# Patent

## Title
Peptides and methods for producing them

## Abstract

The present invention relates to the fields of life sciences and food, feed or pharmaceutical industry. Specifically, the invention relates to novel peptides, pilus structures, polynucleotides as well as vectors, host cells, products and pharmaceutical compositions comprising the polynucleotides, peptides or pilus structures. The invention also relates to gene clusters and antibodies. Furthermore, the present invention relates to methods for producing the peptides or pilus structures or producing the products comprising the peptides or pilus structures. Furthermore, the present invention relates to treatments as well as uses and methods for screening bacterial strains, for reducing or inhibiting the adhesion of pathogenic bacteria, promoting the adhesion of bacterial cells to the mucus and for modifying immune response in a subject. Still, the present invention relates to methods for detecting probiotic bacterial strains or pathogen strains.

## Background
FIELD OF THE INVENTION
The present invention relates to the fields of life sciences and food, feed or pharmaceutical industry. Specifically, the invention relates to novel peptides, proteins, pilus structures, polynucleotides as well as vectors, host cells, products and pharmaceutical compositions comprising the polynucleotides, peptides, proteins or pilus structures. The invention also relates to gene clusters and antibodies. Furthermore, the present invention relates to methods for producing the peptides proteins or pilus structures or producing the products comprising the peptides proteins or pilus structures. Furthermore, the present invention relates to treatments as well as uses and methods for screening of bacterial strains, for reducing or inhibiting the adhesion of pathogenic bacteria, promoting the adhesion of bacterial cells to the mucus and/or epithelium and/or for modifying immune response in a subject. Still, the present invention relates to methods for detecting probiotic bacterial strains or pathogen strains to be identified and/or inhibited.
BACKGROUND OF THE INVENTION
Invasive adherence to host tissues by bacterial pathogens is often facilitated by means of elongated hairlike proteinaceous fibers called pili or fimbriae that protrude outwardly from the microbial cell surface. In Gram-negative pathogenic bacteria the role of pili as colonization agents in pathogenesis is well recognized and the overall mechanism of pilus assembly is clearly defined from over fifty years of research. The most structurally characterized Gram-negative pili are the type I form, found, for example, in the enteropathogenic E. coli, and type IV form, found, for example, in species of Neisseria and Pseudomonas as well as in E. coli. Typically, the Gram-negative pili are long (1 to 4 μm in length) and thin (5 to 8 nm in width), and also display both flexible and robust structural properties. These pili are generally comprised of a series of non-covalently linked multiple protein subunits whose assembly is dependent upon specific chaperone proteins, but independent of any enzymatic activity. Frequently, a protein with adhesive properties is positioned at the tip of the pili. It is generally considered that the intervening length of protein subunits from the microbial surface promotes an unhindered contact between the adhesive tip protein and corresponding host cell receptor sites, which are potentially represented by components of the extracellular matrix (ECM) or specific carbohydrate moieties of glycoproteins and glycolipids (Scott J. R. and Zähner D, 2006, Mol Microbiol 62, 320-330; Telford, J. L., et al. 2006, Nat Rev Microbiol 4, 509-519).
The presence of Gram-positive pilus-like structures was actually first observed in the late 1960's by electron microscopy of Corynebacterium renale (Yanagawa, R. et al. 1968, Jpn J Vet Res 16, 31-37), and in the subsequent years pili have been found in several other Gram-positive bacterial species, including the very recent discovery of pili in the three main invasive disease-causing streptococcal pathogens in humans, i.e., Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae (Telford, J. L., et al. 2006, Nat Rev Microbiol 4, 509-519). The most detailed characterization studies of Gram-positive pili originate from the corynebacteria, streptococci, and bacilli pathogens.
Unlike in the Gram-negative bacteria, the pili in Gram-positive bacteria are much thinner in width (2 to 3 nm) and more difficult to visibly distinguish which also suggests why the presence of these pili may have been over-looked in many species of Gram-positive bacteria (Kang, H. J. et al. 2007, Science 318, 1625-1628). To date, the most thorough description of the pilus-assembly process, that is also generally representative of all Gram-positive pili, has been carried by in vivo characterization studies of pili biogenesis in Corynebacterium diphtheriae (Ton-That, H. and Schneewind, O. 2004, Trends Microbiol 12, 228-234). Structurally, the prototype pili appear as polymers composed of covalently cross-linked protein subunits (called pilins) that are also covalently anchored at the base to the peptidoglycan component of the cell wall, with both of these covalent bonds being enzymatically dependent upon catalysis by different sortase family membrane-bound transpeptidases, i.e., the pilin-specific and the housekeeping sortases, respectively (Mandlik, A. et al. 2008, Trends Microbiol 16, 33-40). The Gram-positive pilus is typically composed of three pilin subunits and, in the case of C. diphtheriae, the genes named as SpaA (sortase-mediated pilin assembly) for the major pilin subunit that exclusively forms the shaft or backbone of the pilus, SpaB for an ancillary minor pilin subunit, and SpaC for another minor pilin subunit with adhesive properties located at the tip of the pilus (FIG. 1). The genes encoding these three pilin subunits are localized within the same loci as a pilin gene cluster along with at least one gene encoding a pilin-specific sortase in close proximity. As well, the genes within the pilin cluster are frequently flanked on both ends by transposable elements suggesting an origin by horizontal gene transfer. The transcription of all these genes is in the same direction and indicative of operon regulatory control (Scott J. R. and Zähner D, 2006, Mol Microbiol 62, 320-330).
The revised model of the overall Gram-positive pilus assembly process, which is dependent upon several different conserved motifs and domains within the primary sequence of each pilin subunit, includes four basic stages (Mandlik, A. et al. 2008, Proc Natl Acad Sci USA 105, 14147-14152; Telford, J. L., et al. 2006, Nat Rev Microbiol 4, 509-519)-(FIG. 1). In the first stage, the pilin proteins, each of which contain a N-terminal signal peptide, are secreted through the bacterial cell membrane by the Sec-dependent pathway and then retained in the cell membrane by the presence of a C-terminal membrane-spanning domain consisting of a hydrophobic region of about 20 residues and a positively charged tail.
In the second stage of the assembly process, the cell wall sorting signal (CWSS), preferably the LPXTG-motif, which also immediately precedes the membrane-spanning domain, becomes available for sortase-dependent cleavage of the cell membrane-anchored pilin proteins. The pilin-specific sortase cleaves this five residue motif between the threonine (T) and glycine (G) residues and forms an acyl-enzyme intermediate involving a covalent thioester bond between the carboxyl group of the threonine residue and a cysteinyl thiol found within the catalytic pocket of the sortase.
The third stage represents the polymerization of the pilin subunits by isopeptide bond formation and involves the cleavage of the thioester bond and the release of the sortase from the pilin subunit by the nucleophilic attack of the ε-amino group from the side chain of a lysine (K) residue conserved in the pilin-motif (WXXXVXVYPKN) of a second pilin subunit. An amide bond is thought to form between the C-terminal carboxyl of the threonine residue in the first pilin subunit and the side chain amino group of the pilin-motif lysine from a second pilin subunit still bound as a covalent thioester with an another pilin-specific sortase (Budzik, J. M. et al. 2008, Proc Natl Acad Sci USA 105, 10215-10220). In this model of pilus assembly, the growing polymeric structure is fed by additional pilin subunits at the base of the pilus and the overall length governed by the amount of available pilin subunits associated with pilin-specific sortases. Since the pilin-motif is a characteristic feature of the major (SpaA) and ancillary minor (SpaB) pilin subunits, but missing in the primary sequence of the minor pilin subunits (SpaC) displaying adhesive properties, this pilin subunit is likely located at the tip of the pilus shaft and the first pilin subunit to initiate pilus polymerization.
The attachment of the polymerized pilus to the cell wall represents the fourth stage of the assembly process. Herein, the ancillary minor pilin subunit (SpaB) signals the cessation of pilus polymerization, but only when presented in association with a housekeeping sortase, whose gene is encoded somewhere else on the genome. In this final stage, the growing polymeric structure of major pilin subunits (SpaA) is transferred from a thioester linkage with a pilin-specific sortase to form an amide bond with the side chain of the lysine in the pilin-motif of SpaB minor pilin subunit, which is coupled as a housekeeping sortase acyl-enzyme intermediate. The nucleophilic attack by the amino group of the pentapeptide of the peptidoglycan lipid II precursor then permits the housekeeping sortase to catalyze the attachment of the SpaB pilin-linked pilus polymer to the cell wall. The E-box represents a third and less characterized conserved primary sequence motif (YXLXETXAPXGY) found between the LPXTG- and pilin-motifs of the pilin subunits from many Gram-positive bacteria.
Thus far, three-dimensional (3-D) structure determinations by x-ray crystallography have revealed structural insights into the assembly and function for only two Gram-positive pilin subunit proteins. Krishnan et al. (2007, Structure 15:893-903) had solved the crystal structure for the minor pilin GBS52 of Streptococcus agalactiae and revealed the presence of two IgG-like domain folds that share a structural similarity with the S. aureus collagen-binding protein Cna which also indicates how this minor pilin subunit could facilitate pilus adherence to a specific host tissue. The crystal structure of the major pilin Spy0128 from Streptococcus pyogenes, solved by Kang et al. (2007, Science 318, 1625-1628), had demonstrated how self-generated intramolecular isopeptide bonds between the side chains of lysine and asparagine residues within the pilin subunit could also complement the sortase-catalyzed intermolecular isopeptide bonds for maintaining the overall strength and stability of pili.
The majority of probiotic microbes are members of the Gram-positive lactobacilli and bifidobacteria and have a long tradition of use in fermented foods and dairy products (Goldin, B. R. and Gorbach, S. L. 2008, Clin Infect Dis 46, S96-S100; Ljungh, A. and Wadstrom, T. 2006, Curr Issues Intest Microbiol 7, 73-89; Salminen, S. et al. 1998, Br J Nutr 80, S147-S171). Pilus structures of probiotic lactobacilli or genes encoding these pilus structures have not been described in the literature. The presence of pilus-like structures or polynucleotides has never been shown in Lactobacillus rhamnosus. 
BRIEF DESCRIPTION OF THE INVENTION
The object of the present invention is to provide novel pilus polypeptides as well as polynucleotides encoding them. Furthermore, the object of the invention is to provide novel pilus structures. Still, the object of the invention is to provide novel methods, uses and products related to the above-mentioned peptides, polypeptides, proteins, pilus structures, and polynucleotides.
The present invention relates to peptides comprising a sequence having at least 94% sequence identity with seq id no 1 (GG00441), at least 94% sequence identity with seq id no 2 (GG00442), at least 84% sequence identity with seq id no 3 (GG00443), at least 91% sequence identity with seq id no 4 (GG00444), at least 83% sequence identity with seq id no 5 (GG02369), at least 94% sequence identity with seq id no 6 (GG02370), at least 93% sequence identity with seq id no 7 (GG02371) or at least 93% sequence identity with seq id no 8 (GG02372), or fragments or variants thereof.
The present invention also relates to a pilus structure comprising at least one of the peptides of the invention, a product comprising at least one peptide or pilus structure of the invention and to a pharmaceutical or nutritional composition comprising at least one peptide or pilus structure of the invention.
Furthermore, the present invention relates to a product comprising at least one peptide or pilus structure of the invention for use as a medicament or for the prevention or treatment of diarrhea, arterial hypertension, vascular diseases, allergies, cancer, atopic diseases, viral diseases, infectious diseases, urinary tract infections, respiratory infections, dental caries, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), mucosal inflammation, gut permeability disorders, obesity, metabolic syndrome, oxidative stress or abdominal pain.
Furthermore, the present invention relates to the use of at least one peptide or pilus structure of the invention in the manufacture of a medicament for treating or preventing diarrhea, arterial hypertension, vascular diseases, allergies, cancer, atopic diseases, viral diseases, infectious diseases, urinary tract infections, respiratory-infections, dental caries, IBS, IBD, mucosal inflammation, gut permeability disorders, obesity, metabolic syndrome, oxidative stress or abdominal pain.
Still, the present invention relates to a polynucleotide comprising a sequence of any one of seq id nos 9-16 or a degenerate thereof, or encoding a peptide of the invention, to a vector comprising the polynucleotide of the invention, to a host cell comprising the polynucleotide or the peptide of the invention, and to a gene cluster comprising at least one polynucleotide of the invention.
Also, the present invention relates to an antibody/antibodies against the peptides of the invention or their functional domains.
The present invention also relates to a method of treating or preventing diarrhea, arterial hypertension, vascular diseases, allergies, cancer, atopic diseases, viral diseases, infectious diseases, urinary tract infections, respiratory infections, dental caries, IBS, IBD, mucosal inflammation, gut permeability disorders, obesity, metabolic syndrome, oxidative stress or abdominal pain comprising administration of at least one peptide or pilus structure of the invention to a subject.
The present invention relates to a method for screening of bacterial strains, which comprise at least one polynucleotide of the invention or a fragment thereof, wherein the method comprises:
i) providing DNA or RNA from bacterial strains;
ii) hybridizing primers or probes specific to the polynucleotide of the invention or a fragment thereof with DNA or RNA from step i) and optionally amplifying the polynucleotide or the fragment thereof;
iii) detecting at least one polynucleotide or a fragment thereof homologous to the polynucleotide of the invention or the fragment thereof.
The present invention relates to a use of at least one polynucleotide of the invention or fragment thereof or at least one antibody of the invention for screening of bacterial strains.
The present invention relates to a method of screening bacterial strains, which comprise at least one peptide or pilus structure of the invention, using at least one antibody of the invention, wherein the method comprises:
i) providing proteins of bacterial strains;
ii) detecting at least one polypeptide, pilus structure or a fragment thereof using the antibody/antibodies.
The present invention relates to a method of reducing or inhibiting the adhesion of pathogenic bacteria to the gastrointestinal tract, to the epithelium or to the mucus of a subject, wherein the method comprises administering at least one peptide and/or pilus structure of the invention to the subject.
The present invention relates to a use of at least one peptide and/or pilus structure of the invention for reducing or inhibiting the adhesion of pathogenic bacteria to the gastrointestinal tract, to the epithelium or to the mucus of a subject.
The present invention relates to a method of promoting the adhesion of a bacterial cell or the adhesion of any other agent to the mucus or epithelium, wherein the method comprises:
i) producing at least one peptide or pilus structure of the invention or a fragment thereof;
ii) displaying the peptide, pilus structure and/or fragment thereof on the bacterial cell or on any other agent;
iii) bringing the bacterial cells or any other agent into contact with the mucus or epithelium.
The present invention relates to a use of at least one peptide or pilus structure of the invention for promoting the adhesion of a bacterial cells or the adhesion of any other agent to the mucus or epithelium.
The present invention relates to a method of modifying immune response in a subject, wherein the methods comprise:
i) producing at least one peptide or pilus structure of the invention or a fragment thereof;
ii) displaying the peptide, pilus structure and/or fragment thereof on a host cell;
iii) optionally bringing the host cell into contact with the mucus or another host cell.
The present invention relates to a use of at least one peptide or pilus structure of the invention for modifying immune response.
The present invention relates to a method of producing a product of the invention, wherein the method comprises a step of generating at least one peptide or pilus structure of the invention to a product.
The present invention also relates to a method of producing at least one peptide or pilus structure of the invention, wherein the method comprises the following steps:
i) providing at least one polynucleotide of the invention;
ii) transforming a host cell with the polynucleotide(s);
iii) culturing the host cell from step ii) to produce the peptide(s) or pilus structure;
iv) optionally recovering the peptide(s) or pilus structure.
In addition, the present invention relates to a method of producing at least one peptide or pilus structure of the invention, wherein the method comprises the following steps:
i) disrupting a cell producing or comprising at least one peptide or pilus structure of the invention;
ii) optionally, recovering the peptide(s) or pilus structure.
Also, the present invention relates to a method of producing at least one peptide of the invention, wherein the method comprises the following steps:
i) providing amino acids;
ii) manufacturing at least one peptide of the invention from the amino acids of step i) with synthetizing at least one peptide.
The present invention relates to a method of detecting potential probiotic bacterial strains by using bioinformatic approaches, wherein the method comprises the following steps:
i) providing a sequence of at least one peptide, polynucleotide or fragment thereof;
ii) comparing the sequence of step i) against sequences of sequence collections;
iii) detecting sequences having biologically congruent fragments to sequences of step i) or having high identity to the sequence of step i).
The present invention also relates to a method of detecting pathogen strains, against which the peptides or pilus structures of the invention are effective, by using bioinformatic approaches, wherein the method comprises:
i) providing a sequence of at least one peptide, polynucleotide of fragment thereof;
ii) comparing the sequence of step i) against sequences of sequence collections;
iii) detecting sequences having biologically congruent fragments to the sequence of step I) or having high identity to the sequence of step i).
The peptides, pilus structures and polynucleotides of the invention provide tools for further developments in food, feed, cosmetics and pharmaceutical industries. The present invention enables rapid and efficient screening methods and reliable and accurate, either qualitative or quantitative analysis of a multitude of bacterial strains. Therefore, the methods and means of the invention enable the discovery of novel probiotic bacterial strains as well as discoveries of new products (incl. ingredients, supplements, and nutritional products), medicaments and therapeutic methods. Furthermore, by the present invention more effective and specific treatments become available.
There is a continued, evident need to offer the consumers new products having clearly demonstrated effects on health and produced in a form that allows them to be used as such or as a part of another product, such as a pharmaceutical or a food or feed product. In accordance with the present invention, products are also applicable as capsules, pills or tablets that allow the use as convenient part or supplement, for example, of the every-day diet or medication.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1a and 1b show the models of pilus assembly and covalent attachment to the cell wall in Gram-positive Corynebacteria.
FIG. 2 shows the Lactobacillus rhamnosus GG (LGG) pili clusters including genes encoding pilin-specific sortases, major pilus shaft protein, minor pilus shaft protein and capping pilus proteins. CWSS indicates a cell wall sorting signal, i.e. a conserved motif found in many Gram-positive bacteria, Pilin Motif and E-box also indicate conserved motifs found in many Gram-positive bacteria.
FIG. 3 shows examples of polyclonal antibodies binding to peptides GG00442, GG00443, GG00444, GG02370, GG02371 and GG02372 of the LGG pilus structure.
FIG. 4 shows a phase contrast Atomic Force Microscope micrograph picture of protruding pili structures of LGG.
FIGS. 5a and 5b show in vitro binding of recombinant histidine-tagged LGG proteins, i.e. SpaA, SpaB, SpaC, SpaD and SpaF pilin proteins, to human intestinal mucus. Resected human intestinal tissue was used as a source of mucus on a polystyrene microtiter plate. The bound proteins were detected by enzyme-linked immunosorbent assay.
FIGS. 6a and 6b show Western blots of cell wall fractions of LGG and as a negative control L. rhamnosus LC705 (LC705) grown in mTSB-medium or MRS+0.6% ox gall bile medium using SpaA and SpaC pilin protein-specific polyclonal antibodies, respectively. FIG. 6a shows the presence of SpaA-containing pili and SpaA monomers in LGG and FIG. 6b shows the presence of SpaC-containing pili and SpaC monomers in LGG. Lane 1: recombinant SpaA/SpaC pilin protein; Lane 2: LGG grown in mTSB; Lane 3: LGG grown in MRS+0.6% ox gall bile; Lane 4: LC705 grown in mTSB, Lane 5: LC705 grown in MRS+0.6% ox gall bile. The antibody used is indicated on a top of each picture. In FIG. 6b, Panel A: lanes 1 to 5 are exposed for 1 second; Panel B: lanes 2-5 are exposed separately for 60 seconds. The positions of the molecular weight standards are indicated on the left as kilodaltons. HMW indicates high molecular weight ladder.
FIGS. 7a and 7b show nucleotide sequences encoding the pili operons presented in FIG. 2. FIG. 7A shows the operon encoding GG00441-GG00444 genes (bold). The putative conserved elements −35 sequence (underlined), −10 sequence (double underlined), ribosomal binding site (underlined italics) and rho terminator (dotted underline). FIG. 7B shows the operon encoding GG02369-GG02372 genes (bold). The putative conserved elements −35 sequence (underlined), −10 sequence (double underlined), ribosomal binding site (underlined italics) and rho terminator (dotted underline).



DETAILED DESCRIPTION OF THE INVENTION
Lactic acid bacteria have been utilized in food industry for a long time and today they are used in various food supplies such as milk products. For example lactobacilli and bifidobacteria are known to have probiotic effects, but the ways by which probiotic bacteria affect the health are not fully understood. Therefore, further investigations of probiotics are warranted.
This invention resides in the finding that also Gram-positive bacteria has pilus structures. Furthermore, the invention resides in the finding of novel pilus peptides and structures in Gram-positive bacteria, specifically in lactobacilli, more specifically in Lactobacillus rhamnosus. 
Peptides of the Pilus Structure
Generally a Gram-positive bacterial pilus extends out from the outer membrane of the bacteria, usually being 1-4 μm long and 2-8 nm wide and appearing in low numbers. Pili is considered to promote adherence of the bacteria to target surfaces. Indeed, as used herein, the expression “pilus structure” refers to an elongated hair or hairlike proteinaceous fiber, comprising multiple protein subunits (preferably more than one subunits). The assembly of these proteins may be dependent on specific proteins, i.e. sortases. A protein having adhesive properties is usually located at the top of the pili. Also the other proteins of the heteromeric pilus structure may be adhesive. As used herein, the expression “part of a pilus structure” refers to any component of a pilus, preferably any protein or any fragment or any variant of the pilus. In a preferred embodiment of the invention, the pilus structure is located on the surface of a microorganism or originates therefrom.
As used herein, the expression “peptide” refers to any peptide such as a dipeptide, polypeptide, protein and/or pilin protein.
In a specific embodiment of the invention, characteristic features of the pilin are major (SpaA), ancillary minor (SpaB) and capping (SpaC) pilin subunits.
Pilin specific sortases act by transferring SpaA to SpaC in a growing polymeric structure of pilin (FIG. 1). In a preferred embodiment of the invention, the peptide comprising a sequence having at least 94% sequence identity with seq id no 1 (GG00441) or a sequence having at least 83% sequence identity with seq id no 5 (GG002369) is a pilin specific sortase (FIG. 2).
SpaA likely forms a back-bone of the pilus structure. The length of the different pilus structures depends on the amount of SpaA in the back-bone (FIG. 1). In a preferred embodiment of the invention, the peptide comprising a sequence having at least 94% sequence identity with seq id no 2 (GG00442) or a sequence having at least 94% sequence identity with seq id no 6 (GG002370) is a major pilus shaft protein, i.e. a major pilin subunit (FIG. 2). GG00442 and GG02370 contain the sortase-recognition site, thus being substrates of the sortases.
SpaB is likely added to the pilus structure at the latest state (terminal stage) of the pilus formation and it forms a link of the pilus to the cell wall (FIG. 1). In a preferred embodiment of the invention, the peptide comprising a sequence having at least 84% sequence identity with seq id no 3 (GG00443) or a sequence having at least 93% sequence identity with seq id no 7 (GG002371) is a minor pilus shaft protein (FIG. 2). GG00443 and GG002371 contain the sortase-recognition site, thus being substrates of the sortases.
SpaC is likely located at the tip of the pilus shaft and the first pilin subunit to initiate pilus polymerization (FIG. 1). In a preferred embodiment of the invention, the peptide comprising a sequence having at least 91% sequence identity with seq id no 4 (GG00444) or a sequence having at least 93% sequence identity with seq id no 8 (GG002372) is a binding pilus protein (FIG. 2). GG00444 protein contains a von Willebrand factor (vWF) domain, and GG00444 and GG02372 contain the sortase-recognition sites, thus being substrates of the sortases.
In a specific embodiment of the invention, the peptide or polypeptide of the invention comprises a sequence having at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9 or 100% identity to amino acid sequence of Seq ID No. 1, 2, 3, 4, 5, 6, 7 or 8, or fragments or variants thereof.
According to a specific embodiment of the invention, the peptide has at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9 or 100% identity to any one of the amino acid sequences of Seq ID No. 1, 2, 3, 4, 5, 6, 7 or 8, or fragments or variants thereof.
In another specific embodiment of the invention the peptide has a sequence shown in any one of the sequences Seq ID No 1, 2, 3, 4, 5, 6, 7 or 8, or fragments or variants thereof.
Identity of any sequence or fragments thereof compared to the sequence of this invention refers to the identity of any sequence compared to the entire sequence of the present invention. Sequence identity may be determined for example by using BLAST (Basic Local Alignment Search Tools) or FASTA (FAST-All). In the searches, setting parameters “gap penalties” and “matrix” are typically selected as default.
As used herein, a fragment or variant of a peptide refers to any part or variant of a peptide, which may have the biological function. A variant refers to a peptide having small alterations in the peptide sequence, e.g. small deletions, mutations or insertions.
In a preferred embodiment of the invention, a peptide having seq id no 2-4 or 6-8 is a part of a pilus structure. In another preferred embodiment of the invention, the pilus structure of the invention comprises at least one of the peptides of the invention, more preferably at least two, or at least three peptides of the invention. Furthermore, in a preferred embodiment of the invention, the pilus structure comprises peptides GG00442 (Seq ID No 2), GG00443 (Seq ID No 3) and GG00444 (Seq ID No 4) and/or peptides GG02370 (Seq ID No 6), GG02371 (Seq ID No 7) and GG02372 (Seq ID No 8).
Gram-Positive and Probiotic Bacteria
The peptides or pilus structures of the invention can be from any bacteria, such as Gram-positive or Gram-negative bacteria. However, in a preferred embodiment of the invention, the peptides or pilus structures are from gram-positive bacteria. Gram-positive bacteria, which may comprise the peptides or pilus structures of the invention, include but are not limited to lactobacilli, lactococci, bifidobacteria, propionibacteria, leuconostoc, streptococci corynebacteria, actinomyces and mycobacteria.
In a preferred embodiment of the invention, the peptide or pilus structure is from probiotic bacteria such as probiotic lactobacilli, lactococci, bifidobacteria, enterococci, propionibacteria, leuconostoc, streptococci or yeast. Probiotics are live micro-organisms, preferably non-pathogenic microbes which, when administered in adequate amounts to man or animal, promote the well being of the host (Fuller, R. 1989, J. Appl. Microbiol. 66:365-378). Probiotics will result in a beneficial health advantage to the host, when consumed as a food or a food supplement in adequate amounts.
Health claims of probiotics in humans or animals include the possible prevention and treatment of many ailments. The health-promoting effects of probiotics include for example the balancing and maintenance of intestinal flora, stimulation of the immune system and anti-carcinogenic activity. The useful effects of probiotics in human intestines are based on several independent factors caused by live bacterial cells, their cell structures and metabolic products.
A bacterium may be referred to as a probiotic if it essentially meets the following requirements (Lee, Y-K and Salminen, S. 1995 Trend Food Sci Technol, 6:241-245): it remains viable in the demanding conditions prevailing in the digestive tract (low pH of the stomach, acids of the digestive system, etc.); attaches to the walls of the intestine; colonizes the GIT; metabolizes in the intestine; is technologically applicable (endures processing); exhibits clinically tested and reported health effects; and is safe to consume.
There are huge differences in microbial content between the different parts of the gastrointestinal tract, about 95% of all the intestinal bacteria appearing in the colon. Over 400 bacterial species have been estimated to thrive in the colon in addition to transient microbes. The dominating species are the following: Bacteroides, Bifidobacterium, Coprococcus, Peptostreptococcus, Eubacterium and Ruminococcus. The number of species Lactobacillus, Streptococcus, Fusobacterium, Veillonella, Propionibacterium and Enterobacteriaceae is slightly less. Some of the species represent useful microbes, whereas others may even be harmful (Tannock, G. W. 1998, Int. Dairy J. 8:527-533). Changes in the composition of the intestinal flora or a sudden reduction in the amount of it (due to severe diarrhea, antibiotics treatment, etc.) increase the infectivity of potentially pathogenic species, which may have serious consequences (outbreak of allergies, intestinal diseases, cancer).
In a preferred embodiment of the invention, the peptide or pilus structure binds to the gastrointestinal tract (GIT), most preferably to the epithelium of the gastrointestinal tract. In another preferred embodiment of the invention, the peptide or pilus structure binds to the mucus. Mucus is a slippery secretory product, a viscous colloid, from mucus-producing cells. Mucus protects epithelial cells for example in the GIT. In addition to antiseptic enzymes and immunoglobulins mucus also contains mucins and inorganic salts. As used herein, gastrointestinal tract refers to a tube from the mouth to the anus, which participates in digesting food. The GIT comprises the mouth, esophagus, stomach, duodenum, jejunum, ileum, small intestine, large intestine (colon), cecum, rectum and anus.
The best-documented probiotics include L. rhamnosus GG, L. johnsonii LA1, L. casei Shirota and Bifidobacterium lactis Bb12. In addition, a number of other probiotics have been described in the literature of the art. In a preferred embodiment of the invention, the peptide or pilus structure is from Lactobacillus rhamnosus, most preferably from Lactobacillus rhamnosus GG (LGG, LGG®) strain, which is a non-pathogenic Gram-positive isolate originally from the USA (U.S. Pat. No. 4,839,281A). LGG strain is isolated from human feces, it is able to grow well in pH 3 and survives even lower pH values as well as high bile acid contents. The strain exhibits excellent adhesion to both mucus and epithelial cells, and colonizes GIT. Lactic acid yield from glucose is good: when grown in MRS broth, the strain produces 1.5-2% of lactic acid. The strain does not ferment lactose and thus it does not produce lactic acid from lactose. The strain ferments following carbohydrates: D-arabinose, ribose, galactose, D-glucose, D-fructose, D-mannose, rhamnose, dulcitol, inositol, mannitol, sorbitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, saccharose, trehalose, melezitose, gentibiose, D-tagatose, L-fucose, and gluconate. The strain grows well at 15-45° C., the optimum temperature being 30-37° C. LGG has been deposited with the depository authority American Type Culture Collection under accession number ATCC 53103.
Pilus Genes
The genes encoding the pilin proteins of a pilus structure are clustered on the same loci in the LGG genome. Altogether two different gene clusters encoding the pilus peptides were found by bioinformatic methods in the LGG genome (FIG. 2).
In one preferred embodiment of the invention, the polynucleotide has a sequence of any one of seq id nos 9-16 or a degenerate or fragment thereof, or it encodes the peptide of the invention or a fragment thereof. A polynucleotide that has a degenerate of a sequence shown in any one of seq id nos 9-16 means that it contains one or more different nucleotides, but still encodes for a same amino acids. A “polynucleotide” as used herein is a sequence of nucleotides such as DNA or RNA sequence, and may be a single or double stranded polynucleic acid. The term polynucleotide encompasses genomic DNA, cDNA and mRNA. Also, the polynucleotide may be isolated DNA.
In another preferred embodiment of the invention, the gene cluster comprises at least one polynucleotide of the invention. In another preferred embodiment of the invention, the gene cluster comprises at least two, at least three or at least four polynucleotides of the invention. Most preferably the gene cluster comprises polynucleotides shown in Seq ID Nos 9-12 or Seq ID Nos 13-16. As used herein, “a gene cluster” refers to a group of at least two genes that encode for peptides/proteins needed for a joint function (concerted action), here e.g. for the pilus structure. The genes of the same cluster are usually grouped together on the same genetic locus.
According to a specific embodiment of the invention, the polynucleotide has at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9 or 100% identity to any one of the nucleotide sequences of Seq ID No. 9, 10, 11, 12, 13, 14, 15 or 16, or fragments thereof.
In another specific embodiment of the invention the polynucleotide has a sequence shown in any one of the sequences Seq ID No 9, 10, 11, 12, 13, 14, 15 or 16.
Products and Pharmaceutical Compositions
In one preferred embodiment of the invention, the product comprises at least one peptide or pilus structure of the invention. The product may also comprise at least two or at least three peptides of the invention. In one preferred embodiment, a product comprises at least one fragment of the peptide of the invention. The products of the invention may be selected from but are not limited to the group consisting of food products, animal feed, nutritional products, food supplements, food ingredients, health food, pharmaceutical products and cosmetics. In one preferred embodiment of the invention, the product is a food or feed product. In another embodiment of the invention the product is functional food, i.e. food having any health promoting and/or disease preventing or treating properties. Preferably a food product of the invention is selected from the group consisting of dairy products, bakery product, chocolate and confectionary, sugar and gum confectionary, cereal products, snacks, berry or fruit based products and drinks/beverages. Dairy products include but are not limited to milk, sour milk, yogurts and other fermented milk products such as cheeses and spreads, milk powders, children's food, baby food, toddler's food, infant formula, juices and soups. In addition to the peptides or pilus structures of the invention, the product may also contain other starters, probiotics etc.
In a preferred embodiment of the invention the product is a pharmaceutical composition. In one preferred embodiment of the invention, the pharmaceutical composition comprises at least one peptide or pilus structure of the invention, and in another embodiment, at least two, or at least three peptides of the invention. The pharmaceutical compositions may be used for example in solid, semisolid or liquid form such as in the form of tablets, pellets, capsules, solutions, emulsions or suspensions. Preferably the composition is for oral administration or for enteral applications.
In addition to at least one peptide or pilus structure of the invention, the pharmaceutical composition may comprise prebiotics, pharmaceutically acceptable carrier(s) (e.g. water, glucose or lactose), adjuvant(s), excipient(s), auxiliary excipient(s), antiseptic(s), stabilizing, thickening or coloring agent(s), perfume(s), binding agent(s), filling agent(s), lubricating agent(s), suspending agent(s), sweetener(s), flavoring agent(s), gelatinizer(s), anti-oxidant(s), preservative(s), buffer(s), pH regulator(s), wetting agent(s) or components normally found in corresponding products.
The product or pharmaceutical composition of the invention comprises the peptide or pilus structure in an amount sufficient to produce the desired effect. Other ingredients as well as other specific components of the products or pharmaceutical compositions are either obtained commercially or prepared by conventional techniques known in the art.
The products or pharmaceutical compositions may be manufactured by any conventional processes known in the art. Generating the peptide or pilus structure to a product means that the peptide or pilus structure may for example be added to any products or mixed with any agents. The peptide or pilus structure may also be generated in a product for example by expression in appropriate conditions. The peptide or pilus structure may be added or mixed either in connection with the preparation or thereafter, during the finishing of the end product. In a preferred embodiment of the invention, the peptide or pilus structure of the invention is added to a product.
Production Methods
The peptide or pilus structure of the invention can be produced for example by synthetic methods e.g. peptide synthesis or by recombinant production with genetically modified organism. In a preferred embodiment of the invention, the peptide or pilus structure is recombinant. As used herein, “recombinant” genetic material refers to a material, which is typically a combination of one or more genetic material, e.g. DNA strands of various origin, and it has been produced by combining or inserting the sequences. Recombinant production enables achieving specific and/or special traits into a gene or gene product or for example into expression of a gene (e.g. over- or underexpression). The polynucleotide of the invention may for example be put under the control of any endogenous or exogenous regulators, such as promoters. Recombinant protein is derived from recombinant DNA.
At least one polynucleotide of interest may be isolated from a cell or produced synthetically. This nucleotide can be transformed to a host cell. A suitable host cell for producing any peptide of the invention may be any eukaryotic cell or micro-organism, preferably bacteria, most preferably lactic acid bacteria such as lactobacilli, lactococci, bifidobacteria, enterococci, leuconostoc, and streptococci, or propionibacteria or yeast.
As used herein, “transformation” refers to a genetic alteration of a cell by foreign genetic material, preferably DNA, resulting in expression of this genetic material. The foreign genetic material can be introduced as such or as incorporated into any other genetic material such as vectors, plasmids etc. Any method of genetic engineering or any molecular cloning methods can be used for transforming a host cell with the polynucleotide of the invention. There are various methods of introducing foreign material into a eukaryotic cell. Materials such as polymers (e.g. DEAE-dextran or polyethylenimine), liposomes and nanoparticles (e.g. gold) have been used as carriers for transformation. Genetic material can also be introduced into cells by using for example viruses or vectors as carriers. Other methods for introducing foreign material into a cell include but are not limited to nucleofection, electroporation, conjucation, transfection, sonoporation, heat shock and magnetofection. The use of various transfection reagents such as calcium phosphate or lipofectamine is well known in the art. Preferable method for introducing foreign material into a bacterial cell is electroporation.
The peptide or pilus structure of the invention may also be produced by cells expressing the peptides or pilus structures naturally.
After a natural cell or transformed host cell has produced the peptide of the invention in appropriate conditions, the peptide can for example be purified from the cell or a secreted form of the peptide can be recovered e.g. from culture media. In order to purify the peptide, the cell may be disrupted for example by sonication, radiation, heating, lysis, mechanical agitation (sharing), enzymatic methods, ‘cell pomb’ or chemical agents (hypotonic shock, detergents, and solvents) or mixtures thereof. The peptide or pilus structure is obtainable from growing or metabolically active, i.e. live and/or lyophilized, or non-viable e.g., heat-killed, irradiated or lysed organisms. The peptide or pilus structure is obtainable from a dead cell or a living cell.
The peptide or pilus structure can be produced in one cell and then displayed on the same cell, or the peptide or pilus structure may be produced in another cell than on which it is displayed.
Any known methods such as immunization can be used for producing antibodies against the peptides of the invention. Antibodies can be generated against any epitopes or functional domains of the peptides and they can be either monoclonal or polyclonal. In a preferred embodiment of the invention, the antibodies are polyclonal (FIG. 3). As used herein, “functional domain of a peptide” refers to any part of the peptide, which has a biological function.
Treatments
Bacteria, a large group of unicellular micro-organisms, cause various diseases in eukaryotes, such as human beings, animals and plants. However, it is only within recent years that the presence of pili on the surface of important pathogens has gained interest among researches. Because GIT and its microbiota affect the well being of the subjects, utility of the pili of the bacteria potentiates novel treatments. The peptides, pilus structures or polynucleotides of the invention can be utilized in a method of treating or preventing diseases either caused by micro-organisms, such as bacteria or virus, or caused by any other reason, such as unbalanced nutrition, stressed life style or genetic pre-disposition. Diseases or ailments, which can be prevented or treated with the peptides, pilus structures, polynucleotides or with the pharmaceutical products of the invention include but are not limited to diarrhea such as traveler's diarrhea, arterial hypertension, vascular diseases, allergy, atopic diseases, urinary tract infections, respiratory infections, dental caries, irritable bowel syndrome, inflammatory bowel disease as well as remedying minor bowel discomfort and enhancing/promoting one's overall well-being. The composition of the invention is also useful for the prevention and treatment of gastrointestinal disorders and diseases, and for promoting general health. The disorders or diseases are preferably selected from the group consisting of mucosal inflammation, gut permeability disorders, IBD, IBS, and other gastrointestinal disorders. In a special embodiment of the invention peptides or pilus structures are used as vaccines (immunological response).
The method of reducing or inhibiting the adhesion of pathogenic bacteria to the GIT of a subject results in preventing or alleviating the symptoms caused by the pathogen. The pathogen is displaced from the epithelia or surface of the GIT by competition with the peptide or pilus structure of the invention. The preferred pathogens to be displaced include but are not limited to Escherichia coli, salmonella, bacilli, bacteroides, listeria, staphylococci, enterococci, clostridia and streptococci. As used herein, “pathogenic bacteria” refers to any bacteria causing any disease or any harmful effect. As used herein “adhesion” refers to anchoring of at least two molecules or structures to each other by chemical or physical bonds/forces or without them. Different types of adhesion such as mechanical adhesion, chemical adhesion, dispersive adhesion, electrostatic adhesion and diffusive adhesion are known. Adhesion can be a reversible or irreversible event, but in a biochemical system, adhesion is usually reversible.
Enterococcus faecalis and Enterococcus faecium are intestinal bacteria that are emerging nosocomial pathogens, including vancomycin-resistant enterococci (VRE) that are highly resistant to the important clinical antibiotic vancomycin (de Regt M. J. et al. 2008, J Antimicrob Chemother. 62(6):1401-1406). Recently, it has been described that E. faecium isolates contain surface located pili and remarkably, the vast majority (71%) of the hospital-acquired and an important fraction (43%) of the non-hospital strains of E. faecium contain pilus genes (Hendricks A. P. et al. 2008, Microbiology 154:3212-3223). In a double-blind and placebo-controlled study it has been described that consumption of Lactobacillus rhamnosus GG effectively cleared enterococci from infection in VRE-positive patients (Manley K. J. et al. 2007 Med J Aust. 186(9):454-457). Molecular support for the competition between pili-containing Lactobacillus rhamnosus GG and VRE originates from binding studies that showed that Lactobacillus rhamnosus has 20-130 fold higher binding to human gastrointestinal mucus than vancomycin-resistant E. faecium (Pultz N J. et al. 2006 Curr Microbiol. 52(3):221-224). Surprisingly, in a binding assay of this invention, the purified His-Tag labelled LGG proteins SpaA, SpaB and SpaC inhibit pathogens e.g. vancomycin-resistant E. faecium from binding to the mucus.
The method of reducing or inhibiting the adhesion of pathogenic bacteria to the gastrointestinal tract, to the epithelium or to the mucus of a subject may comprise the following steps: i) producing at least one peptide of the invention or fragment thereof or pilus structure; ii) displaying the peptide and/or pilus structure on the cell or mucus.
In addition to reducing adherence of harmful or pathogenic bacteria, the present invention also offers the possibility to promote the adhesion of beneficial cells or other agents such as enzyme(s), recombinant cells, microcapsule, nanocapsule or medicament(s) to the GIT. The method of promoting the adhesion of a bacterial cell to the mucus and to the GIT or a use of a peptide or a pilus structure of the invention for promoting the adhesion of a bacterial cell to the gastrointestinal mucus relates to a surprising ability of the novel peptides or pilus structures to adhere to the GIT in vivo, ex vivo or in vitro. The pilus peptide or structure functions as a tool for linking a cell or any other agent such as medicaments, enzyme(s), micro-organism(s), recombinant cells, microcapsule or nanocapsule to the GIT.
The method of modifying the immune response in a subject and use of the peptides or pilus structure for modifying the immune response are based on a surprising finding that the peptides or the pilus structure of the invention cause changes in the immune response. Immune response refers to a response to an antigen in the body, in ex vivo or in vitro system or to a response to another modulator. This response can be mediated by lymphocytes and/or recognition of antigens by specific antibodies. One goal of the immune response is to destroy the antigen, which usually is of foreign origin, or to neutralize it. As used herein, “modifying” refers to any alteration of the immune response such as increase or decrease. Alterations of an immune response can be monitored by any suitable medical, physiological or biological test including but not limited to those, which are based on detecting activation of signalling pathways as well as detecting a transcription or translation level of marker genes or the amount of proteins, e.g. antibodies or receptors. A single marker is not currently available for determining the immune response in a cell or organism. However, preferable markers include but are not limited to tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), IL-10, IL-1 β, and interferon alpha (IFN-α). Other possible markers are IL-1α, IL-6, IL-18, IFN-γ, IL-4, TGF-β, IL-I Ra and IL-18BP. In a preferred embodiment of the invention, the marker(s) is/are selected from a group consisting of TNF-α, Th1 cytokines, IL-10 and IL-12.
Alterations of immune response can be checked by in vitro, ex vivo or in vivo tests from any biological sample or subject. The properties of probiotic strains may be investigated in cell cultures (in vitro) utilizing for example peripheral blood mononuclear cells (PBMC), human monocytes, macrophages and dendrite cells. Examples of ex vivo experiments include determination of phagocytosis of neutrophils and monocytes, oxidative burst i.e. superoxide generation of neutrophils and monocytes, NK cell activity, lymphocyte proliferation and production of cytokines by peripheral blood mononuclear cells, monocytes or lymphocytes. In vivo experiments include but are not limited to determination of a response to vaccines (e.g. vaccine specific antibodies or vaccine-specific antibody forming cells), delayed type hypersensitivity and response to attenuated pathogens.
As an alternative to probiotic effects, the peptides or pilus structures of the invention may cause any other effects in a cell or a subject. These other effects may also occur alone or in addition to probiotic effects. Probiotic effect may be a combination of other immunomodulator(s) and peptides or pilus structures.
In the present invention, the subject for treatments or preventions can be any eukaryotic organism, preferably a human being or an animal, especially pets and production animals. The animal may be selected from a group consisting of production animals and pets, such as cows, horses, pigs, goats, sheep, poultry, dogs, cats, rabbits, reptiles and snakes.
Screening Methods
Any polynucleotide of the invention or any fragment thereof can be used for screening bacterial strains having similar pilus structures. In the method of screening bacterial strains, at least one polynucleotide or fragments thereof encoding for pilus peptides or fragments thereof can be determined for example by PCR based methods, such as conventional PCR and sequencing or minisequencing; hybridisation methods, such as Southern or Northern hybridizations; any bioinformatic methods utilizing different programs and parameters; and any antibody based methods by using antibodies against peptides of the invention, flow cytometry, immunoprecipitation co-immunoprecipitation, immunohisto-chemistry, immunofluorescence, ELISA and ELISPOT techniques. Therefore, in a preferred embodiment of the invention new bacterial strains having pilus structures are screened by PCR using primers designed on LGG pilus-genes. In another preferred embodiment of the invention, new bacterial strains having pilus structures are screened by Southern hybridization using amplification products of LGG genes of the invention as probes.
Stringent hybridisation conditions for primers or probes are preferred in the methods for screening homologous sequences or fragments to the polynucleotide of the invention. As used herein “homologous sequence” or “sequence having high identity” refers to a sequence, which may be identical but does not have to be identical to the other sequence. However, the sequences are similar and they have high identity %.
In another preferred embodiment of the invention, new bacterial strains and meta-populations having pilus structures are screened by computational approaches from existing or newly created sequence listings or data-bases.
The sample to be screened can be taken from any organism or any matter, and may be e.g. bacterial culture, tissue sample, blood sample (serum or plasma sample), food sample or environmental sample. In a preferred embodiment of the invention the bacterial strain to be screened is a potential probiotic bacterial strain.
In the present invention, screening can be carried out in vivo, in vitro, in silico or ex vivo conditions.
The present invention is illustrated by the following examples, which are not intended to be limiting in any way.
Example 1
Cloning, Expression, and Purification of Recombinant LGG Pilin Proteins
The coding sequences for SpaA (GG00442), SpaB (GG00443), SpaC (GG00444), SpaD (GG02370), SpaE (GG02371), and SpaF (GG02372), excluding the region encoding the N-terminal signal peptide and the C-terminal cell wall sorting signal (CWSS), were PCR amplified from LGG genomic DNA using pairs of flanking 5′- and 3′-end oligonucleotide primers, one containing an EcoRI site (a Sacl site for GG02372) and another with a Xhol site (see Table 1). The amplified PCR fragments were cleaved with EcoRI (or Sacl for GG02372) and Xhol restriction endonucleases, then ligated into the corresponding sites in the T7-regulated expression vector pET28b+, and the resulting recombinant plasmids (pKTH5319 for GG00442, pKTH5320 for GG00443, pKTH5321 for GG00444, pKTH5324 for GG02370, pKTH5379 for GG02371, and pKTH5341 for GG02372) propagated in the E. coli strain BL21 (DE3) pLysS for the expression of intracellular C-terminal hexahistidine-tagged proteins. Established procedures were employed in all DNA manipulations using standard protocols. For protein production, E. coli was grown at 37° C. to midlog phase in Luria-Bertani medium supplemented with 50 μg/ml kanamycin, protein expression induced for three hours by 1 mM IPTG, the cells harvested by centrifugation, and the cell pellet resupended in lysis buffer [50 mM NaH2PO4(pH 8.0), 300 mM NaCl, 10 mM imidazole]. The cells were disrupted by sonication, clarified by centrifugation, and the cell-free lysates passed through a 0.45 μm filter. The hexahistidine-tagged pilin proteins were then purified by Ni2+-chelating affinity chromatography. Briefly, the cell-free lysates were each applied to a column of Ni-NTA agarose (Qiagen), washed with wash buffer [50 mM NaH2PO4(pH 8.0), 300 mM NaCl, 20 mM imidazole], and the proteins eluted from the column with elution buffer [50 mM NaH2PO4(pH 8.0), 300 mM NaCl, 250 mM imidazole]. Column fractions containing purified proteins were pooled, buffer-exchanged to 10 mM Tris-HCl (pH 8.0) for the SpaA (GG00442), SpaC (GG00444), SpaD (GG02370), SpaE (GG02371), and SpaF (GG02372) proteins and to 50 mM sodium acetate (pH 5.1) for the SpaB (GG00443) protein using a BioRad EconoPac 10 DG desalting column, and concentrated using a 30 kDa Microsep filter (Pall Life Sciences). The purity of the recombinant pilin proteins were monitored by SDS-PAGE and the protein concentrations estimated by A280 measurements.
Example 2
Generation of Recombinant LGG Pilin Protein-Specific Polyclonal Antibodies
Rabbit polyclonal antibodies specific for the SpaA (GG00442), SpaB (GG00443), SpaC (GG00444), SpaD (GG02370), SpaE (GG02371), and SpaF (GG02372) pilin proteins were produced according to the immunization protocol described by Johnston B. A. et al. (1991, Laboratory of Animal Science 41: 15-21). In brief, a subcutaneous (SC) injection (1 ml) of a 1:1 mix of 400 μg purified recombinant pilin protein in Freud's complete adjuvant was initially administered, followed by three sets of booster injections (SC) of 1:1 mixes of 200 μg protein in Freud's incomplete adjuvant at three-week intervals. The final blood collection was made two weeks after the last booster injection. The preparation of anti-sera from the blood was carried out using standard protocols.









TABLE 1





Gene
Forward oligonucleotide primer*
Reverse oligonucleotide primer**


















SpaA (GG00442)
5′-TCGGGTTCAGAATTCTACGAATGATACGAC
5′-TGCCAGTACCACCCTCGAGTGGCAGAATAC



 


SpaB (GG00443)
5′-GCAGACACAGAATTCAACTGTGCCGACC
5′-CAACTGTATCACCCTCGAGTGGCAACAATTGACG


 


SpaC (GG00444)
5′-CAGTTCAGTTGTGAATTCCACTGATAACATTCG
5′-AGCCCTGACCACCCTCGAGCGGCAAAATTGC


 


SpaD (GG02370)
5′-ACCCGTACAGAATTCGACAACGACTGTG
5′-GTCCGATTCCGCCCTCGAGCGGCAATAATTG


 


SpaE (GG02371
5′-CCACATTGGGTTCAGAATTCTGATCAAACTG
5′-TGCGCCAATCGGACTCGAGCGGCAAATAAC


 


SpaF (GG02372)
5′-GCAAATTGGCAGGAGCTCGGTCCCGGTAG
5′-CCGCTACCACCCTCGAGCGGTAGGAGTG





and


**Restriction endonucleases, EcoRI and SacI in the forward and XhoI in the reverse oligonucleotide primers, are underlined and in boldfaced type






Example 3
Prediction of Protein-Encoding Sequences by Bioinformatic Methods
Prediction of protein-encoding sequences was accomplished using Glimmer3 (Delcher A. L. et al. 2007, Bioinformatics. 23:673-679) and analysing the completed genome sequence of LGG. Glimmer3 was applied using the iteration-mode script (g3-iterated.csh) with following modifications to default parameters: minimum gene length (150 bp) and maximum over lap (50 bp). Start sites of the initial predictions were rectified using BLAST (Altschul S. F. et al. 1997, Nucleic Acids Res. 25(17):3389-3402) and searching for putative ribosomal binding sites. The Glimmer3 predictions for GG00441, GG00442, GG00443, GG00444, GG02369, GG02370 and GG02371 were accepted as such, whereas the prediction of GG02372 was manually rectified to start 21 bp more downstream. Rho-dependent stops sites were predicted using TransTermHP (Kingsford C. L. et al. 2007, Genome Biol. 8:R22.) which showed that GG00441, GG00442, GG00443 and GG00444; GG02369, GG02370, GG02371 and GG02372 are transcribed as single transcript and thus form own operons.
Annotations were obtained by converting the predicted protein-encoding sequences to protein sequences and by performing a homology search against the public sequence database (Wheeler D. L. et al. 2008, Nucleic Acids Res. 36: D13-21). Annotations were accepted only from those sequences of which local alignments between the query had >=35% amino acid identity and covered >=80% of the sequence of the subject. Based on this search, GG00441 and GG02369 were annotated as sortase-enzymes; GG00444 as a von Willebrand factor domain containing protein; GG02370 and GG02371 as a conserved hypothetical protein and GG02372 as an outer membrane protein. No annotations were obtained for GG00442 and GG00443.
Further annotation and information about the sequences were obtained by integrating information of InterPro and COG analyses (Mulder N. J. et al. 2007, Nucleic Acids Res. 35:D224-D228; Tatusov R. L. et al. 2000, Nucleic Acids Res. 28:33-36) and doing specific domain analyses. The specific domain searches were performed using Hmmsearch tool of the Hmmer-package and using sortase associated domain models, obtained from public databases of PFAM and TIGRFAM (Finn R. D. et al. 2008, Nucleic Acids Res. 36:D281-288; Haft D. H. et al. 2003, Nucleic Acids Res. 31:371-373). Following models were used to search for sortase-recognition sites: TIGR01167, TIGR03063, TIGR03065, TIGR03068 and PF00746 and the following models to search for sortases: TIGR01076, TIGR03064, PF04203 and PF07170. Both fs- and Is-models of the PFAM models were searched and the full length models of the TIGR models. Both search-types, the sequence and the domain search, were used. Matches scoring higher than the recorded trusted cut-off given by the database were considered significant. In cases, where the sequence-model was significant, every domain hit was accepted. These searches indicated that GG00441 and GG02369 are sortase-enzymes and that GG00442, GG00443, GG02370 and GG02372 contain the sortase-recognition site, thus being their likely substrates. Sortase-recognition sites were also searched for using regular expression searches (with the patterns LPXTG and LVNTG (Ton-That H et al. 2004, Mol Microbiol. 53:251-261), where X denotes any amino acid) revealing following matches: GG00442 and GG00443, GG00444, GG02370, GG02371 and GG02372. E-boxes were searched using YXXXETXXPX(G/N)X as the regular expression that was derived from the original YXLXETXAPXGY-pattern (Ton-That H et al. 2004, Mol Microbiol. 53:251-261). The E-box search revealed hits on GG00442, GG00443, GG00444, GG02370 and GG02372 verifying the likeliness of these sequences to be sortase-substrates. The existence of possible secretion signals was tested using SignalP3-tool using both the hidden Markov model and the neural network methods. In all cases both methods predicted that the peptide sequences of GG00441, GG00442, GG00443, GG02370, GG02371 and GG02372 contained a signal suitable for the secretion.
Example 4
Bioinformatic Screens on Public Databases
Peptide sequences, fragments thereof, variants thereof, polynucleotide sequences, fragments thereof or variants thereof according to the present invention can be used for performing computational searches against public and private sequence collections and thereby for detecting bacterial strains comprising similar peptide sequences, polynucleotide sequences or pilus structures. Another preferred use of bioinformatic screening methods is for selecting bacterial communities enriched by the peptide sequences, polynucleotide sequences or pilus structures. Bioinformatic searches offer a plausible method for the detection of strains having sequences, which are in public sequence collections but have never been annotated or curated by an expert.
Bioinformatic searches are performed using algorithms such as BLAST (Altschul S. F. et al. 1997, Nucleic Acids Res. 25(17): 3389-3402) or FASTA (Pearson W R, 1990, Methods Enzymol 183:63-98) (preferably default parameters are used). BLAST and FASTA algorithms are used to compare the selected sequences against a set of other sequences and to report statistically significant hits. Peptide sequences, polynucleotide sequences or pilus structures are searched from, for example, the following public sequence collections offered by the National Center for Biotechnology Information (NCBI): non-redundant protein sequences, environmental samples, whole-genome shotgun read and Genomic survey sequences; or preferably from a private sequence collection generated, for example, using high-throughput sequencing methods.
The peptide sequences of seq id no 1-8 or fragments thereof are used to screen for significant matches of peptides by performing a standard Blast search against the non-redundant protein sequence collection of NCBI. When a significant peptide match is found, a bacteria encoding this peptide of interest is classified as a putative probiotic strain or as a putative pathogen, against which the peptide is effective.
Example 5
Atomic Force Microscopy Showing LGG Pili
LGG strain was grown on a MRS (LabM) agar plate at 37° C. for 20 hours anaerobically. Bacterial cells were diluted in sterile water, fixed to Mica i slide and air dried. Both topographic and phase contrast figures of bacteria were obtained by Nanoscope IIIa Multimode AFM (Atomic force microscope, Digital Instruments, Santa Barbara)—microscope and J scanner (FIG. 4).
Example 6
Binding of Recombinant LGG Proteins to Human Intestinal Mucus as Assessed by Non-Quantitative ELISA-Assay
The binding of recombinant hexahistidine-tagged SpaA, SpaB, SpaC, SpaD, SpaF pilin proteins to human intestinal mucus was assessed in vitro. Resected human intestinal tissue was used as a source of mucus. The use of resected human intestinal tissue was approved by the joint ethical committee of the University of Turku and Turku University Central Hospital (both in Turku, Finland) and informed written consent was obtained from the patients. The mucus was isolated from the healthy part of tissue obtained from patients undergoing colonic surgery e.g. due to colorectal cancer. The processing of intestinal tissue and the isolation of mucus was done as described previously (Vesterlund S. et al 2005; Res Microbiol. 156(2):238-244; J Microbiol Methods 60(2):225-233?). Mucus was passively immobilized on a polystyrene microtiter plate (Maxisorp, Nunc, Denmark) by overnight incubation at 4° C. The wells were washed three times with phosphate-buffered saline (PBS; pH 7.2) and blocked with 0.5% (w/v) bovine serum albumin (Sigma A7030) in PBS for 1 h at room temperature. The blocking solution was removed and 0.5 or 0.05 nmol of the hexahistidine-tagged pilin proteins in BSA-PBS was added followed by 1 h incubation at 37° C. After incubation and washes the bound proteins was detected by enzyme-linked immunosorbent assay. The pilin proteins was detected by a mouse Tetra-His antibody (Qiagen, 34670) and a goat anti-mouse IgG Fab specific alkaline phosphatase conjugate (Sigma, A1293) as the secondary antibody. Dilutions 1:2000 and 1:5000 (v/v) were used for the primary and secondary antibodies, respectively. The substrate 4-nitrophenyl phosphate disodium salt (pNPP, Sigma, A7030) in diethanolamine-MgCl-buffer (Reagena, 170057, Finland) was added in concentration of 2 mg/ml and the color development was measured after 1 h at 405 nm. Results are average ±stdev from three parallel measurements (FIGS. 5a-b).
Example 7
Extraction of Cell Wall-Associated Pilus Proteins and Western Blot
Fresh 10 h cultures of LGG and LC705 (negative control) cells in MRS (LabM) were inoculated (1%) in mTSB medium (15 g/l TSB medium, BD Biosciences) enriched with 20 g/l Bacto peptone (Difco), or MRS medium supplemented with 0.6% ox gall bile (Sigma) and cultivated at 37° C. Growth was monitored by measuring optical density (OD600) and cells in stationary growth phase were collected by centrifugation.
The fractionation of the bacterial cells was done essentially as described elsewhere (Åvall-Jääskeläinen S. et al. 2003, Appl Environ Microbiol 69:2230-2236). Briefly, the bacteria (109 cfu) were washed once with PBS and homogenized by beating three times for two minutes with glass beads in a cell mill (Bühler Vibrogen-Zellmühle). The bacterial homogenates were resuspended in 500 μl PBS and centrifuged five minutes at 1,000 g. The supernatant was centrifuged at 16,000 g for 30 minutes at +4° C. to collect the cell walls. The resulting pellets were resuspended in 50 μl of 50 mM Tris-Cl (pH 8.0) supplemented with 5 mM MgCl2, 5 mM CaCl2, 10 mg/ml lysozyme, and 42 U/ml mutanolysin. The resuspended cell wall pellets were incubated 3 hours at 37° C. to release the cell wall associated polypeptides.
The enzymatically treated cell wall fractions were run on a 4-15% gradient gel (Bio-Rad) and transferred to a Immobilon-P PVDF membrane (Millipore). The membrane was subjected to Western analysis with the ECL AdvanceTM Western Blotting Detection Kit (Amersham) according to manufacturer's instructions. The SpaA, SpaB, and SpaC pilin protein-specific polyclonal primary antibodies were diluted 1:25,000, and the Goat Anti-Rabbit IgG (H+L)-HRP-Conjugate (Bio-Rad) secondary antibody was diluted 1:100,000.
The pili in gram-positive bacteria are composed pilin subunits covalently linked to one another. The monomeric pilin subunits are added to the growing pili one by one by the action of sortases, and as a consequence, at a given time point each individual cell carries pili of different lengths on its surface (Scott J. R. and Zahner D. 2006, Mol Microbiol 62:320-330). Thus, a classical way to show the existence of pili is to subject a mutanolysin/lysozyme treated cell wall fraction to Western analysis: if pili are present, a high molecular weight ladder (HMW) will be detected on the blot, and in many instances also a pilin monomers will be observed (Scott J. R. and Zahner D. 2006, Mol Microbiol 62:320-330). The presence of SpaA and SpaC containing pili in LGG is clearly evident from FIGS. 6a and 6b, since both the monomeric SpaA and SpaC pilin subunits and HMWs can be identified from the LGG cell wall extracts using SpaA and SpaC-specific antibodies, whereas LC705 cells are deficient of SpaA and SpaC moieties. The exposure time needed to record chemiluminescent signal from the SpaC blot was 60 seconds, whereas exposure time of 1 second was sufficient for the SpaA blot, implying the SpaA to be present at higher numbers in the pili as SpaC. This difference in relative numbers might suggest the SpaA to be the shaft forming pilin subunit, whereas SpaC could serve as a pilus tip adhesin. Also of notice is that pili are found in LGG cells grown in a medium supplemented with bile, indicating that pili might be expressed in the human gastrointestinal tract.
Example 8
Screening of New Probiotic Strains Having Pili Structures by PCR
Lactobacilli are grown anaerobically in MRS broth at +37° C. for 10 hours. The genomic DNA is isolated as follows. 1 ml of the culture is centrifuged at 14 000 g for 2 min. The collected cells are resuspended in 480 μl of 50 mM EDTA, 100 μl of 50 mg/ml lysozyme (Amresco, Solon, Ohio, USA) and 20 μl of 50 U/μl mutanolysine (Sigma) is added and the mixture is incubated at 37° C. for 1 h. The mixture is centrifuged for 2 min at 14 000 g, the supernatant is discarded and the bacterial pellet is extracted with a Wizard® Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. The purified DNA is suspended in 200 μl of Tris-EDTA (TE) buffer. About 200 ng of genomic DNA is used as a template in PCR reaction. PCR is performed using Dynazyme polymerase (Finnzymes, Espoo, Finland) and oligonucleotide primers based on sequences GG00442, GG00443, GG00444 and GG02370, GG02371, GG02372 genes. The PCR reaction is performed with a PCT-200 apparatus (MJ Research, Waltham, Mass., USA) and contains 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl and 0.1% Triton-X 100 (pH 8.8). The primers are used at 1-μM and the deoxynucleotides at 200-μM concentrations. Initial denaturation is at 94° C. for 2 min. The first cycle is 1 min each at 95° C., 65° C. and 72° C., the next five cycles are 1 min each at 95° C., 60° C. and 72° C., and the last 25 cycles are 1 min each at 95° C., 55° C. and 72° C. To terminate cycling the reaction mixture is maintained at 72° C. for 5 min and at 4° C. for 15 min. The amplified DNA bands are separated in 0.7% agarose gel by gel electrophoresis.









TABLE 2





Gene
Forward oligonucleotide primer
Reverse oligonucleotide primer


















SpaA (GG00442)
5′-TCTCGGGTTTAATGGCACTC
5′-TCTGTATTGGCAGCAGCATC



 


SpaB (GG00443)
5′-TCCTTCCGTCCGTTAGTGAT
5′-CGTTTGTGGCAACAATTGAC


 


SpaC (GG00444)
5′-CCAAATTGGCAACAGACCTT
5′-GCCATCTGGTGCTTTTGTTT


 


SpaD (GG02370)
5′-CGGACGCCTTTTACCAATTA
5′-AACAGGTTTCGTACCGCATC


 


SpaE (GG02371)
5′-TATGACGCGTAAGCAAGCAC
5′-TGGCCGTCAATTAACACAAA


 


SpaF (GG02372)
5′-CTACCGGAGCATGTCGAGTT
5′-GGCCATTTTCATCAGTCGTT









Example of the primers for amplification of pili genes are shown in Table 2, but are not limited to those. The sizes of the amplified PCR-products using L. rhamnosus GG DNA as a template and Table 2 primers are 780 bp, 612 bp and 801 bp for SpaA, SpaB, SpaC, respectively, and for SpaD, SpaE and SpaF 688 bp, 705 bp and 799 bp.
Example 9
Screening of New Probiotics Having Pili Genes by Southern Hybridization
New probiotic strains having pili structures are screened by Southern hybridization using LGG amplification products from Example 8 as probes. Hybridization conditions can be adjusted to stringent, enabling probe hybridization only to identical sequences, or to low stringent, allowing some amount of sequence discrepancy. The PCR amplification products of SpaA, B, C, D, E and F are purified in NuSieve low melt agarose (FMC Bioproducts, Rockland, Me., USA) and labelled with DIG-system (Roche Diagnostics). The total DNA of the bacterial strains is digested with EcoRI and the resulting fragments are separated in 0.7% agarose gel. The DNA fragments in agarose are blotted onto nylon membranes and hybridized according to standard procedure of DIG-system. Stringent hybridization is performed at 68° C., washes are twice in 2×SSC-0.1% SDS at room temperature, and twice in 0.1×SSC-0.1% SDS for 15 minutes at 68° C. Hybridization with lower stringency is performed at 60° C., and the last two washes are in 0.5×SSC-0.1% SDS at 50° C. for 15 minutes. Hybridization is detected with alkaline-phosphatase-conjugated antibody and NBT/BCIP color reaction (DIG-system, Roche).
Example 10
Immunomodulation by Purified LGG Pilus Proteins
Human macrophages are isolated from blood of healthy volunteers (buffy coat fraction) as documented previously (Miettinen M. et al. 2000, J Immunol 164:3733-3740; Miettinen M. et al. 2008, J Leuk Biol 84:1092-1100). Essentially, this is done using freshly collected, leukocyte-rich buffy coats from 4 healthy blood donors (supplied by the Finnish Red Cross Blood Transfusion Service, Helsinki Fla.) and isolating peripheral blood monocuclear cells (PBMCs) by Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala SE) gradient centrifugation. Monocytes are purified from PBMCs by adherence on six-well plastic plates (Falcon Becton Dickinson, Franklin Lakes N.J., US) and cultured for 7 days in macrophage—serum-free medium (Gibco Invitrogen, Grand Island N.Y., US) in the presence of 10 ng/ml recombinant human (rh) GM-CSF (Leucomax, Schering-Plough, Innishannon, IRL) to obtain macrophages. Macrophages are incubated at a concentration of approximately 4 million cells per well in a 6-well microtiter plate and stimulated with an equivalent number of live bacteria (LGG and Streptococcus pyogenes T1M1) or approximately 3, 100, 3000 or 10000, etc., fmol of purified His-Tag labelled LGG proteins SpaA and SpaC. After incubation for 6 h and 24 h, the modulation of the amounts of immune markers or activation of signalling pathways or receptor expression is determined as described previously (Miettinen M. et al. 1996, Infec immunol 64:5403-5405; Miettinen M. et al. 2000, J Immunol 164:3733-3740; Miettinen M. et al. 2008, J Leuk Biol 84:1092-1100).
Typically, cells of the probiotic LGG and the pathogen S. pyogenes T1M1 show immunomodulatory activities and induce a specific Th1-like response in PBMCs or macrophages (Miettinen M. et al. 2000, J Immunol 164: 3733-3740; Veckman V. et al. 2003, J Leuk Biol 74:395-402). Remarkably, the purified LGG pili proteins also induce a response in macrophages, demonstrating their functionality in immunomodulation. Moreover, these experiments show that the LGG pili proteins signal to human host cells.
Example 11
Competition Assay with LGG Pilus Proteins
The processing of intestinal tissue and the isolation of mucus was done as described in Example 6.
The competition assay is carried out according to Vesterlund S. et al. 2006 (Microbiology 152(6):1819-1826). Mucus (Sigma), at a concentration 0.5 mg/ml, is passively immobilized on a polystyrene microtiter plate (Maxisorp, Nunc, Denmark) by overnight incubation at 4° C. The wells are washed three times with phosphate-buffered saline (PBS; pH 7.2) and blocked with 0.5% (w/v) bovine serum albumin (Sigma A7030) in PBS for 1 h at room temperature. The blocking solution is removed and 5.0 or 0.05 nmol of the histidine-tagged pilin protein or pili structure in BSA-PBS is added followed by 1 h incubation at 37° C. The unbound pili proteins or pili structures are washed away as described above. The pathogenic bacterial cells are added to the wells in a volume of 100 μl, four parallel wells are used in each experiment. Bacteria are allowed to adhere for 1 h at 37° C. and the wells are washed three times with 250 μl PBS to remove the nonadherent bacteria. The bacteria bound to mucus are released and the genomic DNA extracted by Wizard Genomic kit (Promega). The number of bacteria in a sample is determined by quantitative PCR using species specific primers and SYBR Green detection. The adhesion ratio (%) of bacteria is calculated by comparing the number of adhered bacteria to that of added bacteria. LGG pili proteins and pili structures inhibit adhesion of pathogenic bacterium to the mucus.

## Claims

1. An isolated peptide comprising a sequence having at least 94% sequence identity with SEQ ID NO: 4 (GG00444).

2. The peptide according to claim 1, which is a recombinant peptide.

3. The peptide according to claim 1, which is from bacteria.

4. The peptide according to claim 3, which is from Lactobacillus rhamnosus.

5. The peptide according to claim 4, which is from Lactobacillus rhamnosus GG (LGG) strain.

6. The peptide according to claim 1, which binds to the gastrointestinal tract.

7. The peptide according to claim 1, which binds to the mucus.

8. A product comprising the peptide according to claim 1.

9. The product according to claim 8, which is a food or feed product.

10. The food product according to claim 9, wherein the food product is selected from the group consisting of a dairy products, a bakery product, a chocolate, a confectionary, a sugar confectionary, a gum confectionary, a cereal product, a snack, a berry based product, a fruit based product, a drink and a beverage.

11. The food product according to claim 10, wherein the food product is selected from the group consisting of a milk product, a sour milk product, a yogurt, a cheese, a spread, a milk powder, a children's food, a baby food, a toddler's food, an infant formula, a juice and a soup.

12. A pharmaceutical composition comprising the peptide according to claim 1.

13. A method of treating a disorder selected from the group consisting of diarrhea, arterial hypertension, vascular disease, allergy, cancer, atopic disease, viral disease, infectious disease, urinary tract infection, respiratory infection, dental caries, irritable bowel syndrome, inflammatory bowel disease, mucosal inflammation, gut permeability disorder, obesity, metabolic syndrome, oxidative stress and abdominal pain in a subject in need thereof, comprising administering the peptide of claim 1 to the subject.

14. A method of reducing or inhibiting the adhesion of pathogenic bacteria to the gastrointestinal tract, to the epithelium or to the mucus of a subject, comprising administering the peptide according to claim 1 to the subject.

15. The peptide according to claim 1 comprising a sequence having at least 95% sequence identity with SEQ ID NO:4 (GG00444).

16. The peptide according to claim 1, which is a purified peptide.