COMPOSITIONS COMPRISING RECOMBINANT PROBIOTIC BACTERIA AND METHODS OF USE THEREOF

Abstract

The invention features probiotic bacteria expressing Clostridium difficile SlpA, or fragment thereof, and its use for the treatment or prevention of Clostridium difficile infection and gut colonization.

Claims

1. An isolated polypeptide comprising a bacterial secretion signal, a C. difficile SlpA variable domain, and a Lactobacillus SlpA cell wall binding domain.

2. The isolated polypeptide of claim 1, wherein the SlpA variable domain has the amino acid sequence: TABLE-US-00010 AAPVFAATTGTQGYTVVKNDWKKAVKQLQDGLKDNSIGKITVSFNDGVVG EVAPKSANKKADRDAAAEKLYNLVNTQLDKLGDGDYVDFSVDYNLENKII TNQADAEAIVTKLNSLNEKTLIDIATKDTFGMVSKTQDSEGKNVAATKAL KVKDVATFGLKSGGSEDTGYVVEMKAGAVEDKYGKVGDSTAGIAINLPST GLEYAGKGTTIDFNKTLKVDVTGGSTPSAVAVSGFVTKDDTDLA

3. The isolated polypeptide of claim 1, wherein the SlpA cell wall binding domain is a Lactobacillus acidophilus or Lactobacillus casei SlpA cell wall binding domain.

4. The isolated polypeptide of claim 1, wherein the SlpA cell wall binding domain has the amino acid sequence: TABLE-US-00011 SNTNGKSATLPVVVTVPNVAEPTVASVSKRIMHNAYYYDKDAKRVGTDSV KRYNSVSVLPNTTTINGKTYYQVVENGKAVDKYINAANIDGTKRTLKHNA YVYASSKKRANKVVLKKGEVVTTYGASYTFKNGQKYYKIGDNTDKTYVKV ANFR

5. The isolated polypeptide of claim 1, wherein the bacterial secretion signal is a Lactococcus, Lactobacillus, Lactobacillus acidophilus, or Lactobacillus casei secretion signal.

6. The isolated polypeptide of claim 1, wherein the bacterial secretion signal has the amino acid sequence: TABLE-US-00012 MKKNLRIVSAAAAALLAVAPVAASAVSTVSA

7. The isolated polypeptide of claim 1, having the amino acid sequence: TABLE-US-00013 MKKNLRIVSAAAAALLAVAPVAASAVSTVSAAAPVFAATTGTQGYTVVKN  50 DWKKAVKQLQDGLKDNSIGKITVSFNDGVVGEVAPKSANKKADRDAAAEK 100 LYNLVNTQLDKLGDGDYVDFSVDYNLENKIITNQADAEAIVTKLNSLNEK 150 TLIDIATKDTEGMVSKTQDSEGKNVAATKALKVKDVATEGLKSGGSEDTG 200 YVVEMKAGAVEDKYGKVGDSTAGIAINLPSTGLEYAGKGTTIDFNKTLKV 250 DVTGGSTPSAVAVSGFVTKDDTDLASNTNGKSATLPVVVTVPNVAEPTVA 300 SVSKRIMHNAYYYDKDAKRVGTDSVKRYNSVSVLPNTTTINGKTYYQVVE 350 NGKAVDKYINAANIDGTKRTLKHNAYVYASSKKRANKVVLKKGEVVTTYG 400 ASYTEKNGQKYYKIGDNTDKTYVKVANFR*

8. An isolated nucleic acid molecule encoding the polypeptide of claim 1.

9. The isolated nucleic acid molecule of claim 8, comprising a sequence optimized for expression in Lactococcus, Lactococcus lactis, Lactobacillus, Lactobacillus acidophilus, or Lactobacillus casei.

10. The isolated nucleic acid molecule of claim 8, having the nucleic acid sequence: TABLE-US-00014 GGATCCATGAAGAAAAATTTAAGAATCGTTAGCGCTGCTGCTGCTGCTTT ACTTGCTGTTGCTCCAGTTGCTGCTTCTGCTGTATCTACTGTTAGCGCTG CTGCACCTGTATTTGCTGCAACCACTGGTACACAAGGCTATACGGTGGTT AAGAATGATTGGAAAAAGGCTGTCAAACAATTACAAGATGGACTTAAAGA TAATAGTATTGGTAAGATTACGGTCAGTTTCAATGATGGTGTGGTAGGAG AAGTAGCACCTAAATCAGCGAATAAGAAAGCAGATCGAGATGCAGCCGCA GAAAAGTTGTATAATCTTGTAAATACACAATTAGACAAATTAGGCGATGG CGATTATGTAGATTTTTCTGTTGATTACAATCTAGAGAATAAGATTATCA CCAATCAAGCCGATGCCGAAGCTATTGTTACTAAATTGAATTCGTTAAAT GAAAAGACGCTAATTGATATTGCAACTAAAGATACGTTTGGAATGGTGTC TAAAACGCAGGATTCTGAAGGAAAGAATGTTGCGGCAACAAAAGCGTTAA AAGTAAAAGATGTGGCAACTTTTGGCTTAAAGAGTGGAGGTAGTGAAGAT ACCGGATATGTTGTCGAAATGAAAGCGGGTGCTGTTGAAGATAAGTATGG TAAAGTAGGTGATTCTACAGCTGGTATTGCAATCAATCTTCCATCAACAG GTTTAGAATATGCAGGCAAAGGAACAACTATTGATTTCAACAAAACCCTT AAAGTTGATGTAACTGGTGGTAGTACACCGAGTGCAGTTGCCGTAAGTGG TTTGTGACTAAAGATGATACAGATTTAGCATCAAATACTAATGGTAAGTC AGCTACTTTGCCAGTAGTTGTTACTGTTCCTAATGTTGCTGAGCCAACTG TAGCCAGCGTAAGCAAGAGAATTATGCACAACGCATACTACTACGACAAG GACGCTAAGCGTGTTGGTACTGACAGCGTTAAGCGTTACAACTCAGTAAG CGTATTGCCAAACACTACTACTATCAACGGTAAGACTTACTACCAAGTAG TTGAAAACGGTAAGGCTGTTGACAAGTACATCAACGCTGCAAACATCGAT GGTACTAAGCGTACTTTGAAGCACAACGCTTACGTTTACGCATCATCAAA GAAGCGTGCTAACAAGGTTGTATTGAAGAAGGGTGAAGTTGTAACTACTT ACGGTGCTTCATACACATTCAAGAACGGCCAAAAGTACTACAAGATCGGT GACAACACTGACAAGACTTACGTTAAGGTTGCAAACTTTAGATAATAAAG ATCTTCGAATTCCCGCGGCCGC

11. A vector comprising the nucleic acid molecule of claim 8.

12. The vector of claim 11, wherein the vector is a Lactococcus, Lactobacillus, Lactobacillus acidophilus, or Lactobacillus casei expression vector, and wherein the vector comprises a Lactococcus or Lactobacillus origin of replication.

13. (canceled)

14. The vector of claim 11, wherein the vector is selected from the group consisting of pMGM10, pMGM11, pTRK848, or pTRK882.

15. The vector of claim 11, comprising a first sequence identical to a sequence of a first fragment in a Lactobacillus genome, wherein the first fragment is located at the 5′ or 3′ terminus of a thyA gene, or within the thyA gene of Lactobacillus, and a second sequence identical to a second fragment in the Lactobacillus genome, wherein the second fragment is located at the 5′ or 3′ terminus of the thyA gene.

16. (canceled)

17. (canceled)

18. An isolated cell comprising the isolated nucleic acid of claim 8 or the vector of claim 11.

19. (canceled)

20. The isolated cell of claim 18, wherein the cell is selected from the group consisting of a Lactococcus, Lactococcus lactis, Lactobacillus, Lactobacillus acidophilus, or Lactobacillus casei cell.

21. The isolated cell of claim 18, wherein the isolated nucleic acid molecule is integrated into the chromosome of the isolated cell.

22. A method of treating or preventing Clostridium difficile infection in a subject, the method comprising administering Lactococcus or Lactobacillus expressing a chimeric SlpA polypeptide to the gut of the subject, wherein the chimeric SlpA polypeptide comprises a bacterial secretion signal, C. difficile SlpA variable domain, and Lactobacillus SlpA cell wall binding domain, thereby treating or preventing Clostridium difficile infection in the subject.

23. (canceled)

24. (canceled)

25. The method of claim 22, wherein the subject has undergone or is undergoing treatment with antibiotics, wherein the antibiotic is one or more of a cephalosporin, metronidazole, fluoroquinolone, moxifloxacin, vancomycin, and fidaxomycin.

26. (canceled)

27. (canceled)

28. A composition comprising an effective amount of Lactococcus or Lactobacillus expressing a chimeric SlpA polypeptide, wherein the chimeric SlpA polypeptide comprises a bacterial secretion signal, C. difficile SlpA variable domain, and Lactobacillus SlpA cell wall binding domain.

29. (canceled)

30. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIGS. 1A-1E depict the generation of plasmid constructs and the biological sequences used. FIG. 1A depicts the construction of plasmid pMGM10. FIG. 1B depicts the construction of plasmid pMGM11. FIG. 1C depicts the codon-optimized slpA chimera nucleic acid sequence that was cloned into the plasmids pMGM10 and pMGM11. FIG. 1D depicts the amino acid sequence of the SlpA chimera polypeptide. FIG. 1E depicts the promoter sequences used in constructing the plasmids: fructooligosaccharides (Fos) promoter in plasmid pTRK848/pMGM11 and phosphoglycerate mutase (pgm) promoter pTRK882/pMGM10.

[0063] FIG. 2 are immunofluorescence images showing the surface expression of C. difficile SlpA in Lactobacillus casei compared to pre-immune serum and vector only controls.

[0064] FIG. 3 is a graph showing mean time to death in Syrian Golden hamsters challenged with virulent Clostridium difficile and treated with recombinant Lactobacillus expressing a chimeric SlpA (CHI) or Lactobacillus carrying an empty vector (EV) or undergoing antibiotic treatment (Antibiotics), compared to animals that were unchallenged (Unchallenged).

[0065] FIG. 4 provides a schematic diagram of a plasmid encoding SlpA chimeric protein. The internal fragment of a thyA directs the chimera integration into the genome and concomitantly insertionally inactivates the thyA, which encodes an essential enzyme thymidylate synthase (ThyA). Lactobacillus casei and Lactobacillus acidophilus specific constructs were made. A gene catP encodes an enzyme Chloramphenicol acetyltransferase, which is an effector of chloramphenicol resistance in bacteria. A region including an oriR gene and a repA.sup.ts gene constitutes a temperature sensitive broad host replicon based on a pWVO1 plasmid. YtvA is an anaerobic fluorescent protein used to confirm transformation of Lactobacillus sp and monitor colonization.

[0066] FIG. 5 provides a schematic diagram of a plasmid encoding SlpA chimeric protein. A 5′ seq and a 3′ seq are two fragments located at 5′ and 3′ terminus of a thyA gene, respectively, and direct the double homologous recombination to replace the thyA gene in the bacterial chromosome with a Slp A chimeric protein encoding sequence and a YtvA fluorescent reporter encoding sequence. Lactobacillus casei and Lactobacillus acidophilus specific constructs were made. The thyA gene encodes an essential enzyme thymidylate synthase (ThyA). The YtvA is an anaerobic fluorescent protein used to confirm transformation of Lactobacillus sp and monitor colonization. A gene catP encodes an enzyme Chloramphenicol acetyltransferase, which is an effector of chloramphenicol resistance in bacteria. A region including an oriR and a repA.sup.ts genes constitutes a temperature sensitive broad host replicon based on pWVO1 plasmid.

[0067] FIG. 6 provides a partial sequence of a plasmid used for a thyA directed integration of a SlpA chimeric protein encoding sequence into a bacterial genome and concomitantly insertionally inactivates thyA in Lactobacillus casei.

[0068] FIG. 7 provides a partial sequence of a plasmid used for double homologous recombination-based system for replacing a thyA gene in a bacterial chromosome with a Slp A chimeric protein encoding sequence and a YtvA fluorescent reporter encoding sequence in Lactobacillus casei.

[0069] FIG. 8 provides a partial sequence of a plasmid used for a thyA directed integration of a SlpA chimeric protein encoding sequence into a bacterial genome and concomitantly insertionally inactivates thyA in Lactobacillus acidophilus.

[0070] FIG. 9 provides a partial sequence of a plasmid used for double homologous recombination-based system for replacing a thyA gene in a bacterial chromosome with a Slp A chimeric protein encoding sequence and a YtvA fluorescent reporter encoding sequence in Lactobacillus acidophilus.

DETAILED DESCRIPTION OF THE INVENTION

[0071] The invention provides a probiotic bacteria (e.g., Lactobacillus, Lactococcus) expressing the Clostridium difficile surface protein SlpA, or a fragment or chimeric polypeptide thereof, and its use for colonizing the gut or digestive tract of a subject. The invention also provides a method of treating or preventing Clostridium difficile infection and colonization. The invention features use of the probiotic bacteria of the invention for the replacement of a gut microbiome associated with disease.

[0072] The invention is based, at least in part, on the discovery that expression of Clostridium difficile SlpA (e.g., chimeric SlpA), or fragment thereof, in Lactobacillus or Lactococcus is effective for colonizing the gut with the recombinant bacteria. It was also found that recombinant Lactobacillus protected the gut from virulent Clostridium difficile challenge. These findings indicate that administration of gut recombinantly expressing Clostridium difficile SlpA, or fragment thereof can be used to treat or prevent Clostridium difficile infection and colonization.

Clostridium difficile

[0073] Clostridium difficile is a gram-positive, anaerobic, spore-forming bacterium, and causes the antibiotic-associated diarrheal disease, C. difficile infection (CDI). It is also a leading cause of bacterial healthcare-associated infections in hospitals in the United States. Like many enteric pathogens, Clostridium difficile must associate with the intestinal mucosa to begin the process of host colonization.

[0074] Multiple C. difficile adhesins have been described, including the flagellin FliC, the flagellar cap protein FliD, fibronectin-binding proteins, a heat-shock protein, GroEL, the surface associated, heat-shock-induced adhesin, Cwp66, and the surface layer protein, SlpA. SlpA contains two biologically distinct entities, the high-molecular weight (HMW) and the low molecular weight (LMW) subunits, which are derived via Cwp84-mediated cleavage of a single precursor protein, and assemble on the bacterial surface into a paracrystalline lattice. The two subunits associate with high affinity through the N-terminus of the HMW protein and the C-terminus of the LMW protein.

[0075] Cwp66 and SlpA are encoded by two genes in a 17-gene cluster that encodes many surface-associated proteins. Such S-layer proteins (SLPs) provide structural integrity to the cells, act as molecular sieves, bind to host tissues and extracellular matrix proteins, and contribute to host cell adhesion and immune evasion.

Surface-Layer Protein A (SlpA)

[0076] Many gram-positive bacteria including C. difficile possess a surface-layer that covers the peptidoglycan-rich cell wall. This “S-layer” consists of many proteins that form a paracrystalline lattice around the bacterial cell. The most abundant S-layer protein in C. difficile is SlpA, a major contributor of adhesion to, and colonization of, intestinal epithelial cells. (Merrigan et al. PLoS ONE 8(11): e78404). Individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduce C. difficile attachment. SlpA-mediated adherence-interference correlates with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA is derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.

Therapeutic Compositions

[0077] The invention features probiotic bacteria expressing Clostridium difficile SlpA or fragment thereof (e.g., chimeric SlpA). In particular, Clostridium difficile SlpA, or fragment thereof, is expressed in Lactococcus (e.g., Lactococcus lactis) or Lactobacillus cells (e.g., Lactobacillus acidophilus or Lactobacillus casei). In additional embodiments, one or more strains of probiotic bacteria expressing a chimeric SlpA polypeptide are administered or formulated as a therapeutic composition. In certain embodiments, the SlpA expressed is a chimeric SlpA comprising a C. difficile SlpA variable domain and Lactobacillus (e.g., Lactobacillus acidophilus or Lactobacillus casei) SlpA cell wall binding domain. The SlpA additionally includes a bacterial secretion signal that is appropriate for surface expression in its host cell (e.g., Lactococcus, Lactobacillus, Lactobacillus acidophilus or Lactobacillus casei). In various embodiments, the invention also includes nucleic acid molecules and vectors encoding a chimeric SlpA polypeptide. Vectors encoding the chimeric SlpA polypeptide can be used to direct or regulate the expression of the chimeric SlpA by the cell (see e.g., Duong et al., Microbial Biotechnoloy 2010, 4(3): 357-367 which is herein incorporated in its entirety by reference). Such vectors contain one or more origin of replication sequences that can be used by a bacterial cell, promoter sequences for expression in a bacterial cell (e.g., constitutive or inducible), genetic markers for selection (e.g., antibiotic resistance); origin of transfer sequences for bacterial conjugation (e.g., traJ), and may also be codon optimized for protein expression.

[0078] Alternatively, nucleic acid sequences encoding chimeric SlpA may be integrated into the Lactobacillus or Lactococcus genome. In certain embodiments, nucleic acid sequences encoding the chimeric SlpA can be integrated into the Lactobacillus or Lactococcus genome through recombination between vectors comprising the nucleic acid sequence encoding the chimeric SlpA poly peptide and bacterial chromosome. Such techniques are well known in the art (see e.g., Leenhouts, et al., Appl. Environ. Microbiol. 1989, 55(2): 394-400, and Gaspar, et al., Appl. Environ. Microbiol. 2004, 70(3): 1466-74 which are herein incorporated in their entirety by reference).

[0079] Probiotic strains may also be engineered with auxotrophic selection, for example requiring thiamine or thymine supplementation for survival.

Methods of the Invention

[0080] The present invention provides methods of treating diseases or symptoms thereof associated with the presence of one or more undesirable bacteria in the gut of a subject. Accordingly, the invention provides compositions and methods for treating a subject having or at risk of developing a disease associated with undesirable changes in the gut microbiome, the method involving administering a therapeutically effective amount of a composition comprising a probiotic bacteria of the invention to a subject (e.g., a mammal, such as a human). In particular, the compositions and methods of the invention are effective for treating or preventing Clostridium difficile infection, colonization, or diseases and symptoms thereof (e.g., diarrhea). Without being bound to theory, Lactobacillus or Lactococcus expressing Clostridium difficile SlpA or fragment thereof (e.g., chimeric SlpA) colonize the gut and compete with Clostridium difficile for binding and colonization. Accordingly, the method includes the step of administering to a mammal a therapeutic amount of an amount of a composition comprising one or more probiotic bacteria strains of the invention sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.

[0081] Identifying a subject in need of treatment for a disease associated with the gut microbiome can be in the judgment of a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). In certain embodiments, the subject has undergone or is undergoing treatment with antibiotics. As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0082] The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of a composition comprising the probiotic bacteria of the invention to a subject (e.g., human) in need thereof. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider. The compositions herein may be also used in the treatment of any other disorders in which a microbial imbalance in the digestive tract may be implicated.

Methods of Delivery

[0083] Compositions comprising the probiotic bacteria of the invention may be administered orally, rectally, or enterally. Preferably compositions administered to a subject in tablet form, by feeding tube, by enema, or by colonoscopy. Preferably, the probiotic bacteria of the invention are diluted in a suitable excipient (e.g., saline solution). An effective dose may include 10.sup.6-10.sup.9 colony forming units of bacteria per day).

Expression of Recombinant Polypeptides

[0084] In order to express the fusion protein of the invention, DNA molecules obtained by any of the methods described herein or those that are known in the art, can be inserted into appropriate expression vectors by techniques well known in the art. For example, a double stranded DNA can be cloned into a suitable vector by restriction enzyme linking involving the use of synthetic DNA linkers or by blunt-ended ligation. DNA ligases are usually used to ligate the DNA molecules and undesirable joining can be avoided by treatment with alkaline phosphatase.

[0085] Therefore, the invention includes vectors (e.g., recombinant plasmids) that include nucleic acid molecules (e.g., genes or recombinant nucleic acid molecules encoding genes) as described herein. The term “recombinant vector” includes a vector (e.g., plasmid, phage, phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the recombinant vector was derived. For example, a recombinant vector may include a nucleotide sequence encoding an SlpA chimeric polypeptide operatively linked to regulatory sequences, e.g., promoter sequences, terminator sequences, and the like, as defined herein. Recombinant vectors which allow for expression of the genes or nucleic acids included in them are referred to as “expression vectors.”

[0086] In some of the molecules of the invention described herein, one or more DNA molecules having a nucleotide sequence encoding one or more polypeptides of the invention are operatively linked to one or more regulatory sequences, which are capable of integrating the desired DNA molecule into a prokaryotic host cell. Cells which have been stably transformed by the introduced DNA can be selected, for example, by introducing one or more markers which allow for selection of host cells which contain the expression vector. A selectable marker gene can either be linked directly to a nucleic acid sequence to be expressed, or be introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins described herein. It would be apparent to one of ordinary skill in the art which additional elements to use.

[0087] Factors of importance in selecting a particular plasmid or viral vector include, but are not limited to, the ease with which recipient cells that contain the vector are recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.

[0088] Once the vector(s) is constructed to include a DNA sequence for expression, it may be introduced into an appropriate host cell by one or more of a variety of suitable methods that are known in the art, including but not limited to, for example, transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.

[0089] After the introduction of one or more vector(s), host cells are usually grown in a selective medium, which selects for the growth of vector-containing cells. Expression of recombinant proteins can be detected by immunoassays including Western blot analysis, immunoblot, and immunofluorescence. Purification of recombinant proteins can be carried out by any of the methods known in the art or described herein, for example, any conventional procedures involving extraction, precipitation, chromatography and electrophoresis. A further purification procedure that may be used for purifying proteins is affinity chromatography using monoclonal antibodies which bind a target protein. Generally, crude preparations containing a recombinant protein are passed through a column on which a suitable monoclonal antibody is immobilized. The protein usually binds to the column via the specific antibody while the impurities pass through. After washing the column, the protein is eluted from the gel by changing pH or ionic strength, for example.

Kits

[0090] The invention provides kits for colonizing probiotic bacteria of the invention in the gut of a host. The invention also provides kits for the treatment or prevention of Clostridium difficile infection or colonization. In particular embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition comprising the probiotic bacteria of the invention; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0091] The kit preferably contains instructions that generally include information about the use of the composition for the expansion of the microbial consortia in the gut of the subject. The kit further contains precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0092] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0093] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: Probiotic Bacterial Strains Expressing Chimeric SlpA are Effective at Gut Colonization and Protecting Against C. difficile Challenge

[0094] Developing novel interventions that avoid the use of antibiotics is important in the treatment of C. difficile infection (CDI). Many gram-positive bacteria including C. difficile possess a surface-layer that covers the peptidoglycan-rich cell wall. This “S-layer” consists of many proteins that form a paracrystalline lattice around the bacterial cell. Surface layer protein A (SlpA), an adhesin and a major component of the cell surface layer (or S-layer) of C. difficile, facilitates gut colonization. Novel probiotic organisms were designed and engineered to express C. difficile SlpA on their cell surface (FIGS. 1A-1E). Without being bound to a particular theory, the engineered probiotic colonizes gut niches specifically occupied by virulent infecting C. difficile strains, thus preventing disease.

[0095] A chimeric SlpA polypeptide was created that included C. difficile SlpA sequences for bacterial adherence. The chimeric SlpA polypeptide was designed with a L. acidophilus cell-wall binding domain and secretion signal sequence and the host-cell binding domain of C. difficile. Plasmid vectors were constructed to express the slpA chimera either constitutively or in response to a fructose oligosaccharide inducer. Plasmid vectors encoding SlpA chimeras were designed and introduced into probiotic bacterial strains Lactococcus lactis and Lactobacillus acidophilus via three methods, electroporation, bacterial conjugation (pMTL82151 with pTRK848 comprising traJ; pMTL82151 with pTRK882 comprising traJ), and protoplasting. Transformants were assessed for stable carriage of introduced plasmids, and tested for SlpA surface expression. Western blot analysis and cell surface immunofluorescence showed that the chimeric SlpA was expressed by the probiotic bacterial cells (FIG. 2).

[0096] To test the ability of the recombinant bacteria expressing the chimeric SlpA to occlude C. difficile competitively, a study was performed in Syrian Golden hamsters (FIG. 3). Animals were treated with recombinant Lactobacillus expressing a chimeric SlpA or Lactobacillus carrying an empty vector. Another group of animals underwent treatment with antibiotics. Animals that were administered Lactobacillus expressing chimeric SlpA showed extended survival and overall survival at the conclusion of the experiment. By comparison, none of the animals that were treated with antibiotics or administered Lactobacillus with empty vector survived to the end of the experiment. All animals that were not challenged with virulent C. difficile survived to the end of the experiment. These results show that Lactobacillus expressing the chimeric SlpA colonized the gut and was able to protect against virulent C. difficile challenge. Thus, this indicates that administration of probiotic bacteria expressing chimeric SlpA has the potential to be an effective treatment or preventative for C. difficile infection and/or colonization.

Example 2: A Chimeric slpA Polynucleotide can be Integrated into the Lactobacillus Genome

[0097] Probiotic organisms can include biocontainment features for eventual clinical use and, concomitantly, obviating the requirement of antibiotics for in vivo plasmid maintenance and stable SlpA expression. The SlpA expression may also be controlled. The biological containment can be achieved via a two-step mechanism, where both SlpA expression and Lactobacillus sp. survival can be controlled. Novel vectors were designed to allow probiotic organisms to express chimeric SlpA stably and under control (FIGS. 4 and 5). In one embodiment, the nucleic acid sequence encoding a chimeric SlpA peptide can be integrated into a bacterial chromosome to allow stability of SlpA expression in the absence of selection pressure. The chimeric SlpA expression can be expressed under the control of a fructo-oligosaccharide (FOS) promoter. FOSs are well-tolerated, safe and widely-used supplements in humans and agriculturally-relevant animals.

[0098] Probiotic organisms expressing the chimeric SlpA protein, such as the lactic acid bacterium, including both L. casei and L. acidophilus, can be genetically modified such that complete lethality of the probiotic organisms occurs in the absence of thymine supplementation. This “thymineless death” is predicated on the absolute requirement of deoxy-thymidine triphosphate (dTTP) for DNA synthesis in all living organisms. Of the two pathways for dTTP synthesis in most bacteria, the de novo pathway involves conversion of dUMP to dTMP by the essential enzyme thymidylate synthase (ThyA). The less-used “salvage” pathway involves the conversion of supplemented thymidine into dTMP by thymidine kinase. dTMP is then converted to dTTP. Disruption or mutation of thyA in bacteria results in immediate auxotrophy and, in vitro, can be tolerated only by addition of exogenous thymidine that is utilized by the salvage pathway. Withdrawal of thymidine results in rapid, total cell death. Free thymidine is not abundant or bio-available in vivo (in the gut), and is unable to support growth of thyA auxotrophs. Therefore, the probiotic organisms with thyA gene disrupted will necessarily be lost from the gut unless continually administered.

[0099] The nucleic acid sequence encoding the chimeric SlpA can be integrated into the Lactobacillus sp. through a single homologous recombination at a single site (FIG. 4) or a double homologous recombination (FIG. 5). For the single homologous recombination, one vector is constructed for each species. Thus, a vector is constructed for specific use in L. casei, such that the vector includes a nucleic acid sequence that is identical to a nucleic acid sequence of a fragment of the thyA gene in L. casei. Another vector is constructed for specific use in L. acidophilus, such that the vector includes a nucleic acid sequence that is identical to a nucleic acid sequence of a fragment of the thyA gene in L. acidophilus. The sequence of the vector for the specific use in L. casei for single recombination is shown in FIG. 6 and the sequence of the vector for the specific use in L. acidophilus for single recombination is shown in FIG. 8.

[0100] For the double homologous recombination, one vector is constructed for each species. Thus, a vector is constructed for specific use in L. casei, such that the vector includes a first nucleic acid sequence that is identical to a nucleic acid sequence of a fragment located at the 5′ of the thyA gene in L. casei and a second nucleic acid sequence that is identical to a nucleic acid sequence of a fragment located at the 3′ of the thyA gene in L. casei. Another vector is constructed for specific use in L. acidophilus, such that the vector includes a first nucleic acid sequence that is identical to a nucleic acid sequence of a first fragment located at the 5′ of the thyA gene in L. acidophilus and a second nucleic acid sequence that is identical to a nucleic acid sequence of a second fragment located at the 3′ of the thyA gene in L. acidophilus. The sequence of the vector for the specific use in L. casei for double recombination is shown in FIG. 7 and the sequence of the vector for the specific use in L. acidophilus for double homologous recombination is shown in FIG. 9.

Other Embodiments

[0101] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0102] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0103] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.