucleotide sequence indicated in the Figure 5 or fragments of said sequence. Preferably is used the entire nucleotide sequence of the coding sequence of the slpA gene.
  • the fragments used as hybridization probes can also be synthetic fragments obtained by conventional synthesis techniques and the sequence of which is substantially identical to that of the nucleic acid molecules according to the invention.
  • hybridizing DNA molecule includes fragments, derivatives and allelic variants of the above-described nucleic acid molecules that code for the above-described protein (or its equivalent) or a biologically active fragment thereof. Fragments are understood to be parts of nucleic acid molecules long enough to code for the described protein (or its equivalent) or a biologically active fragment thereof.
  • derivative means in this context that the nucleotide sequences of these molecules differ from the sequences of the above-described nucleic acid molecules in one or more positions and are highly homologous to said sequence.
  • the present invention is directed also to DNA molecules which are homologous with the
  • DNA molecules contained in the coding sequences of any of SEQ LD 1 to SEQ LD 6 or with 11 the DNA molecules encoding a polypeptide having the amino acid sequence contained in any of SEQ LD 7 to SEQ ID 12 or with the degenerated forms of these DNA molecules.
  • "Homology” is understood to refer to a sequence identity of at least 50%, preferably more than 70% and still more preferably more than 90% on the length of at least 300 nucleotides. The deviations from the nucleic acid molecules described above can be the result of deletion, substitution, insertion, addition or combination.
  • Homology furthermore means that the respective nucleotide sequences or encoded proteins are functionally and/or structurally equivalent.
  • the DNA molecules that are homologous to the DNA molecules described above and that are derivatives of said DNA molecules are regularly variations of said molecules which represent modifications having the same biological function. They may be naturally occurring variations, such as sequences of other organisms or mutations. These mutations may occur naturally or may be achieved by specific mutagenesis. Furthermore, these variations may be synthetically produced sequences.
  • the present invention furthermore relates to DNA molecules, the sequences of which have an amino acid sequence which shows at least 40 % identity, preferably at least 50 % identity, to a sequence contained above and which code for a polypeptide capable of binding to human and/or animal epithelial cell types, like intestinal, urogenital and/ or endothelial cells.
  • the amino acid sequences of this invention show identity of less than 36% compared to any known amino acid sequence.
  • an amino acid sequence that is an "equivalent" of a specific amino acid sequence is meant an amino acid sequence that is not identical to the specific amino acid sequence, but rather contains at least some amino acid changes (deletion, substitutions, inversions, insertions, etc.) that do not essentially affect the biological activity of the protein as compared to a similar activity of the specific amino acid sequence, when used for a desired purpose.
  • the biological activity of a polypeptide means here the capability of binding to epithelial cells. 12
  • an "equivalent" amino acid sequence contains at least 40% - 99% identity at the amino acid level to the specific amino acid sequence, most preferably at least 50%, more preferably at least 60% and in an especially highly preferable embodiment, at least 95% identity, at the amino acid level.
  • binding is used here to mean the adherence of a cell, protein, protein region or polypeptide reasonably firmly to an epithelial cell, like intestinal, urogenital and/ or endothelial cell type .
  • the binding capacity has been measured by determining the binding of polypeptides encoded by DNA molecules expressed as gene fusions in the variable region of the 7/C H7 gene of Escherichia coli.
  • the resulting chimeric flagella were assessed by indirect immunofluorescence for binding to human intestinal and human urinary bladder cells.
  • the binding capacity was visually characterized to be very strong (++++), strong (+++), weak (+) or no binding at all (-).
  • binding is meant the adherence of proteins or cells to the epithelial cells, particularly to the surface of the cell.
  • the binding was specific: chimeric flagella harbouring inserts that represented the N- terminal part of the S-layer protein bound to intestinal as well as urinary bladder cell types, whereas the C-terminal part of the S-layer protein did not confer binding on the chimeric flagella.
  • the S-layer expressing bacterium Lactobacillus brevis ATCC 8287 efficiently adhered to the human small intestinal cell line and to the human urinary bladder cell line. Bacterial adhesiveness to both cell lines was completely abolished after removal of the S- layer protein (SlpA) from the bacterial surface by guanidine hydrochloride extraction.
  • Table I Binding of different strains of lactic acid bacteria to human epithelial cell line Intestine 407.
  • lactic acid bacteria are meant all Gram-positive; anaerobic, microaerophilic or aero- tolerant; catalase negative; rods or cocci; most importantly they all produce lactic acid as sole, major or important product from the energy-yielding fermentation of sugars.
  • genuine members of lactic acid bacteria include at least the following genera: Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Carnobacterium, Sporolactobacillus, Streptococcus, Enterococcus, Aerococcus, Vagococcus, Tetragenococcus and Atopium.
  • Many characteristics typical to genuine LAB are also common to the genus Bifidobacterium which consists of important health-promoting intestinal bacteria. 14 Transfer of the binding property to new host cells
  • nucleotide sequences of this invention it is possible with the nucleotide sequences of this invention to modify or improve the binding capability of various prokaryotic or eukaryotic hosts to human and/or animal epithelial cell types, like intestinal, urogenital and/or endothelial cell types.
  • the hosts cells are improved or modified to have the binding capability by transferring the host cells by at least one of the DNA molecules of this invention.
  • the binding capability may be transferred to any suitable bacterial host, for example to strains of lactic acid bacteria or bifidobacteria or to a fungal strain, like to a yeast strain.
  • a nucleotide sequence of this invention may be inserted into a DNA vector with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phophatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are described in Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • nucleotide sequences of this invention may be operably linked to the transcriptional and secretory regulatory elements in an expression vector, and introduced into a host cell to produce desired protein under the control of such sequences.
  • a DNA molecule is said to be capable of expressing a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are operably linked to the nucleotide sequence which encodes the desired polypeptide.
  • An operable linkage is a linkage in which a sequence is connected to a regulatory sequence in such a way as to place expression of the sequence under the influence or control of the regulatory sequence.
  • sequences functional in the host cell may be substituted as necessary.
  • the vectors of the invention may comprise other operable linked functional elements such as DNA elements which confer antibiotic resistance on a host cell, and which provide for an origin of replication, or for insertion of a desired sequence into the chromosome of a host cell.
  • Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow selection of host cells which contain the expression vector in the chromosome, for example the marker may provide resistance to antibiotics.
  • the selectable marker gene (that can be later removed by methods well known in the art) can either be directly provided on the same vector as that providing the desired DNA gene sequences to be expressed, or such markers may be introduced into the same cell by co-transformation.
  • Factors of importance in selecting a particular plasmid or phage vector include the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells, which do not contain the vector and the number of copies of the vector which are desired in a particular host.
  • recipient cells After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the nucleotide sequences of this invention result in the production of the desired protein, or the production of a fragment of this protein and/or a host having the desired properties.
  • DNA molecules including the entire gene of L. brevis slpA can be 16 expressed on the surface of other selected well known probiotic microbes in order to enhance and target their adhesion to the host cells to allow more efficient colonization.
  • the degree of the adhesion mediated by the polypeptides of this invention can be varied by modifying their expression by different copy numbers, promoter strength regulation or by changing the length and amino acid composition of the binding domain with genetic engineering. With amino acid modifications encoded by the DNA molecules of this invention their host specificity may be affected if required.
  • Probiotics have been defined as live micro-organisms which beneficially affect the health of the host (human or animal ) by improving its intestinal balance. To date, the term probiotic has been widened to include also live preparations used genitourinary to prevent infections and restore the disturbed microbiological ecological balance. Different characteristics associated with potentially health affecting bacteria may include e.g. i) acid and bile stability, ii) adherence to intestinal cells, iii) colonization of the intestinal tract, iv) production of antimicrobial substances, v) antagonism against pathogenic bacteria and vi) safety in food and clinical use.
  • the probiotic effects of the hosts having (or modified to have) the binding capacity to epithelial cells can be enhanced by genetic means.
  • Various hosts of this invention can be used to colonize the human or animal gastrointestinal or urogenital tract and to exclude pathogens by binding to cell receptors, which would otherwise be bound by pathogens or by growing to cell surface areas, which would otherwise be colonized by pathogens.
  • polypeptides of this invention As an example of establishing a surface expression of the polypeptides of this invention, one can apply well conserved cell wall binding anchors (e.g. C-terminal sequence of PrtP from L. lactis or other lactic acid bacteria, staphylococcal protein A, Streptococcus pyogenes M6 protein, yeast anchor sequences etc.), fused to a spacer region and a DNA molecule of this invention which is preceded by a suitable promoter and signal sequence (see 17 e.g. Piard et al. J. Bacteriol. 179:3068-3072 (1997) and Steidler et al Appl. Environ. Microbiol 64: 342-345 (1998).
  • C-terminal sequence of PrtP from L. lactis or other lactic acid bacteria, staphylococcal protein A, Streptococcus pyogenes M6 protein, yeast anchor sequences etc. fused to a spacer region and a DNA molecule of this invention which is
  • DNA molecules including the entire gene of L. brevis slpA can be transferred to another microorganism which could be for example a live vaccine carrier or a new putative host developed for vaccine purpose to enhance their adhesion properties.
  • the degree of adhesion can be varied by modifying the expression of the DNA molecules of this invention by different copy numbers, promoter strength regulation or by changing the length and amino acid composition of the binding domain with genetic engineering.
  • amino acid modifications encoded by the DNA molecules of this invention their host specificity may be affected if required.
  • the portals of entry of many pathogens are mucosal surfaces and most infections also begin at some mucosal site.
  • the natural immune response to a pathogen is also likely to begin at the mucosal site of entry.
  • a similar route for its delivery may be regarded as a rational approach.
  • Most vaccines available to date are, however, injected parenterally giving systemic response alone which may not be long lasting and protective enough.
  • Oral vaccines are most likely to prevent intestinal diseases as they stimulate mucosal associated lymphoid tissue in the gastrointestinal tract directly.
  • S- layers function as effective adjuvants.
  • the C-terminal part of the SlpA gene can be modified to carry desired antigen epitopes and to efficiently present them as multiple copies at the S-layer formed by the SlpA subunits. Either a heterogenous or uniform S-layer can be formed depending on whether the host carries both an antigen-expressing and wild-type slpA gene or only the modified antigen-expressing gene. By this the amount of antigen on the cell surface may be affected.
  • the vaccine antigen can also be expressed on the cell surface apart from a S-layer which in such a case functions as adhesion factor and adjuvant only.
  • Preferably expression hosts for oral vaccine carriers are strains of lactic acid bacteria and bifidobacteria, more preferably L. brevis species, in particular the strain L. brevis ATCC8287.
  • polypeptides of this invention responsible of the binding property in living vaccine carriers, also non-living applications for these structures are fully possible.
  • the desired antigen polypeptides and shorter antigen epitopes having been modified to have the binding property of this invention can be expressed in the host chosen and administered after isolation and purification as pure protein preparations. If required, such preparations may be protected against degradation by using inert particles such as biodegradable microparticles, liposomes and cochelates (O'Hagan, Novel Delivery Systems or Oral Vaccines. CRC Press, Florida (1994)). 19
  • DNA molecules of this invention are their use in bifunctional molecules i.e. by creating fusion constructs where the binding region mediates the adhesion to mucosal cells and the other domain functions as an active target-specific molecule.
  • Such bifunctionally acting molecules could be formed for example by combining the DNA molecules of this invention with DNA sequences encoding enzymes, single chain antibodies or pharmaceutical proteins or toxins.
  • Targeted enzymes could be applied for example in the gut for degradation of (i) lactose by ⁇ -galactosidase to decrease adverse effects in lactose intolerance or ii) milk proteins by proteases and peptidases e.g. to release bioactive peptides or to increase tolerance against milk allergy.
  • Certain toxins bound specifically to the polypeptides of this invention could also be used for destruction of pathogens at different mucosal sites.
  • the delivery of the bifunctional molecule could be by direct spray or liquid preparations (nasal mucosa), by melting capsules (vaginal mucosa) or by inert particles described above for vaccine delivery systems (gastrointestinal tract).
  • Lactobacillus brevis as a probiotic host
  • L. brevis as such may function as a novel probiotic strain particularly due to its highly efficient binding capacity both in the gut and urinary tract even though its use in practical applications is yet unraveled . Its probiotic effect can be further enhanced for example by introducing genes encoding i) production of selected 20 bacteriocin(s) and the corresponding immunity and ii) other antimicrobial substances to antagonist pathogens and iii) enzymes increasing its metabolic activity towards available substrates to strengthen its competetiveness in the chosen niche.
  • the degree of the SlpA mediated adhesion can be varied by modifying its expression by different copy numbers, by promoter strength regulation or by changing the length and amino acid composition of the binding domain with genetic engineering. With amino acid modifications of the SlpA binding domain their host specificity may be affected if required.
  • L. brevis ATCC8287 In addition to the demonstrated slpA encoded binding capacity ofJ. brevis ATCC8287, this strain is also highly resistant in low pH and bile thus possessing the key characteristics of a probiotic strain. L. brevis strains have also been shown to have antagonistic effects against intestinal pathogens. Furthermore, L. brevis can be regularly found in the intestine of man and animals as well as in a variety of fermented food products.
  • the guanidine hydrochloride-treated cells were washed once with 2 M guanidine hydrochloride and then twice with PBS; the control cells were washed twice with PBS.
  • the bacterial cells were suspended in the cell culture medium used with the target epithelial cell lines (see below). 21
  • Bacterial adherence assays The bacterial adhesion to cultured human epithelial cells was evaluated essentially as described earlier ( Tarkkanen et al, Infect. Immunol. 65:1546-1549 (1997)).
  • the human small intestine Intestine 407 (ATCC CCL6) cells were cultivated to confluence in RPMI 1640 medium (Gibco Laboratories, Grand Island, N.Y.) supplemented with 10 % (w/v) fetal calf serum (PAA Laboratories Gmbh, Linz, Austria), 1 % (w/v) L- glutamine (Life Technologies,), 1 % (w/v) nonessential amino acids (Gibco Laboratories, Grand Island, N.
  • the human urinary bladder transitional T24 cells (ATCC HTB-4) were cultered in McCoy's 5A medium (Life Technologies) supplemented with fetal calf serum, L-glutamine, and gentamycin as above.
  • the cell lines were cultured on diagnostic glass slides (Knittel Glassbearbeitungs GmbH, Braunschweig, Germany). Before the adhesion assays, the cells were washed once with PBS.
  • the bacteria were suspended in RPMI 1640 medium at the concentrations ranging from 5 x 10 7 to 10 9 cells/ml, and 40 ⁇ l of the suspension per well was added to the epithelial cells and the slides were incubated for 1 h at 37 °C in a moist chamber. The slides were washed five times at room temperature with PBS for five min each and fixed for 10 min with methanol. The cells with adherent bacteria were then examined in a BX50 microscope (Olympus Optical Co., Hamburg, Germany) either directly by Nomarski interference optics (for photographing) or, for quantitative analysis, stained for 5 min with 10 % v/v Giemsa stain and analyzed by light microscopy.
  • Flagella display The principle of the flagella display system used here was recently described (Westerlund-Wikstrom et ⁇ /. , Prot. Engin. 10: 1319-1326 (1997)). Fragments 22 representing different parts of the slpA gene were amplified by polymerase chain reaction (PCR) with Pfu polymerase and using chromosomal DNA from the L. brevis strain ATCC 8287 as the template. The primers were designed on the basis of the nucleotide sequence of sip A ( Vidgren et al. J. Bacteriol. 9:7419-7427 (1992)) and contained anAccI restriction site at the 5 ' termini.
  • PCR polymerase chain reaction
  • the primers encoded the SlpA peptide sequences 31-245 (SEQ LD NO. 10; this peptide sequence is encoded by the nucleotide sequence SEQ LD NO. 4, which represents the nucleotide residues from 91-735), 31-300 (SEQ LD NO. 11 this peptide sequence is encoded by the nucleotide sequence SEQ LD NO. 5, which represents the nucleotide residues from 91-900), 96-200 (SEQ LD NO. 7; this peptide sequence is encoded by the nucleotide sequence SEQ LD NO. 1, which represents the nucleotide residues from286-600), 96-245 (SEQ LD NO.
  • this peptide sequence is encoded by the nucleotide sequence SEQ LD NO. 2, which represents the nucleotide residues from286-735), 96-370 (SEQ LD NO. 9; this peptide sequence is encoded by the nucleotide sequence SEQ LD NO. 3, which represents the nucleotide residues from 286-1110), or 239-447 (SEQ LD NO. 13; this peptide sequence is encoded by the nucleotide sequence SEQ LD NO. 14, which represents the nucleotide residues from 715-1341), where the residue numbers include the 30-mer signal sequence of the SlpA peptide.
  • the slpA fragments were cloned into the Accl site in the plasmid pFHC H7 ⁇ deleted for 174 bp in the variable region of fliC and expressed in trans in ZX coli that isfliC::TnlQ andf ⁇ mA::cat (Westerlund-Wikstr ⁇ m et al Prot. Eng. 10: 1319-1326(1997)).
  • the flagellar filaments were extracted and, after a sodium dodecylsulfate gel electrophoresis (SDS-PAGE), adjusted to an equal concentration of the FliC peptide as described recently (Westerlund-Wikstr ⁇ m et al, Prot. Engin. 10: 1319-1326 (1997)).
  • the flagella lacking an insert, i.e. the ⁇ FliC filaments were available from previous work (Westerlund-Wikstr ⁇ m et ⁇ /.(1997)).
  • Binding tests with chimeric flagella The binding of the chimeric flagella onto the epithelial cells was assessed by indirect immunofluorescence as detailed recently (Westerlund- Wikstr ⁇ m et al. (1997)). Briefly, the epithelial cells were washed at room temperature with
  • control assays included staining of the epithelial cells as above but using the ⁇ FliC flagella lacking an insert, or omitting the flagellar extract, or the flagellar extract and the immunoglobulins in the staining procedure.
  • the grids were washed in PBS containing 1 mg/ml BSA, and the bound antibodies were detected with AuroprobeTMEM Protein A-conjugate (Amersham, Amersham Place; Little Chalfont, Buckinghamshire, UK; diluted 1/40).
  • the grids were examined in a Jeol JEM-IOOCX transmission electron microscope at an operating voltage of 60 kV.
  • flagellar preparations were analyzed by SDS-PAGE using a 1 % (w/v) stacking gel and a 10 % separating gel. Polypeptides were transferred onto a nitrocellulose membrane using a semi-dry transfer apparatus at 0.9 mA/cm 2 membrane for 2 h at 4 °C.
  • the membrane was quenched with PBS containing 20 mg/ml BSA for 16 h at 20 °C and washed with PBS.
  • Polypeptides were visualized by staining with diluted polyclonal anti-flagella antibodies or anti-SlpA antibodies and alkaline-fosfatase-conjugated secondary antibodies as described (Westerlund-Wikstr ⁇ m et al. (1997)).
  • a phosphatase substrate solution containing nitroblue- tetrazolium (162 ⁇ g/ml) and 5-bromo-4-chloro-3-indolyl-l -phosphate (370 ⁇ g/ml) was used.
  • Adherence ofL. brevis ATCC 8287 to intestinal cells We initially assessed the adhesiveness 24 of the L. brevis strain ATCC 8287 to the human small intestine cell line Intestine 407. The strain showed an efficient adhesion to the intestinal cells (Fig. 1 A).
  • the cells of L. brevis ATCC 8287 express the S-layer protein SlpA as their major cell surface protein (Vidgren et al. J. Bacteriol. 9:7419-7427 (1992)), and we therefore extracted the S-layer from the bacterial surface and determined how this affected the adhesion. Extraction of cells with 2M guanidine hydrochloride is a routine procedure to remove bacterial S-layers, and the treatment does not lyse the bacterial cells.
  • Binding of chimeric flagella to epithelial cells-We next analyzed by an indirect immunofluorescence assay the binding of the chimeric flagella to Intestine 407 cells representative examples of the assays are shown in Fig. 4.
  • Hybridization was performed in the following buffer: 5xSSC, 1% Blocking Reagent (Boehringer Mannheim), 0,02% SDS, 0, 1 % layryl sarcosine at 68°C overnight using the full length L. brevis slpA gene labelled with DIG (digoxigenin- dUTP, Boehringer Mannheim) as probe. Hybridization was followed by washing steps in descreasing salt concentration and increasing temperature. The final wash step was in O.lxSSC, 0.1% SDS at 68°C for 15 min twice. The negative control used was and equal amount of E.coli or calf thymus DNA to that of test DNA.
  • All L. brevis strains tested gave a positive hybridization signal except the strain VK3 from TNO and the Japanese strain Yasui 0296961015. These two hybridization negative L. brevis strains were also shown to lack the S-layer protein by SDS-PAGE analysis. Furthermore, the other above mentioned S-layer expressing lactobacilli not belonging to the L. brevis and/or L. buchneri group remained negative in the hybridization test.
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO : 1:
  • GAAGGTTCAT TATACTATCA
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO : 3:
  • GAAGGTTCAT TATACTATCA
  • GCTACTAAGA CTCGTGAAGG TTCATTATAC TATCACGTAA CTGCTACTAA CGGTAGTGGT 540 ATTAGTGGTT GGATTTACGC TGGTAAGGGC TTCAGTACTA CTGCTACTGG TACACAAGTA 600
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GCT ACT GGT ACT AAC GCT TTA TAC ACG AAG 374 Ala Thr Arg Asn Val Glu Ala Thr Gly Thr Asn Ala Leu Tyr Thr Lys 50 55 60
  • GGT GAA AAG GGT CAA GTT GTC ACA TTA ACT GCC
  • ATC GAT ACT GAT TTG 1382 Gly Glu Lys Gly Gin Val Val Thr Leu Thr Ala He Asp Thr Asp Leu 385 390 395

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Abstract

L'invention concerne une molécule d'ADN codant pour un polypeptide se liant à des types de cellules épithéliales humaines et/ou animales. On a découvert que différents fragments de protéine couche S SlpA de lactobacillus brevis présente des propriétés adhésives à l'égard des types de cellules épithéliales. Il est possible de modifier ou améliorer la capacité de liaison de plusieurs cellules procaryotes ou eucaryotes aux types de cellules épithéliales humaines et/ou animales, comme les types de cellules endothéliales, intestinales et/ou uro-génitales au moyen de structures à surfaces lactobacillaires. Il est possible avec les séquences nucléotidiques d'améliorer les propriétés de liaison d'une cellule hôte présentant des effets probiotiques agissant sur les types de cellules épithéliales humaines et/ou animales.
PCT/FI1999/000290 1998-04-03 1999-04-06 Une region proteique responsable de la liaison avec des types de cellules epitheliales et une sequence d'adn codant pour cette region WO1999051631A1 (fr)

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FI980782A FI980782A7 (fi) 1998-04-03 1998-04-03 Epiteelisoluihin sitoutuva proteiinialue ja sitä koodaava DNA-sekvenssi

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085774A1 (fr) * 2000-05-12 2001-11-15 Alimentary Health Limited Produit a base d'adhesine probiotique obtenue a partir de $i(lactobacillus)
US7195906B2 (en) 1999-01-15 2007-03-27 Enterprise Ireland (Trading As Bioresearch Ireland) Bifidobacterium in the treatment of inflammatory disease
WO2015182470A1 (fr) * 2014-05-29 2015-12-03 カルピス株式会社 Agent pour favoriser l'incorporation de substances dans le tractus intestinal
CN115850384A (zh) * 2022-08-10 2023-03-28 丁辰 一种微生物来源的活性肽及其制备方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000581A1 (fr) * 1992-06-30 1994-01-06 Viagen Oy Systeme d'expression de lactobacillus utilisant des sequences geniques de proteine capsidique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000581A1 (fr) * 1992-06-30 1994-01-06 Viagen Oy Systeme d'expression de lactobacillus utilisant des sequences geniques de proteine capsidique

Non-Patent Citations (4)

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Title
CHEMICAL ABSTRACTS, Volume 126, No. 13, 31 March 1997, (Columbus, Ohio, USA), SAVIJOKI KIRSI et al., "High Level Heterologous Protein Production in Lactococcus and Lactobacillus Using a New Secretion System Based on the Lactobacillus Brevis S-Layer Signals", page 1, Abstract No. 167032; & GENE, 1997, 186(2), 255-262. *
FEMS MICROBIOLOGY REVIEWS, Volume 20, 1997, HUBERT BAHL et al., "IV. Molecular Biology of S-Layers", pages 47-98. *
JOURNAL OF APPLIED BACTERIOLOGY, Volume 74, 1993, C. SCHNEITZ et al., "Adhesion of Lactobacillusacidophilus to Avian Intestinal Epithelial Cells Mediated by the Crystalline Bacterial Cell Surface Layer (S-Layer)", pages 290-294. *
JOURNAL OF BACTERIOLOGY, Volume 174, No. 22, 1992, GABRIELE VIDGREN et al., "S-Layer Protein Gene of Lactobacillus Brevis: Cloning by Polymerase Chain Reaction and Determination of the Nucleotide Sequence", pages 7419-7427. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7195906B2 (en) 1999-01-15 2007-03-27 Enterprise Ireland (Trading As Bioresearch Ireland) Bifidobacterium in the treatment of inflammatory disease
WO2001085774A1 (fr) * 2000-05-12 2001-11-15 Alimentary Health Limited Produit a base d'adhesine probiotique obtenue a partir de $i(lactobacillus)
WO2015182470A1 (fr) * 2014-05-29 2015-12-03 カルピス株式会社 Agent pour favoriser l'incorporation de substances dans le tractus intestinal
JPWO2015182470A1 (ja) * 2014-05-29 2017-06-01 アサヒグループホールディングス株式会社 腸管における物質取り込み促進剤
CN115850384A (zh) * 2022-08-10 2023-03-28 丁辰 一种微生物来源的活性肽及其制备方法和应用

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