JP2016512428A - Diffocin and its use - Google Patents

Abstract

The present disclosure relates to the discovery and isolation of an entire gene cluster encoding an R-type high molecular weight bacteriocin that specifically kills a dangerous pathogen, Clostridium difficile bacteria. The present disclosure is also a disclosure of the method for producing the R-type bacteriocin in harmless aerobic cells. The present disclosure is also the disclosure of small molecules, non-ORF 1374 receptor binding domains (RBDs), which are incorporated into diffocins to form heterologous diffocins with genetically modified or altered bactericidal spectra. The heterologous diffocins provided in the present disclosure may comprise a heterogeneous RBD and its cognate substrate junction region (BPAR), or fused BPAR. The present invention is a powerful fungicide with improved thermal and pH stability to selectively kill Clostridium difficile bacteria in the gastrointestinal tract environment, where severe injury and even death of infected patients or livestock occur And a method for producing the same.

Description

FIELD OF THE INVENTION This application relates to the identification and isolation of gene clusters sufficient to produce bacteriocin, and more particularly to R-type high molecular weight bacteriocins that specifically kill Clostridium difficile and the sterilization thereof. It relates to methods of modifying, producing and using specificity.

Grand Information This invention was accomplished with government support under grant number R43AI098186-01 awarded by the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The United States government has certain rights in this invention.

Background of the Invention Clostridium difficile is an obligately anaerobic, spore-forming gram-positive bacterium and is a notorious pathogen for humans and other mammals (Bartlett et al., 1977, Bartlett et al., 1979,). Keel et al., 2007, Sunenshine & McDonald, 2006). Clostridium difficile, if low in density, settles harmlessly in the mammalian gastrointestinal tract (GI). However, during growth, antibiotic administration often reduces commensal bacteria, producing enough exotoxin to cause disease ranging from mild diarrhea to characteristic pseudomembranous colitis. These toxins are life-threatening and cause significant complications, especially in older humans and other mammals (Bartlett, 2002).

  Spores formed by the spread of this pathogen are difficult to inactivate or eradicate in hospitals and chronic disease care facilities, so the probability of patients with Clostridium difficile increases rapidly throughout such facilities ( Bartlett, 2007). In fact, BI / NAP1 / 27, a relatively new strong toxin-producing strain of Clostridium difficile and causing severe disease due to a large epidemic, has been well documented recently (Spigaglia et al., 2002; (Pepin et al., 2004, McDonald et al., 2005, Muto et al., 2005, Loo et al., 2005, Belmares et al., 2009). Increasing cases of Clostridium difficile-associated disease (CDAD) in children, which previously remained at low risk, have increased significantly (Benson et al., 2007, Zilberg et al., 2010).

  Prophylactic elimination of pathogens by the administration of antibiotics in asymptomatic carriers or carriers is unavoidable, as it induces Clostridium difficile-related diseases.

  R-type bacteriocins made by Gram-negative bacteria have been described, and R-type bacteriocins that kill other competing Gram-negative strains are utilized, even in the context of other species or genera of Gram-negative bacteria. (Kageyama et al., 1964, Kageyama et al., 1964a, Kingsbury, D, 1966, Blackwell and Law, 1981, Blackwell et al., 1982, Campagnari et al., 1994, Strauch et al. Jabrane et al., 2002). The fusion of the R-type pyocin to the heterologous receptor binding domain (RBD) of the substrate junction region (BPAR) is described and the result is the creation of a new R-type pyocin with novel bactericidal properties against gram-negative bacteria (Williams et al., 2008, Scholl et al., 2009).

  Other high molecular weight bacteriocins or R-type bacteriocins have been described as gram-positive bacteria (Coetzee et al., 1968, Thompson and Pattee, 1981, Zink et al., 1995). However, little is known about the structure of R-type high molecular weight bacteriocin produced by Gram-positive bacteria. Moreover, while such bacteria have been described, none have been characterized at the genetic level, or none have been treated in a symptomatic and essential way to develop effective drugs. As high molecular weight bacteriocin, two kinds of Clostridium bacteria, Clostridium botulinum, and Clostridium perfringens have been described (Ellison and Kautter, 1970, Anastasio et al., 1971, Nieves et al., 1981). There is no description of those produced by Clostridium difficile or those that kill Clostridium difficile.

SUMMARY OF THE INVENTION The present invention codes for a C. difficile-specific R-type bacteriocin (referred to herein as diffocin) that is bactericidal against other strains of Clostridium difficile. Based on the isolation of all gene loci or clusters. That is, for the expression of diffocin gene cluster and the production of diffocin in aerobic bacteria, that is, for the discovery of the bactericidal spectrum of diffocin against Clostridium difficile strain determined by the open reading frame (ORF) 1374 of the diffocin gene cluster. Based. The present invention produces novel diffocins and provides a direct or indirect preparation and administration method for eliminating Clostridium difficile bacteria that settle there from the gastrointestinal tract (GI) of animals, including humans, Provide experimental means to alter diffocin specificity by genetic engineering. Administration of diffocin treats or prevents the development of Clostridium difficile infection (CDI) without damaging the symbiotic gastrointestinal (GI) bacteria that are very necessary for treatment and health, as is the case with conventional antibiotics. can do.

  According to the present invention, an isolated nucleic acid molecule encoding an R-type high molecular weight (hmw) bacteriocin is provided. In one embodiment, the nucleic acid molecule is isolated from the genome of a Clostridium difficile strain and is a nucleic acid molecule encoding an R-type high molecular weight (hmw) bacteriocin, and the R-type hmw bacteriocin is SEQ ID NO: 4. A R-type hmw bacteriocin consisting of at least 80% of the polypeptide selected from the group consisting of -16, 18, 19, and 66-80, and the R-type hmw bacteriocin is at least It has a receptor binding domain (RBD) that binds one other Clostridium difficile receptor and therefore has bactericidal activity against other strains or Clostridium difficile strains. In certain embodiments, the nucleic acid molecule is derived from the genome of a C. difficile strain selected from the group deposited with Cd4, Cd16, Cd19108, Cd19123, Cd19126, Cd19145, and ATCC Deposit Number 43593. In some embodiments, the strain comprises Cd16 and the nucleic acid molecule comprises SEQ ID NO: 1, or the strain comprises Cd4 and the nucleic acid molecule comprises SEQ ID NO: 61.

  In other embodiments, an isolated nucleic acid molecule encoding an R-type high molecular weight (hmw) bacteriocin is provided. Wherein the nucleic acid molecule is derived from the genome of a first strain of Clostridium difficile and comprises a sequence of a first polynucleotide that is at least 80% identical to a polynucleotide encoding SEQ ID NOs: 66-77. And wherein the nucleic acid molecule is a second strain of Clostridium difficile or a receptor-binding domain (RBD) of a prophage or prophage remnant from a RBD genome of a bacteriophage that infects Clostridium difficile Wherein the R-type hmw bacteriocin is a first substrate wherein at least 50 contiguous amino acids are linked at the amino terminus and are at least 80% identical to the polypeptide SEQ ID NO: 78. A junction region (BPAR) polypeptide, and wherein Wherein R type hmw bacteriocin has bactericidal activity against at least one of Clostridium difficile strains.

  In another embodiment of the invention, there is provided an isolated R-type bacteriocin encoded by a nucleic acid molecule of the invention. In one embodiment, the R-type bacteriocin is expressed in aerobic producing bacteria. In one embodiment, the R-type bacteriocin can be orally administered to animals and can be excreted in the feces in a state of bactericidal activity. In a particular embodiment, the R-type bacteriocin retains some bactericidal activity after incubation at 25 ° C. for 30 minutes at pH 2.5 and pH 10.6. In one embodiment, the R-type bacteriocin retains certain bactericidal activity even after culturing at pH 3.4 and pH 9 at 25 ° C. for 30 minutes. In another embodiment, the R-type bacteriocin retains certain bactericidal activity after incubation at 45 ° C. for 60 minutes.

  In another embodiment of the invention, an isolated R-type high molecular weight (hmw) bacteriocin with bactericidal activity is provided. Here, the R-type hmw bacteriocin includes a substrate binding region (BPAR) of a first strain of a first bacterium of a Clostridium gene, and a receptor binding domain (RBD) of a second strain of the first bacterium. Or a second bacterial receptor binding domain (RBD) of a bacteriophage that infects the Clostridium gene or Clostridium species. Here, the bacteriocin has bactericidal activity against at least one strain of Clostridium difficile. In certain embodiments, the BPAR is derived from a first strain of Clostridium difficile, and the RBD is derived from a first strain of Clostridium difficile, or a bacteriophage that infects Clostridium difficile. Is from. In certain embodiments, the BPAR is at least 80% identical to one or more corresponding segments of SEQ ID NOs: 16, 54-56 and 78. In one embodiment, the BPAR is a polypeptide having 50 or more amino acids of SEQ ID NO: 16 or 78 linked, or a polypeptide having 50 or more amino acids of SEQ ID NOs: 54-56 linked. It is at least 80% identical. In one embodiment, the BPAR is a fusion of the amino terminus of the first BPAR that is at least 80% identical to SEQ ID NO: 78 and the C-terminus of the second BPAR that is homologous to the heterologous RBD.

  In some embodiments, the RBD is at least 80% identical to one or more corresponding segments of SEQ ID NOs: 17 and 49-56. In other embodiments, the encoded RBD is at least relative to an RBD selected from the group of SEQ ID NOs: 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 116. 80% identical.

  In another embodiment of the invention, an expression cassette comprising the nucleic acid molecule of the invention is provided. The expression cassette may be contained within an expression vector such as a plasmid, or may be contained on the producer cell's chromosome. In some embodiments, the expression cassette comprises a heterologous promoter linked by genetic recombination to a nucleic acid molecule encoding the R-type bacteriocin. The promoter can be inducible, repressive, or constitutively active. The promoter is inducible in one embodiment and repressive in another embodiment. In some embodiments, the promoter is induced by adding or removing a small molecule inducer (inducer), repressor (repressor), or derepressor (delipressor). In one embodiment, the promoter is induced by a small molecule inducer or delipressor. In one embodiment, the small molecule inducer or depressor is a reactive oxygen product (ROS) or a ROS generator. In one embodiment, the ROS is a peroxide that is non-toxic to humans and other animals. In one embodiment, the peroxide is hydrogen peroxide. In certain embodiments, the expression of the cassette is achieved by manipulating the recA gene encoding a RecA constitutively active protein and placing a heterologous promoter responsive to a small molecule inducer or depressor under control. Control.

  In yet another embodiment of the invention, a producer cell comprising the expression cassette of the invention is provided. The expression cassette may be contained in an episomal expression vector in the producer cell. Alternatively, the producer cell may contain a nucleic acid molecule or an expression cassette of the invention in the chromosome. In certain embodiments, the producer cell is non-pathogenic and not an obligate anaerobe. In some embodiments, the non-pathogenic and non-obligate anaerobic bacterium is a species from a bacterial genus selected from the group consisting of Neisseria gonorrhoeae, lactic acid bacteria, and Listeria. In one embodiment, the non-pathogenic and non-biased anaerobic bacterium is selected from the genus Bacillus. In some embodiments, the bacterium is Bacillus subtilis. In a particular embodiment, the Bacillus subtilis is defective in the PBSX gene cluster. In other embodiments, the producer cell is an obligate anaerobic but non-pathogenic bacterium.

  In yet another embodiment of the present invention, a method for producing the R-type hmw bacteriocin of the present invention is provided. The method is effective in inducing the expression of the R-type bacteriocin in a producer cell comprising the nucleic acid sequence of the present invention genetically linked to an inducible promoter or a derepressed promoter sensitive to an inducing agent or derepressor. Exposure to an appropriate concentration of the formulation and purifying the expressed R-type bacteriocin. In some embodiments, the nucleic acid molecule encoding the R-type bacteriocin is heterologous to the producer cell's genome. In certain embodiments, the nucleic acid molecule is contained in a chromosome of the producer cell or is contained in an extrachromosomal expression vector of the producer cell. In certain embodiments, the producer cell is non-pathogenic and not an obligate anaerobic bacterium. In some embodiments, the non-pathogenic and non-biased anaerobic bacteria are species derived from bacterial genes selected from the group consisting of Bacillus subtilis, lactic acid bacteria, and Listeria. In one embodiment, the non-pathogenic and non-biased anaerobic bacteria are derived from the Bacillus subtilis gene. In certain embodiments, the bacterium is Bacillus subtilis. In certain embodiments, the Bacillus subtilis does not dissolve when induced to produce the R-type bacteriocin. In a further embodiment, the Bacillus subtilis is defective in the PBSX gene cluster.

  In a further embodiment of the invention, a method for killing pathogenic bacteria is provided. The method involves contacting a pathogenic bacterium with the R-type bacteriocin of the present invention, wherein the R-type bacteriocin binds to and kills the pathogenic bacterium. In one embodiment, the pathogenic bacterium is Clostridium difficile. In one embodiment, the Clostridium difficile is in an animal body and a bactericidal amount of the R-type bacteriocin is administered to the animal.

  In other embodiments, a method of treating or preventing Clostridium difficile infectious disease in an animal is provided. The method includes direct administration of a bactericidal amount of the R-type bacteriocin of the present invention to an animal, indirect reagent administration by administration of producer cells, or a naturally occurring diffocin that is genetically modified to produce no toxin. Includes administration of difficile bacterial spores. In certain embodiments, an animal infection with Clostridium difficile is treated by producing a bactericidal amount of bacteriocin by administering the required amount of the producer cells of the invention to the animal. In one embodiment, the bacteriocin-encoding nucleic acid is under the control of a lactose promoter and the animal is administered lactose. In some embodiments, the animal is a mammal. In one embodiment, the mammal is a human.

Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Nucleic acid or amino acid sequences of SEQ ID NOs: 1-80 are provided. Fig. 2 shows the bactericidal activity of diffocin in clinical isolation of Clostridium difficile. The indicator bacterial number is indicated along the upper part of the matrix, that is, the strain with an asterisk is the NAP1 / 027 / BI strain. The origin identification of the Clostridium difficile tested with diffocin is shown along the left edge. The strains are LC Fortier (4 and 16), ATCC (43593), or RM Alden Research Lab, Culver City, CA. Obtained from A scanning electron micrograph of diffocin 16 is shown. Note the organ of the flower-like tail fiber. It is a photograph of the spot assay result of diffocin 4 against the target strain Cd19135. Purified diffocin was serially diluted 10-fold and 5 μl of the diluted specimen was dropped onto the target Clostridium difficile flora. After overnight anaerobic culture at 37 ° C., clarification of the microbial flora showed defocin bactericidal properties. Shown are silver-stained SDS-PAGE photographs of diffocin 4 (“4”) and diffocin 16 (“16”) sieving (“F”) and non-sieving (“UF”) effect specimens. The arrows indicate the bands that were excised and identified by mass spectrometry. Schematic representation of the diffocin gene cluster from Cd630 and Cd16 is shown. The loci, ORF1362 to ORF1375, consisting of ORFs encoding typical structural and assembly proteins of myovirus phage tail organs are shown in the following map. These adjacent genes are genes that encode putative phage-like regulatory proteins. The arrow at the bottom of the figure indicates the location where the ORF is transcribed, and the estimated function of the ORF is shown at the top of the map. FIG. 5 is an image of a spot assay of diffocin 16 produced by Bacillus subtilis BDR123-488 (sector 1) and BDR123-491 (sector 2) tested on the Clostridium difficile strain 19099. The results of 1W cluster analysis of partial amino acid sequences (SEQ ID NOs: 81-86, in the order of appearance of each) encoded by the ORF1374 gene from each of five strains of Clostridium difficile producing active diffocin are provided. The asterisk “ ^* ” at the bottom of each sequence represents the identity of the amino acid at the position encoded by all 5 strains, and the “:” is an amino acid similar to the high position at the position encoded by all 5 strains. "·" Indicates that it is a somewhat similar amino acid at the position encoded by all 5 strains, and a blank at a known position in the sequence is a similar amino acid encoded by all 5 strains It means that there is no. The results of 1W cluster analysis of partial amino acid sequences (SEQ ID NOs: 81-86, in the order of appearance of each) encoded by the ORF1374 gene from each of five strains of Clostridium difficile producing active diffocin are provided. The asterisk “ ^* ” at the bottom of each sequence represents the identity of the amino acid at the position encoded by all 5 strains, and the “:” is an amino acid similar to the high position at the position encoded by all 5 strains. "·" Indicates that it is a somewhat similar amino acid at the position encoded by all 5 strains, and a blank at a known position in the sequence is a similar amino acid encoded by all 5 strains It means that there is no. The results of 1W cluster analysis of partial amino acid sequences (SEQ ID NOs: 81-86, in the order of appearance of each) encoded by the ORF1374 gene from each of five strains of Clostridium difficile producing active diffocin are provided. The asterisk “ ^* ” at the bottom of each sequence represents the identity of the amino acid at the position encoded by all 5 strains, and the “:” is an amino acid similar to the high position at the position encoded by all 5 strains. "·" Indicates that it is a somewhat similar amino acid at the position encoded by all 5 strains, and a blank at a known position in the sequence is a similar amino acid encoded by all 5 strains It means that there is no. The results of 1W cluster analysis of partial amino acid sequences (SEQ ID NOs: 81-86, in the order of appearance of each) encoded by the ORF1374 gene from each of five strains of Clostridium difficile producing active diffocin are provided. The asterisk “ ^* ” at the bottom of each sequence represents the identity of the amino acid at the position encoded by all 5 strains, and the “:” is an amino acid similar to the high position at the position encoded by all 5 strains. "·" Indicates that it is a somewhat similar amino acid at the position encoded by all 5 strains, and a blank at a known position in the sequence is a similar amino acid encoded by all 5 strains It means that there is no. The results of 1W cluster analysis of partial amino acid sequences (SEQ ID NOs: 81-86, in the order of appearance of each) encoded by the ORF1374 gene from each of five strains of Clostridium difficile producing active diffocin are provided. The asterisk “ ^* ” at the bottom of each sequence represents the identity of the amino acid at the position encoded by all 5 strains, and the “:” is an amino acid similar to the high position at the position encoded by all 5 strains. "·" Indicates that it is a somewhat similar amino acid at the position encoded by all 5 strains, and a blank at a known position in the sequence is a similar amino acid encoded by all 5 strains It means that there is no. It is an image of the spot assay of “diffocin 4” and “diffocin 4-16” produced by BDR123-580. The latter was produced by B. subtilis BDR123-587 in that diffocin 4 ORF 1374 (SEQ ID NO: 49) was replaced with diffocin 16 ORF 1374 (SEQ ID NO: 17). These two diffocins produced by Bacillus subtilis were examined on both the 19137 and 19145 flora as seen in the figure. The bactericidal specificity of “diffocin 4-16” was obtained by converting the specificity of diffocin 4 into the specificity of diffocin 16. It is the plot figure which compared the load by the Clostridium difficile bacteria of the mouse | mouth which administered the heterologous diffocin with the load of the non-administration mouse | mouth. A new, diffocin containing non-1374 based RBD functioned to reduce CDI in the body. In comparison with non-administered mice challenged with Clostridium difficile spores ("Diff"), new diffocins containing non-1374 base RBD were further orally challenged with Clostridium difficile spores. Mice (“+ Diff”) showed a statistically significant (Student t test, p <0.05) significant decrease in the excretion of Clostridium difficile into feces. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided. The nucleic acid sequence or amino acid sequence of SEQ ID NOs: 87-163 is provided.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, R-type high molecular weight (hmw) bacteriocin is simply known as R-type bacteriocin, and R-type protein, diffocin, monocin, enterocolate. It includes lysine, meningosine, or other high molecular weight (hmw) bacteriocins that are structurally or genetically related to the myovirus family of bacteriophages. R-type bacteriocins include modified forms of R-type protein, diffocin, monocine, enterocoliticin, meningosine (Williams et al., 2008, Strauch et al., 2001, Kingsbury, 1966, Zink et al., 1995). ).

  As used herein, the term “diffocin” refers to an R-type high molecular weight bacteriocin isolated or derived from Clostridium difficile, and includes native spheres obtained from Clostridium difficile. At the same time, a spheroid obtained through expression of the diffocin gene cluster in an artificial production cell is also included. The diffocin may be a recombinant spheroid consisting of a polypeptide encoded by a gene derived from one or more strains of Clostridium difficile, and SEQ ID NOs: 2-23, 49 and 62- It consists of a polypeptide that is 80% or more identical to 80 one or more polypeptides.

  The R-type bacteriocin of the present invention exhibits thermal instability, weak acid resistance, trypsin resistance, sedimentation in a centrifuge at about 65,000 xg and can be analyzed by electron microscopy (Kageyama et al., 1962, Bradley, 1967, Daw et al., 1996, Jabrane et al., 2002, Fortier et al., 2007). Often, the genetically modified R-type bacteriocin disclosed herein will have one or more of these properties in any combination. The attendant properties commonly found in the R-type bacteriocins disclosed herein do not include nucleic acids and are possible with many bacteriophages but cannot be reproduced by themselves after or during the killing of the target bacteria. Replication deficiency. The R-type bacteriocin of the present invention is purely a protein and not an organic organism.

  The R-type bacteriocin disclosed herein is a complex molecule constituting a plurality of proteins or polypeptides, a subunit of the myovirus family, and similar to the tail structure of a bacteriophage. R-type bacteriocins that appear in nature are subunit structures encoded by bacterial genomes such as Clostridium difficile or Pseudomonas aeruginosa and formed by R-type bacteriocins to provide a natural defense against other bacteria. (Kageyama, 1975). Sensitive target bacteria are typically killed by simple R-type bacteriocin molecules (Kageyama et al., 1964, Kageyama et al., 1964a, Morse et al., 1980, Strauch et al., 2001).

  A “target bacterium” or “target bacterium group” refers to a bacterium or group of bacteria that is bound to the disclosed R-type bacteriocin and / or whose growth is exacerbated, persists, or replication is suppressed. In some embodiments, the target bacterium is selected from the genus Clostridium. In certain embodiments, the bacterium is Clostridium difficile. In one embodiment, one or more strains of Clostridium difficile are targeted. A typical strain of Clostridium difficile is included, but is not limited to NAP1 / BI / ribotype 027, as described in FIG.

  As used herein, the term “growth inhibition” or mutation refers to slowing or stopping the rate of cell division of a bacterium or pausing cell division, or killing the bacterium or group of bacteria.

  The term “nucleic acid” or “nucleic acid molecule” as used herein typically refers to deoxyribonucleotides or ribonucleotide polymers (purified or mixed) that form single or double strands. . The term may be a nucleotide analog or a nucleic acid containing a modified backbone residue or linkage, which may have been synthesized, naturally occurring and artificially generated, and has a binding state, structure similar to the referenced nucleic acid. Or may have functional properties and may have been metabolized in a manner similar to the referenced nucleotide and may include them. Examples of such analogs include, but are not limited to, phosphorothioates, oxazaphospholidines, methyl acids, chiral methyl acids, 2-0 methyl ribonucleotides, and peptide nucleic acids (PNAs). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide in some contexts.

  Certain nucleic acid sequences cover conservatively altered mutated sequences (in ways such as degenerate codon substitutions) and complementary sequences, as well as suggested sequences. In particular, degenerate codon substitution may be achieved by a gene sequence that is in the third position (“fluctuation”) of one or more (or all) codons that are substituted with mixed bases and / or deoxyneusin residues. . Thus, the nucleic acid sequences encoding the protein sequences disclosed herein also encompass modified heterologous species as described herein.

  As used herein, the term “segment” as used in reference to an amino acid sequence refers to a contiguous sequence of amino acids that is 10, 12, 15, 20, 25, 50 or 100 amino acid residues in length. .

  As used herein, the term “heterologous” is used in reference to the location of a protein or nucleic acid sequence, but the sequences are not normally found in a similar relationship to each other in the natural state. Refers to a sequence containing one or more substitutions. In one embodiment, heterologous sequences are produced from different species of bacteria. In another example, the heterologous sequence is produced from different strains of the same species of bacteria. In one embodiment, the heterologous sequence is produced from different strains of Clostridium difficile. In other embodiments, the heterologous sequence is produced from a bacterial group and bacteriophage or prophage, or from an artificial sequence of bacterial group and synthesized DNA.

  The terms “polypeptide”, “peptide” and “protein” as used herein refer to terms that are typically interchangeable in relation to amino acid residue polymers. Amino acids are quoted by three letter symbols that are widely known or by one letter proposed by the International Biochemical Union Nomenclature Committee (IUPAC-IUB).

  A virulence factor is a molecule involved in virulence regardless of the general survival rate of living organisms. Thus, the absence of virulence factors does not reduce the pathogenicity of living organisms and reduce survival. Pathogenic agents have several functions, such as gene expression suppression, colonization or mobility donation, toxin donation, toxin infusion, elimination of antibiotics, or formation of protective coatings including biofilms.

  A fitness factor is a molecule involved in the general survival rate, growth rate, or competitiveness of its environment. Thus, the lack of a matching factor can cause the organism to become less viable or competitive and indirectly reduce pathogenicity as a result of such a compromise. Matching agents also have a number of functions, such as the acquisition of nutrients, ions or water, the formation of cell membrane or cell wall components and protective layers, the replication, repair, or mutagenesis of nucleic acids, the protection or attack against environmental or competitive injury. Have some of.

  As used herein, the term “producer cell” refers to a cell that encodes diffocin and is capable of producing or expressing a non-naturally occurring nucleic acid molecule. Producer cells can survive and grow in the presence of oxygen, and a vector containing a nucleic acid molecule encoding the diffocin can be integrated into the producer cell's chromosome or become an episome, transforming the producer cell's traits To do. The producer cell is a gram positive bacterium. In certain embodiments, the producer cell may be a bacterium selected from the genus Neisseria, Lactobacillus, Lactococcus, Clostridium, or Listeria. In a preferred embodiment, the producer cell is a bacterium selected from the genus Neisseria, Lactobacillus, Lactococcus, or Listeria. In some embodiments, the bacterium is one of the koji molds selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus megateriun. In one embodiment, the bacterium is Bacillus subtilis. In certain embodiments, the producer cell is a Bacillus subtilis strain that lacks the PBSX gene cluster. In another embodiment, the bacterium is a member of the genus Lactobacillus selected from the group of lactic acid bacteria, casei bacteria, and Bulgarian lactobacilli. In yet another embodiment, the bacterium is Listeria. In other embodiments, the non-pathogenic producer cell may be a cell of an E. coli or Clostridium gene.

Detailed Description of Implementation Modes of the Present Disclosure R-type bacteriocin isolated from Clostridium difficile has bactericidal activity.
Two Clostridial difficile strains, Cd4 and Cd16 (obtained from LC Fortier) through cell culture in a medium to long-term strict anaerobic environment and exposure to 3 μg / ml mitomycin C medium for bactericidal activity testing A lysate was prepared. After lysis of bacteria, the lysate particles were concentrated and purified with a high-speed centrifuge (Example 1). These samples were shown by electron microscopy to contain a concentrate of phage-like particles without heads (FIG. 3). After concentration and purification, the samples were subjected to the target Clostridium difficile flora in a strict anaerobic environment in order to assess the bactericidal activity of the samples in a spot assay. As most phage tail bacteriocins target different strains from their producing bacteria, as an early stage we tested a target strain consisting of a panel of 29 clinically isolated Clostridium difficile . After overnight culture, the presence of the bactericidal activity of the test specimen was evaluated by clarification of the Clostridium difficile flora to which the purified substance was spotted. FIG. 4 shows a typical spot assay in which diffocin (dif4) produced from Cd4 is dropped onto a Clostridium difficile strain, Cd19135.

  Dif4 (Cd4 producing diffocin) and dif16 (Cd16 producing diffocin) showed different bactericidal spectra in the spot assay results (FIG. 2). Dif4 showed bactericidal activity in 10 of 29 clinically isolated Clostridium difficile strains, and dif16 had activity in 8 of 29 strains. There were several strains that were redundantly affected by both diffocins, and the two diffocins had distinct bactericidal spectra. The diffocins produced from Cd108 and Cd43593 had a very similar bactericidal spectrum and killed at least 9 out of 10 NAP1 / 027 / BI highly virulent strains in a panel of 29 Clostridium difficile. In further studies, dif 43593 killed all 18 additional, single NAP1 / 027 / BI isolates. Thus, dif43593 killed all 28 test strains of NAP1 / 027 / BI type Clostridium difficile (FIG. 2).

Identification of the diffocin locus The diffocin particles, isolated and purified from the Cd4 strain, were denatured and the protein components were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and detected by silver staining (Fig. 5). Two independent bands, ˜200 kD and ˜40 kD, were excised and subjected to mass spectrometry. Peptides from the 40 kD band are the open reading frames of two Clostridium difficile strains among a variety of Clostridium difficile strains with the complete gene sequence, including the reference strain Cd630 (GenBank Acc. No. NC — 09089.1). It was found to be consistent with the predicted material encoded by ORF). The first material was a 39,192 dalton phage-like protein corresponding to ORF 1363 (SEQ ID NO: 5). The second predominant polypeptide in this band corresponds to ORF 1371 (SEQ ID NO: 14) and was a 39,565 dalton phage-like substrate protein. These proteins happened to be very close in molecular weight and migrated to the same band on SDS PAGE. The 200 kD band produced a major protein corresponding to the C-terminal protein of ORF 1374 (SEQ ID NO: 17) immediately downstream of the other two phage-like ORFs.

  Since these ORFs are in very adjacent positions within the Cd630 gene, their surrounding regions were analyzed. One prophage-like element containing ORF1360A-1379 (FIG. 6 and SEQ ID NOs: 1-23) was found between 1574593 bases and 1596384 bases. Of particular note is that the structural genes encoded in this region corresponded only to the components of the tail structure of typical myovirus phages. That is, no capsid-corresponding gene, capsid-containing protein, or portal protein was found. Furthermore, there were no ORFs that encoded putative DNA replication or DNA package structures. Thus, there was no doubt that this locus was the gene sequence of one R-type (myovirus phage tail) bacteriocin. Adjacent structural genes were multiple ORFs encoding a putative phage-like suppressor protein. Due to the fact that the ORF of this locus encoded a polypeptide found in the diffocin particle and the gene cluster was similar to that of the R-type bacteriocin, we found that this region was the diffocin locus. Specified.

  The structural gene was encoded in one chromatid and transcribed in the same direction. The gene organization was similar to that of R-type pyocin and many myovirus phages. Some of the structural proteins showed sequence similarity with known Clostridium difficile phages, including phage Φ119 and ΦC2 (Goh et al., 2007, Govind et al., 2006). Clostridium difficile strain Cd630 is known to encode two intact prophages. Some of the diffocin ORFs have sequence similarity to C. difficile strain Cd630 prophages 1 and 2, suggesting that these C. difficile myovirus phages and diffocins share a common lineage. To do.

  Of particular note is the ORF1374 (SEQ ID NO: 17) gene of the diffocin. This gene, ORF 1373, encoded a large polypeptide immediately downstream of SEQ ID NO: 16. The position of the gene cluster showing that ORF1374 is part of the R-type bacteriocin tail fiber, ie, the receptor binding domain (RBD). An electron micrograph of what was designed as diffocin revealed a large flower-like structure in the tail fiber region, making it apparent that the structure contained a huge protein. ORF1373 (SEQ ID NO: 16) encodes the substrate junction region of the diffocin tail fiber, BPAR, and is apparently an abbreviation for similar ORF1373 in Clostridium difficile phage that encoded RBD along with BPAR. Became. Such phage lack ORF1374. Thus, the naturally occurring diffocin tail fiber consists of two proteins, ORF 1373 and ORF 1374, forming a bound tail fiber. There, the Clostridium difficile bacteriophage has a tail fiber composed of a somewhat longer ORF 1373 that provides the functions of the single-stranded proteins B. difficile BPAR and RBD. Accompanying this finding, in the present invention, a nucleic acid encoding either ORF1373 (SEQ ID NO: 16) or ORF1374 (SEQ ID NO: 17) was examined as a substrate for obtaining a novel RBD-specific function by genetic recombination.

Diffocin is pervasive to isolated Clostridium difficile.
A single series of clinical isolates of Clostridium difficile was used to assess the ability to produce diffocin particles that appear to have different bactericidal spectra. Clinically isolated Cd19123, Cd19145, Cd19126, Cd19108 (obtained from RM Alden Research Laboratory, Culver City, Calif.) And ATCC Cd43593 were induced to mitomycin C under a strict anaerobic environment. The produced particles were then purified and concentrated. The purified product was spotted separately on other Clostridium difficile flora collected clinically to detect bactericidal activity. The results indicated that each of these isolates produced particles (the diffocins from each of clinical isolates Cd19123, Cd19145, Cd19126, Cd19108, and ATCC Cd43593 were converted to dif123, dif145, dif126, dif108, and dif43593, respectively. And a different bactericidal spectrum (FIG. 2). Dif108 and dif43593 show a very similar broad spectrum, while the Cd19145 strain produced particles with a narrow bactericidal spectrum. Several isolates, including Cd19113 and Cd19150 that were induction tested, lysed after exposure to mitomycin, but did not produce detectable diffocin particles.

  In this test, it was found that the genomic sequence test strain Cd630 encoded the diffocin locus. In fact, this genome was used to identify the diffocin gene. However, on the other hand, the Cd630 strain lysed after induction with mitomycin C did not produce detectable diffocin particles. Instead, small amounts of prophage 1 and prophage 2 were produced.

Identification and cloning of diffocin genes Cd630 did not produce detectable diffocin particles upon lysis induction with mitomycin C. Therefore, the diffocin gene cluster derived from Cd16 was cloned in exchange for it. An overview of the Cd16 gene sequence was obtained for the first time. A diffocin locus similar to the Cd630 locus was identified and annotated. The entire gene cluster of diffocin (SEQ ID NO: 1) from the Cd16 producer strain was cloned into a cloning vector (BAC) and then all genes required to produce active diffocin for non-pathogenic microorganisms that are not obligate anaerobes. Expressed to include. Since Clostridium difficile is a pathogenic and obligate anaerobic bacterium, it is a production cell contrary to common sense for naturally occurring diffocin or diffocin modified by artificial DNA recombination. Isolated diffocin gene clusters can also be processed by genetic engineering techniques and controlled for expression by a non-toxic small molecule inducer or a heterologous promoter corresponding to a depressor added to the gene cluster. Such small molecules include, but are not limited to, their non-metabolic analogs such as IPTG replacing tetracycline, anhydrous tetracycline, lactose, arabinose, xylose and lactose.

Diffocin Diffocin is an R-type hmw bacteriocin isolated from Clostridium difficile and is bactericidal against other Clostridium difficile strains. Diffocin may be isolated from a Clostridium difficile strain that grows in an anaerobic environment in the presence of mitomycin C. In some embodiments, the diffocin is a clinically isolated strain of Clostridium difficile Cd4, Cd16, Cd19123, Cd19145, Cd19126, Cd19108, or ATCC Cd43593 (dif4, dif16, dif123, dif145, dif126, respectively). dif108 and dif43593). In one embodiment, the diffocin is isolated from Cd4 and in another embodiment is isolated from Cd16.

  In another embodiment of the invention, an isolated nucleic acid molecule encoding a diffocin derived from a Clostridial genome is provided. In one embodiment, the nucleic acid molecule comprises the gene cluster of SEQ ID NO: 1. In other embodiments, the nucleic acid molecule comprises the gene cluster of SEQ ID NO: 61. In other embodiments, the nucleic acid molecule encodes a diffocin comprising one or more polypeptides selected from the group consisting of SEQ ID NOs: 2-23, 49, 62-80. In still other embodiments, the nucleic acid molecule encodes a diffocin comprising one or more polypeptides selected from the group consisting of SEQ ID NOs: 4-16, 18, 19, and 66-80. In one embodiment, the nucleic acid molecule encodes the polypeptide of SEQ ID NOs: 2-23. In other embodiments, the nucleic acid molecule encodes the polypeptides of SEQ ID NOs: 49 and 62-80.

  Further provided is a mutant diffocin. Mutant diffocins include diffocins having an amino acid sequence that is at least 80% identical to a polypeptide selected from the group consisting of SEQ ID NOs: 4-16, 18, 19, and 66-80. In other embodiments, the mutant diffocin is at least 85%, 88%, 89%, 90%, 95 to a polypeptide selected from the gene group consisting of SEQ ID NOs: 4-16, 18, 19, and 66-80. %, 96%, 97%, 98%, or 99% have the same amino acid sequence.

  In some embodiments, the mutant diffocin may comprise a heterologous substrate junction region (BPAR), wherein the BPAR is at least in one or more corresponding segments of SEQ ID NOs: 16, 54-56, and 78. 80% identical. In other embodiments, the BPAR is at least 85% identical to one or more corresponding segments of SEQ ID NOs: 16, 54-56, and 78. In other embodiments, the BPAR is at least 89% identical to one or more corresponding segments of SEQ ID NOs: 16, 54-56, and 78. In other embodiments, the BPAR is at least 90% identical to one or more corresponding segments of SEQ ID NOs: 16, 54-56, and 78. In yet other embodiments, the BPAR is at least 95% identical to one or more corresponding segments of SEQ ID NOs: 16, 54-56, and 78. In yet other embodiments, the BPAR is at least 98% identical to one or more corresponding segments of SEQ ID NOs: 16, 54-56, and 78. In a further embodiment, the BPAR is at least 99% identical to one or more corresponding segments of SEQ ID NOs: 16, 54-56, and 78.

  In some embodiments, the mutant diffocin comprises at least 50 contiguous amino acids at the amino terminus of the native BPAR polypeptide. In a further embodiment, the mutant diffocin comprises at least 100 contiguous amino acids at the amino terminus of the native BPAR polypeptide. In certain embodiments, the linking chain of at least 50 amino acids is at the amino acid terminus of BPAR that is at least 80% identical to the polypeptide of SEQ ID NO: 78. In some embodiments, the linking chain of at least 50 amino acids is at the amino acid terminus of BPAR that is at least 85%, 90%, 95%, or 98% identical to the polypeptide of SEQ ID NO: 78. In other embodiments, the binding chain of at least 100 amino acids is at least 85%, 88%, 89%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polypeptide of SEQ ID NO: 78. Is at the amino acid terminus of BPAR.

  In other embodiments, the mutant diffocin comprises a BPAR with a cognate RBD. As used herein, “BPAR cognate to RBD” or “cognate BPAR” refers to a pair of BPAR and RBD that appear simultaneously in a native diffocin, Clostridium difficile genome, bacteriophage, or prophage. In certain embodiments, the RBD and its cognate BPAR are heterologous other than the expressed diffocin molecule. In one embodiment, the cognate BPAR is fused to the amino terminus of the native BPAR of diffocin to form a “fusion BPAR”. Thus, in some embodiments, the mutant diffocin comprises a fusion BPAR. In certain embodiments, the mutant diffocin comprises a heterologous RBD and its cognate BPAR. In some embodiments, the fusion BPAR is at least a sequence selected from the gene group consisting of SEQ ID NOs: 88, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 115. 80% identical. In another embodiment, the fusion BPAR is at least 85 in a sequence selected from the gene group consisting of SEQ ID NOs: 88, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 115. %, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

  In further embodiments, the mutant diffocin may comprise a heterologous receptor binding domain (RBD), wherein the RBD is at least 80% in one or more corresponding segments of SEQ ID NOs: 17 and 49-53. Or at least 80% identical to a polypeptide comprising a receptor binding domain (RBD) region of a polypeptide selected from the group of genes consisting of SEQ ID NOs: 54-56. In other embodiments, the RBD is at least 85% identical to one or more corresponding segments of SEQ ID NOs: 17 and 49-53, or is selected from the group of genes consisting of SEQ ID NOs: 54-56. It is at least 85% identical to a polypeptide comprising the receptor binding domain (RBD) region of the peptide. In other seedling forms, the RBD is at least 89% identical to one or more corresponding segments of SEQ ID NOs: 17 and 49-53 or is selected from the group of genes consisting of SEQ ID NOs: 54-56. It is at least 89% identical to a polypeptide comprising the receptor binding domain (RBD) region of the peptide. In another embodiment, the RBD is at least 90% identical to one or more corresponding segments of SEQ ID NOs: 17 and 49-53, or selected from the group of genes consisting of SEQ ID NOs: 54-56. It is at least 90% identical to a polypeptide comprising the receptor binding domain (RBD) region of the peptide. In other embodiments, the RBD is at least 95% identical to one or more corresponding segments of SEQ ID NOs: 17 and 49-53, or selected from the group of genes consisting of SEQ ID NOs: 54-56. It is at least 95% identical to a polypeptide comprising the receptor binding domain (RBD) region of the peptide. In other embodiments, the RBD is at least 98% identical to one or more corresponding segments of SEQ ID NOs: 17 and 49-53, or is selected from the group of genes consisting of SEQ ID NOs: 54-56. It is at least 98% identical to a polypeptide comprising the receptor binding domain (RBD) region of the peptide. In other embodiments, the RBD is at least 99% identical to one or more corresponding segments of SEQ ID NOs: 17 and 49-53, or is selected from the group of genes consisting of SEQ ID NOs: 54-56. It is at least 99% identical to a polypeptide comprising the receptor binding domain (RBD) region of the peptide. In some embodiments, the receptor binding domain (RBD) region comprises amino acid residue 51 linked to the carboxy terminal residue of SEQ ID NO: 54, 55, or 56.

  In yet other embodiments, the RBD is produced from a Prophage insert or Prophage residue contained in a Clostridium difficile genome, bacteriophage, Clostridium difficile. A “prophage residue” or prophage element or site refers to a sequence that encodes only the position of a phage or isolated phage protein, rather than a complete phage molecule. Thus, in some embodiments, prophage residues may include, for example, sequences encoding RBD and its cognate BPAR, and substrate genes. In some embodiments, the RBD is at least 80% identical to an RBD selected from the gene group consisting of SEQ ID NOs: 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 116. is there. In other embodiments, the RBD is at least 85%, 88% of RBD selected from the gene group consisting of SEQ ID NOs: 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 116. 89%, 90%, 95%, 96%, 97%, 98% and 99% are identical.

  In another embodiment, the diffocin can have a modified bactericidal spectrum by fusing the phage tail RBD to the product of diffocin ORF 1373 by genetic recombination. ORF1374 encodes an initial spectral determinant or native diffocin RBD, while this large protein is complexed with the ORF1373 protein and the ORF1373 protein provides BPAR, ie, the RBD of the ORF1374 protein is joined to the diffocin substrate structure. Let ORF 1373 is similar to the tail fiber genes of myovirus bacteriocin such as ΦCD2 (SEQ ID NO: 54), ΦCD119 (SEQ ID NO: 55), and ΦCD27 (SEQ ID NO: 56) as well as the tail fiber of R-type pyocin. And has the same amino acid sequence. The diffocin ORF 1373 (eg, SEQ ID NO: 16 or 78) is characteristic of the first 160 amino acids, particularly in the N-terminal protein or BPAR, but of the Clostridium difficile myovirus phage, ΦCD2 (SEQ ID NO: 54), It has an arrangement very similar to the tail fiber. However, their phage tail fibers are longer than diffocin ORF 1373 proteins and contain a C-terminal RBD to distinguish their target bacteria. The diffocin ORF 1373 protein does not contain this latter domain, and the RBD function has been replaced by an isolated polypeptide encoded by ORF 1374. Thus, ORF1374 can be deleted together from the RBD of phage tail fibers, such as the diffocin gene cluster and the tail fiber of ΦCD2, and can therefore be fused to the diffocin BPAR encoded by ORF1373. A diffocin having a phage tail protein and having a bactericidal spectrum associated with the host region of the donor phage can be generated. Importantly, since the region of the amino acid sequence is homologous between the ORF 1373 protein that successfully functions and fuses with the Clostridium difficile phage tail-like fiber, one is mutated or non-mutated from the Clostridium difficile phage to the host region. Isomers can be selected, and the area is to be a new source of RBD to create modified diffocins with a new bactericidal spectrum.

  In one embodiment of a recombinant diffocin, the RBD provides a diffocin that is replaced with an RBD from another bacteriophage that infects other Clostridium difficile strains or Clostridium difficile. In one embodiment, the diffocin nucleic acid molecule comprises a sequence encoding SEQ ID NO: 16 or 78, and does not include one corresponding to a native RBD (ie, a sequence encoding SEQ ID NO: 17 or 49, respectively). In other words, the native RBD is replaced with an RBD encoding the isomeric sequence. In certain embodiments, the nucleic acid molecule comprises a heterologous sequence encoding a receptor binding domain (RBD) of an R-type bacteriocin of different strains of Clostridium bacteria. In one embodiment, the nucleic acid molecule is a polypeptide selected from the gene group consisting of the sequence encoding SEQ ID NO: 16 or 78 and the gene group consisting of SEQ ID NO: 49-53 or the polypeptide selected from the gene group consisting of SEQ ID NO: 54-56 Contains a sequence encoding a receptor binding domain. In other embodiments, the nucleic acid molecule comprises an RBD from a polypeptide selected from the group consisting of SEQ ID NOs: 2-16 and 18-23 or SEQ ID NOs: 62-80, and the gene group consisting of SEQ ID NOs: 17 and 49-56 Consisting of a heterologous sequence encoding

  In other embodiments of diffocin by genetic recombination, the RBD of the diffocin may be replaced with a modified form of native RBD. “Native RBD” refers to an RBD having an amino acid sequence specific to an RBD isolated or cloned from a Clostridium difficile strain or a bacteriophage that infects Clostridium difficile. Examples of native RBDs from multiple Clostridium difficile strains include SEQ ID NOs: 17 and 49-53. Examples of native RBD from bacteriophages infected with Clostridium difficile include SEQ ID NOs: 54-56 (eg, amino acid residue 51 linked to the carboxy terminal residue). In some embodiments, the modified RBD comprises a change in the amino acid sequence of the RBD that is related to the native RBD. Non-limiting examples of amino acid sequence alterations include the insertion (or addition) or removal of one or more amino acids. In a further embodiment, the diffocin is disclosed in patent applications US 2006-0121450, Jun. Substitution with an RBD derived from an organism that diversifies its structure through the development of a retreoment that creates diversity (DGR), or its insertion, as described in the 8,2006 publication (incorporated herein by reference in its entirety) including.

  In some embodiments, the modified RBD has a different bactericidal spectrum than the unmodified or native RBD. In certain embodiments, the modified RBD is at least 80% identical to the native RBD. In another embodiment, the RBD is in a receptor binding region of a polypeptide selected from the group consisting of SEQ ID NOs: 17 and 49-53, or a polypeptide selected from the group consisting of SEQ ID NOs: 54-56. At least 85%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical, and the modified RBD has a bactericidal spectrum different from that of an unmodified or native RBD. Have.

  In some embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a cognate chaperone of the RBD. In one embodiment, the chaperone is selected from the gene group consisting of SEQ ID NOs: 89, 90, 113, 114, and 117.

The target bacterium Clostridium difficile strain, due to its pathogenicity, was isolated from a very wide range of patients by pulse gel electrophoresis. The abnormally grown toxins of BI / NAP1 or ribotype 027 strains are highly toxic as a result of the lack of the function of the tcdC gene that suppressively regulates the expression levels of toxin A and toxin B (McDonald et al., 2005).

  In fact, Clostridium difficile strains harboring a tcdC mutagenesis gene have been spreading in medical facilities and between facilities. These epidemic highly toxic strains are particularly important target bacteria, and it is important that they can be prophylactically eliminated from the gastrointestinal tract of carrier patients by oral administration before or immediately after the start of conventional antibiotic treatment. Clostridium difficile bacteria that produce wild-type levels of toxin A and toxin B are potentially lethal and are very important target pathogens, especially complications for patients over 50 years old (BartlettJG , 2002).

  Factors such as virulence that can be attached to the target surface of Clostridium difficile strains, adaptation factors such as S-layer proteins, and epidemics, regardless of whether or not the strain is epidemic, Such pathogens provide an attractive means of resolving their virulence or adaptation factors if they appear as a counter-force against the targeted R-type bacteriocin. Because of the high specificity of diffocin RBD, life forms other than Clostridium difficile are not targets, and the symbiotic bacteria of the gastrointestinal tract—as they do not cause collateral damage to normal digestive functions and bacteria necessary for health, These points are the specific and powerful advantages of diffocin.

  “Infection” refers to the growth of bacteria in a site or tissue or non-bacterial cell, where the bacteria cause, or potentially cause, a disease or symptom in that site or tissue or non-bacterial cell. Infection treatment may include prophylactic treatment with spores of detoxifying Clostridium difficile producing a diffocin such as a drug, material, producer cell, or diff43593. Subjects to be treated include, but are not limited to, donated organs, tissues, and cells, medical devices such as ventilators and dialysis machines, and wounds during or after surgery. Other uses include the removal of target bacteria that cause further growth problems. In additional embodiments, hmw bacteriocin is used to treat food, plant, or harvest of bacterial infections or contaminated plants, or to treat environmental developments in locations such as target bacterial medical or commercial facilities. The

  As a description of the present disclosure, an antibacterial R-type bacteriocin may be used to inhibit the growth, persistence, or replication of specific bacterial populations. The bacterial group may be a pathogenic or environmentally harmful strain or may be treated in a prophylactic manner. In general, pathogenic microorganisms cause disease, sometimes only under certain circumstances.

Preparation and use of diffocin Diffocin is a particle of approximately 10 million daltons and creates differential centrifugation, differential filtration, aqueous two-phase separation, polyethylene glycol (PEG) precipitates and / or biopharmaceutical grade oral antimicrobials Can be separated and purified by ion exchange chromatography. R-type bacteriocin has been shown to be stable to freeze-thaw and a stable formulation can be made by dry spray.

  In some embodiments of the invention, methods for producing R-type hmw bacteriocin are provided. The method comprises a nucleic acid sequence genetically linked to an inducible promoter sensitive to an inducing agent at a concentration that induces expression of said R-type bacteriocin, the producer encoding said nucleic acid sequence encoding R-type hmw bacteriocin Methods of exposing the cells and purifying the expressed R-type bacteriocin. In one embodiment, the R-type high molecular weight (hmw) bacteriocin comprises one or more polypeptides selected from the group consisting of SEQ ID NOs: 2-23 or SEQ ID NOs: 49 and 62-80. The nucleic acid molecule is heterologous to the producer cell's natural nucleic acid and may be contained in the producer cell's chromosome or in an episomal expression vector.

  Like the targeted powerful bactericides, diffocin is used to eliminate or non-settle Clostridium difficile from the lower gastrointestinal tract of humans and other animals and consequently prevent CDAD. Animals and humans treated with antibiotics with a broad spectrum of bactericidal spectrum are at high risk of developing potentially fatal CDAD if infected with Clostridium difficile. Non-settlement is a particularly attractive utility of diffocin to preserve healthy gastrointestinal flora. In addition, diffocin is a detoxifier capable of producing producer cells or diffocin to reduce the pathogen burden of acute CDAD and / or reduce the high incidence or recurrence of CDAD or recurrence after successful treatment with other therapies. The Clostridium difficile spores can be administered directly or indirectly.

Mode of Administration R-type bacteriocin delivered to the stomach and upper duodenum is inactivated at an acidity of pH 4.0 or less, which is normally functioning there. However, in order to reach the target pathogenic bacteria that mainly settle in the lower gastrointestinal tract, diffocin must pass through the upper gastrointestinal tract. In such cases, one or more known methods for protecting sensitive agents from acids and proteases in the upper gastrointestinal tract and delivering the active agent to the remote upper or lower gastrointestinal tract In this way, diffocin can be prescribed. In addition, animals can be treated with antihistamines such as cimetidine and proton pump inhibitors to prevent stomach acidification prior to oral administration of R-type bacteriocin. In this way, a properly prescribed diffocin, a producer cell capable of producing diffocin, or a non-toxic Clostridium difficile bacterium producing diffocin, in a normal or drug-resistant human or animal with a gastrointestinal intestinal acidification By orally administering the spore, it is delivered to the settled part of the pathogenic bacteria and the effect is made effective. Depending on the intestinal transit time, the frequency of direct or indirect oral administration of diffocin may be every 6 hours, 12 hours, 18 hours, 24 hours, 1 week, or 1 month. Diffocin may also be administered to CDAD-onset patients or patients who have recently recovered from CDAD at the same or higher frequency. Particularly in the management of onset CDAD, diffocin may be dispensed directly or indirectly and administered orally for each rectal suppository, enema, or colon perfusion.

  The genetically modified diffocins of the present disclosure may be administered to any subject who is injured, diagnosed or suspected of having a bacterial infection or contamination. By way of non-limiting example, such subjects include not only animal (mammals, reptiles, amphibians, birds, fish) species, but also insects, plants, fungi. Non-limiting examples of individual mammals include humans, non-human primates, cattle, pigs, goats, sheep and other agricultural species, mice and rats, rodents, pets, exhibits, shows, etc. Including animals such as dogs, cats, guinea pigs, rabbits, and horses, and work animals such as dogs and horses. Non-limiting examples of individual birds include pets or show birds such as chickens, ducks, geese, parrots and parakeets. Animal subjects to be treated with the recombinant diffocin of the present disclosure may also include quadruped or biped, aquatic animals, vertebrates, invertebrates, insects.

  In some embodiments, the subject to be treated is a still immature human child or other young animal. Thus, the present disclosure includes treatment of pediatric conditions that are infections of bacteria or other microorganisms that are sensitive to the diffocins of the present disclosure.

  The present disclosure provides results for the treatment or prevention of opportunistic infections due to unwanted growth of bacteria present in the microflora of humans or non-human animals. Opportunistic infections are the result of a subject's immune suppression or antibiotic treatment that alters the urogenital (GU) or gastrointestinal (GI) symbiotic flora. The present disclosure also provides the results of treatment or prevention of immunosuppressed subjects and subjects exposed to other pharmaceutical agents. Diffocins with antibacterial activity may be used in combination with other antibacterial or antimicrobial agents, such as, but not limited to, antibiotics and antifungal agents. An “antimicrobial agent” is a formulation or ingredient that can be used for the growth or death of a unicellular organism. Antimicrobial agents include antibiotics, chemotherapeutic agents, antibodies (with or without complement), DNA, RNA, proteins, lipids, or chemical inhibitors of cell wall synthesis or function.

  In some embodiments, diffocin, producer cells, or diffocin-producing detoxified Clostridium difficile spores are formulated with a “pharmaceutically acceptable pharmaceutical additive”, enteric coating or carrier. . Such an ingredient is one that can be suitably used in humans, animals, and / or plants without undue side effects. Non-limiting examples of side effects include toxicity, irritation, and / or allergic reactions. Such pharmaceutical excipients or carriers are typical which correspond to a reasonable benefit / risk ratio. Non-limiting pharmaceutically suitable carriers include sterile aqueous and non-aqueous solutions, suspensions, and emulsions. Non-limiting examples include standard pharmaceutical additives such as phosphate buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. Examples of non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Water soluble carriers include water, alcohol / water solutions, emulsions or suspensions, buffered solvents including saline. Parenteral infusions include sodium chloride solution, Ringer's solution glucose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous fluids include liquid nutritional supplements, electrolytes (such as those based on Ringer's glucose).

  Additional formulations and pharmaceutical ingredients disclosed herein consist of isolated diffocins specialized for bacterial pathogens, 2, 3, 5, 10 or 20 or more different A mixture of diffocins, producer cells or detoxified Clostridium difficile spores with the ability to produce multiple diffocins targeting the same bacterial pathogen and two different bacterial pathogens or different strains of the same bacterial pathogen It consists of detoxified Clostridium difficile spores with the ability to produce multiple diffocins that target a mixture of 3, 5, 10, or 20 or more mixtures.

  Optionally, the components comprising the diffocin or producer cells of the present disclosure may be sprayed by drying or lyophilization using means known in the art.

  Diffocins are usually used in “safe and effective” amounts and concentrations. “Safe and effective” amounts and concentrations refer to an amount sufficient to exhibit the desired therapeutic response without undue side effects as described above. Diffocin is also used in “therapeutically effective” amounts and concentrations. “Therapeutically effective” amounts and concentrations are, but are not limited to, slow down the rate of bacterial cell division, stop bacterial cell division, or stop or reduce bacterial growth rate. Refers to the amount effective to produce the desired therapeutic response. A safe and effective amount or an amount effective for treatment or prevention will vary depending on a variety of factors, but will be readily determined by the skilled practitioner without undue experimentation. Examples of non-limiting factors include the special condition being treated, the physical condition of the subject (the subject to be treated), the type of subject being treated, the duration of the treatment, simultaneous therapy (if any), and adoption Includes specific prescriptions that are being made.

  The subject matter of the present invention that is generally described will be more readily understood with reference to the following examples, provided by way of illustration, and these examples are not intended to be limiting of the present disclosure except as specified. Not intended.

1. Determination of diffocin bactericidal activity Clostridium difficile cultures are subjected to a 10% CO ₂ , 10% H ₂ , 80% N ₂ atmosphere in a strict anaerobic environment in a Forma Scientific environmental chamber. In culture. All media, buffers, and plates were oxygen-free in this atmosphere for at least 24 hours prior to use. The culture was streaked on a Clostridium difficile selective agar plate (BD Diagnostics, BBL Cat. 222228) and cultured at 37 ° C. for 2 days. These plates were stored to maintain anaerobic conditions at ambient temperature.

To induce diffocin, Clostridium difficile was grown at 37 ° C. without shaking in a liquid culture using Brucella solvent (Difco). When the OD ₆₀₀ was about 0.2, mitomycin C was added to a final concentration of 3 μg / ml. The culture was then cultured for 3-16 hours. Bacterial lysis was detected by visual clearing of the culture.

  The culture was removed from the anaerobic chamber and killed bacteria were removed by centrifugation at 5,000 × g. The culture supernatant was passed through a 0.2 μm cellulose acetate syringe filter. The filtrate was centrifuged at 90,000 × g for 2 hours to obtain the diffocin precipitate. The precipitate made up to 1/50 the volume was resuspended in 10 mM Tris buffer pH 7.5, 50 mM sodium chloride solution, 3% mannitol solution.

  The target strain was grown in Brucella culture at 37 ° C. Add 100 μl culture to 5 ml preheated, oxygen-free, agar medium (0.5% agar) covered with Brucella, flow into Brucella plate (1.5% agar) and solidify I let you. A sample of 5 μl of diffocin preparation was spotted onto the plate and exposed to air drying (30 minutes). The plates were cultured at 37 ° C. overnight under anaerobic conditions. Antibacterial activity was determined by confirming the transparency at the location of the flora to which the sample was donated and the location of the spots, or by the presence or absence of bacterial growth.

2. Cloning of the Cd16 diffocin locus A sequence overview of the Cd16 genome of Clostridium difficile was obtained using a DNA genome 454 sequence analyzer. The Dif16 whole locus or gene cluster (SEQ ID NO: 1) was identified by comparison with the Cd630 strain (see above).

  Preparation of backbone BAC vector. The starting vector was pETcoco1 (Novagen). In order to remove the two enzymes XhoI from this vector, it was modified with primers AV1419 (SEQ ID NO: 24) and AV1420 (SEQ ID NO: 25) with the enzyme BbsI ends. For removal, specific regions were amplified from pETcoco1 DNA using these primers, and then the PCR product was cut with BbsI and religated to a longer pETcoco1 vector fragment previously cut with XhoI. This ligation destroyed two XhoI parts of pETcoco1. This latter plasmid was further modified in a similar manner to destroy EcoRI using primers AV1416 (SEQ ID NO: 26) and AV1245 (SEQ ID NO: 27). The resulting vector is called SW251.

  Preparation of a pUC19 vector that accepts a fragment of the diffocin gene cluster. Polylinker pUC19 (New England BioLabs) was modified by degradation with EcoRI and HindIII and ligation with oligos AV1372 (SEQ ID NO: 28), AV1373 (SEQ ID NO: 29), AV1374 (SEQ ID NO: 30), and AV1375 (SEQ ID NO: 31) did. This modification changed the polylinker to NotI-NheI-KpnI-XhoI-EcoRV-BstBI-BbsI-EcoRI-NsiI-SphI-BamHi-AscI. This is called SW232.

  Cloning of the diffocin gene cluster into SW232. Each of the three fragments of the diffocin gene cluster (SEQ ID NO: 1) was amplified by PCR from Cd16 DNA. The 5 'fragment (SEQ ID NO: 32) was amplified with NotI and XhoI terminal primers 1368 (SEQ ID NO: 35) and 1289 (SEQ ID NO: 36), respectively. The intermediate fragment (SEQ ID NO: 33) was amplified with XhoI and EcoRI terminal primers AV1288 (SEQ ID NO: 37) and AV1366 (SEQ ID NO: 38), respectively. The 3 'fragment (SEQ ID NO: 34) was amplified with EcoRI and BamHI end primers AV1367 (SEQ ID NO: 39) and AV1300 (SEQ ID NO: 40), respectively. These three PCR fragments were separated and cloned into SW232. The 5 ′, intermediate, and 3 ′ proteins are referred to as SW241, SW242, and SW243, respectively.

  Cloning of diffocin gene cluster into BAC SW251. Each of the three fragments of the diffocin gene cluster (incorporated into SW241, SW242, and SW243) was propagated by cloning into E. coli and purified and excised from the SW241, SW242, and SW243 vectors. SW241 was decomposed with NotI and XhoI, SW242 was decomposed with XhoI and EcoRI, and SW243 was decomposed with EcoRI and AscI. (Note that the AscI part is part of the modified SW232 polylinker described above)

  These three fragments were incorporated into SW251 which was first digested with NotI and AscI. The resulting plasmid was called DG461 and contained the dif16 whole gene cluster. It was amplified with E. coli.

  Creation of diffocin integration vector for Bacillus subtilis expression. A Bacillus subtilis integration vector, pDR111, containing the cloning / promoter part and the site of the amyE gene adjacent to the spectinomycin resistance gene was used.

  The pDR111 polylinker was modified by digestion with HindIII and SphI and ligation to oligos DG1 (SEQ ID NO: 41) and DG2 (SEQ ID NO: 42). In this process, NotI and AscI parts were added to pDR111. A region including the front and back of the entire amyE gene with the modified polylinker was amplified using primers DG9 (SEQ ID NO: 43) and DG10 (SEQ ID NO: 44), both of which have a BsaI end. This fragment was ligated to the NotI and AscI parts of SW251 (resulting in the destruction of the two sites) and created DG487. It should be noted that new NotI and AscI were introduced into DG487 by insertion of the pDR111-derived modified polylinker.

  After propagation of DG487 in E. coli, the diffocin cluster containing the NotI / AscI fragment from DG461 was activated and cloned into the NotI / AscI part of DG487. This new construct was named DG488 and was used as a vector for causing Bacillus subtilis to produce the entire diffocin gene cluster (see below).

3. Expression of the entire diffocin gene cluster in Bacillus subtilis The natural diffocin producer is Clostridium difficile, an obligate anaerobe. If exposed to oxygen, even in trace amounts, Clostridium difficile bacteria form spores and quickly die. It is difficult to produce even small amounts of diffocin from cultured Clostridium difficile, it is difficult to produce, is toxic, and it is required to produce a diffocin amount effective for prevention and treatment. Not realized even under anaerobic conditions. In an appropriate manner, the entire gene for diffocin from Clostridium difficile is first identified and isolated by molecular cloning, and the cluster is anaerobic Gram-positive hay for further genetic recombination and production Introduced into the fungus.

  A Bacillus subtilis integration vector, DG488, was made as described in Example 2 and included a total of 22,827 bases (SEQ ID NO: 1) of the diffocin locus. This vector was used to reassemble the diffocin gene cluster into the Bacillus subtilis genome.

  The Bacillus subtilis recipient strain was BDR123, which had a chloramphenicol resistance marker inserted into the amyE gene. When this strain was transformed into DG488, recombination occurred before and after the amyE sequence within the vector and the amyE genomic sequence. This was the result of the insertion of the entire sequence between the DG488 amyE gene, including the diffocin locus and the spectinomycin resistance gene, into the BDR123 genome. The successful recombinant gene was spectinomycin resistant but was sensitive to chloramphenicol because it lost the genetic marker as a result of recombination. This Bacillus subtilis strain was named BDR123-488.

  The entire diffocin locus was inserted into the DG488 vector (Example 2) and therefore was inserted in its entirety into BDR123-488. The entire locus of the inserted diffocin contained all the regulatory genes required for normal expression of Clostridium bacteria, and the structural gene of the diffocin particle had these genes and / or regulatory elements controlled. Since Bacillus subtilis and Clostridium are bacteria, it has been speculated that these regulatory elements of diffocin function in the background of Bacillus subtilis and are induced by DNA damage through the mechanism of RecA. In fact, as speculated, production of diffocin particles was induced in BDR123-488 by contact with the DNA damaging agent mitomycin C and killed Cd19099 strain (FIG. 7).

  Diffocin-regulated genes (ORF 1359, 1360, 1361) were in the 5 'region of the locus associated with the structural gene. There were also regulatory genes (ORF 1377 (SEQ ID NO: 20), 1378 (SEQ ID NO: 21), and 1379 (SEQ ID NO: 23)) in the 3 'region of the locus associated with the downstream structural gene. To eliminate the latter such regulatory gene, DG488 was generated from DG488 with a single-stranded three-way linkage. One PCR fragment was made from DG488 by PCR amplification using primers DG13 (SEQ ID NO: 45) and DG14 (SEQ ID NO: 46), and the other fragments were primers DG15 (SEQ ID NO: 47) and DG16 (SEQ ID NO: 48). It was made by PCR amplification using Both PCR fragments were digested with AscI and SphI. DG488 was digested with SphI to produce DG491, and the two degraded PCR fragments were ligated to a long vector fragment derived from SphI digested DG488. DG491 was transformed into BDR123 (BDR123-491) to generate genetically engineered Bacillus subtilis containing the diffocin gene cluster lacking ORFs 1377 (SEQ ID NO: 20), 1378 (SEQ ID NO: 21), and 1379 (SEQ ID NO: 23). Was transformed. As a result of the formation of the wild-type diffocin gene cluster in BDR123-488, these modified diffocin gene clusters lacking the ORF expressed active diffocin upon exposure to mitomycin C (FIG. 7).

4). Characterization of bactericidal spectral determination sequences from various diffocins Cd16 diffocin locus (SEQ ID NO: 1) and Cd630, other Cd sequence strains (QCD-66c26, QCD-23m63, QCD-32g58, QCD-63q42) and Cd4 (sequence) In comparison with the locus of number 61), with one exception, all open reading frames (SEQ ID NOs: 1 and 61) retained 89% -100% amino acid sequence identity. The exception was ORF1374. This exceptional sequence was similar in size but varied among all diffocin sequences, remaining only 30% sequence identity. The position of ORF1374 within the diffocin gene cluster was consistent with the position of the receptor binding domain. The ORF1374 sequence of the isolated active diffocin was determined and the sequence (SEQ ID NO: 17, 49-53) was found to be very diverse. A comparison of these sequences is shown in FIG. Furthermore, the spectrum of isolated diffocin (FIG. 2) reflected the similarity or dissimilarity of the ORF 1374 amino acid sequence of FIG. For example, the ORF1374 sequence of dif16 (SEQ ID NO: 17) and dif126 (SEQ ID NO: 52) differed by only one amino acid and the bactericidal spectra were almost identical. On the other hand, the ORF1374 sequences of dif16 (SEQ ID NO: 17) and dif108 (SEQ ID NO: 50) differed by 188 amino acids, and their bactericidal spectra were very different with little overlap. Conclusion ORF1374 was the only heterologous protein in the gene cluster because ORF1374 is a target recognition determinant and corresponds to the characteristic spectrum of each specific diffocin It was attached.

5. Expression and cloning of Dif4 in Bacillus subtilis The diffocin 4 locus was cloned from Cd4 in the same manner as for diffocin 16. However, some modifications were required due to the deletion of the EcoRI site, SEQ ID NO: 61, within the dif4 gene cluster. The polylinker using oligo DG211, SEQ ID NO: 57, and DG212, SEQ ID NO: 58 has an XhoI site, and plasmid SW251 (see Example 2) was modified to introduce NotI and AscI sites, respectively. . In this way, vector DG577 was created.

  Diffocin from Cd4 DNA was amplified in three fragments. The first fragment used primers DG210 (SEQ ID NO: 59) and AV1288 (SEQ ID NO: 37) to introduce XhoI and NcoI sites. The second fragment used primers DG209 (SEQ ID NO: 60) and DG15 (SEQ ID NO: 47) to introduce NcoI and AscI sites. These two fragments were cloned into DG577 previously cut with XhoI / AscI to create DG578. The third fragment was amplified using AV1368 (SEQ ID NO: 35) and AV1289 (SEQ ID NO: 36) to introduce XhoI and NotI sites, and DG578 previously cut with XhoI and NotI to create DG579. Cloned into. The latter construct containing the Dif4 gene cluster (SEQ ID NO: 61) had characteristics similar to DG491 for dif16. That is, it lacked unnecessary ORF1377, ORF1378, and ORF1379, which are presumed to be repressive sequences downstream of the structural gene of diffocin. An integration vector for introducing the Dif4 gene cluster was created by taking the NotI AscI fragment from DG579 and cloning it into DG487 (see Example 2). The plasmid constructed in this way is DG580.

  In order to express dif4 in Bacillus subtilis, DG580 containing the dif4 locus lacking ORF 1377-1379 (SEQ ID NO: 61) was integrated into the Bacillus subtilis genome by genetic recombination. The Bacillus subtilis receptor BDR123 had a chloramphenicol resistance marker inserted into the amyE gene. When this strain was transformed with DG580, genetic recombination occurred before and after the amyE sequence within the vector and the amyE genomic sequence. This was the result of inserting all the sequences between the DG580 and the amyE region including the diffocin locus and the spectinomycin resistance gene into the BDR123 genome. A successful recombination was spectinomycin resistance but was sensitive to chloramphenicol due to the lack of a chloramphenicol resistance marker as a result of this recombination. This Bacillus subtilis strain was named BDR123-580.

  This genetically modified dif4 contains all the regulatory genes required for normal diffocin expression in Clostridium difficile and, as shown previously for dif16 in Bacillus subtilis, by contact with mitomycin C, Dif4 particles were produced upon induction in BDR123-580, FIG. Thus, the examples of the present disclosure provide for the cloning and expression of both the dif4 and dif16 loci in Bacillus subtilis as an example of a non-pathogenic aerobic producer.

6). Determination of the sterilization spectrum of diffocin by ORF 1374 ORF 1374 encodes a long predicted peptide (˜200 kDa) which is shown as part of the purified diffocin structure by mass spectrometry. Comparing the gene classlers of the main gene products, dif4 and dif16, these clusters, which are presumed to be structural components, are particularly similar in amino acid level. The major amino acid sequence that differs between the two clusters is ORF1374. For this reason and other reasons discussed below, it was speculated that this gene product confers the target specificity of diffocin. To evaluate this, the dif4 ORF 1374 (ie, the sequence encoding SEQ ID NO: 49) was replaced with a Cd16-derived ORF 1374 (ie, the sequence encoding SEQ ID NO: 17) in DG580 to generate DG587. . DG587 was recombined into the Bacillus subtilis genome BDR123 for genetic recombination of BDR123-587, as was done for dif16 and dif4. The resulting BDR123-587 was exposed to mitomycin and treated with a lysate to adjust diffocin. The resulting diffocin particles have bactericidal activity against Clostridium difficile strain 19145 that is sensitive to diffocin 16 and are not capable of killing strain 19137 that is sensitive to diffocin 4 (FIG. 9).

  This experiment was further refined. The construction of DG587 resulted in ORF 1373, a Cd4 and Cd16 chimera. The construct was DG587 ORF 1373 restored to be 100% homologous to its origin Cd4 1374, SEQ ID NO: 78. Thus, a construct was created that was a complete exchange of only ORF 1374, SEQ ID NO: 17. This construct was named DG603. This construct was recombined into Bacillus subtilis genome BDR123 and induced with mitomycin C as described above. The resulting diffocin particles had bactericidal activity against strains 19099 and 19145, and lacked the bactericidal power of 19137. Thus, the bactericidal spectrum of diffocin was determined as expressed here by the protein encoded by ORF 1374 and by a modification of ORF 1374 that alters the bactericidal spectrum of diffocin.

7). Producer cells without PBSX that do not lyse when RecA is activated PBSX prophage is ubiquitous in wild-type Bacillus subtilis. Induced prophage possesses a stunted head structure, contains small random DNA fragments, and is defective. PBSX prophage is under the control of RecA and is thus induced by genes that damage DNA, such as mitomycin C, and other traits that severely stress bacteria. When this prophage is induced, it causes lysis and detaches PBSX. To control the expression of diffocin by modifying the activity of RecA or dinR / lexA, in order to avoid contamination with fungal media containing PBSX particles and to eliminate the lysis of Bacillus subtilis producer cells, Eliminated from B. subtilis BDR11 bacteria.

The PBSX gene knockout (deficiency) was constructed according to the procedure outlined by Liu et al. Briefly, in order to delete the araR gene of the parent strain BDR11 and create BDG2, using the primers and overlap extension PCR technique used in the Liu material, the B. subtilis arabinose promoter, P _araA -neo ^R , In the presence of neomycin / kanamycin resistance gene. This deletion of the araR gene was confirmed by PCR and conferring kanamycin resistance.

  Subsequently, DNA construction for deleting the PBSX locus itself was performed. For this construction, the following 5 PCR products were joined into one large product by overlapping extended PCR. That is, 5 ′ sequence of 1 kb xylB gene amplified from BDR11, 3 ′ sequence of 1 kb xylA gene amplified from BDR11, chloramphenicol resistance gene that is cat gene amplified from plasmid pJW034, araR amplified from BDR11, and This is a 1 kb sequence containing the xylB gene amplified from BDR11. The overlapping extended PCR product was cloned into the XmaI and SpeI sites of pU19. This construct was linearized with SacII and transformed into the BDG2 bacterial strain, which was spread on LB agar plates supplemented with 5 μg / ml chloramphenicol. Fungal colonies were removed from this plate and spotted on LB agar plates supplemented with 5 μg / ml chloramphenicol or 20 μg / ml kanamycin. Strains that are resistant to chloramphenicol and kanamycin were cultured for 4 hours in LB fluid medium without antibacterial selection and spread on LB agar plates supplemented with 20 μg / ml kanamycin. Fungal colonies that grew on these plates were evaluated by colony PCR to confirm the presence of the PBSX gene. By confirming the sequence of the PCR product that crosses the site of the PBSX gene in the BD123 wt strain, the exclusion of the PBSX gene clathler in the BDG9 strain was confirmed. Further analysis revealed that Bacillus subtilis dissimilar strains, BD123 or BDG2, BDG9, did not lyse or produce PBSX particles in the presence of 3 μg / ml mitomycin C.

  A PBSX deficient strain, BDG9, was transformed with plasmid DG580 to create BDG27. Transformation of the Cd4 diffocin gene clathler was confirmed by spectinomycin resistance. BDG27 was grown and induced with mitomycin C as described above. After 16 hours, hoping for diffocin to accumulate in the cells, the cells were collected and lysed with BugBuster (Novagen) to destroy PBSX deficient cells. The cells were lysed with BugBuster, the debris was removed by centrifugation, and the bactericidal activity against strain 19137 was evaluated with the supernatant. Diffocin produced by BDG27 showed resistance to Cd19137 but not Cd19099. Thus, it was proved that diffocin 4 was produced in this undissolved, PBSX-deficient strain.

8). Diffocin expression in Bacillus subtilis by induction of small molecule inducer, hydrogen peroxide In Example 3, mitomycin C was used to induce diffocin production in Bacillus subtilis strain BDR123. Mitomycin C is a DNA damaging gene and a carcinogen, so a low molecular inducer was sought. Hydrogen peroxide (H ₂ O ₂ ) has been shown to elicit SOS sensitivity as well as mitomycin C (Imlay and Linn, 1987). However, hydrogen peroxide is generally recognized as safe by the US FDA (GRAS). Furthermore, it has been shown to cause prophage induction in several aerobic bacterial species, including E. coli and Bacillus subtilis (Imlay and Linn, 1987, Bol and Yasbin, 1990). The effect of hydrogen peroxide on diffocin production in prophage and / or Clostridium difficile is not known, and Clostridium difficile is obligately anaerobic and normally does not contain hydrogen peroxide in the gastrointestinal tract. Slightly present.

  In order to determine whether hydrogen peroxide can induce diffocin production in Bacillus subtilis, a comparative study between mitomycin C producing diffocin and hydrogen peroxide in Bacillus subtilis strain BDG45 was performed. BDG45 contains in its gene the diffocin 4 gene clusterer (SEQ ID NO: 61) linked to chloramphenicol resistance transformed into the amyE sequence. BDG45 cultures were grown overnight, diluted back, and grown until OD600 reached 1.0. At that time, the proliferating bacteria were left or treated with 0.5 ug / ml mitomycin C, 0.2 mM or 1 mM hydrogen peroxide and cultured at 28 ° C. The sample took 21 hours to complete induction. Diffocin preparations were performed as in Example 1 except that the diffocin precipitate was resuspended in 10 mM Hepes pH 7.4, 50 mM NaCl (HN50). The bactericidal activity of the preparations was determined by serial dilution (5 times) with HN50 before spotting on the bacterial flora containing an isolate (19137) sensitive to diffocin 4 as in Example 1. The results of the bactericidal activity assay are shown in Table I. With an induction time of 21 hours, mitomycin C and 1 mM hydrogen peroxide treated samples readily produced diffocin bactericidal activity after 625 and 3125 fold dilutions, respectively. The 0.2 mM hydrogen peroxide treated sample slightly induced diffocin production. Subsequent studies have demonstrated that diffocin production is induced in aqueous hydrogen peroxide from 0.2 mM to 20 mM.

9. Nucleic acids, phages and prophages isolated from the Clostridium difficile genome encoding functional RBD proteins for heterologous diffocins The diffocin preparation obtained from Clostridium difficile isolate M68 has a broad bactericidal spectrum (Table II). ORF1374 (SEQ ID NO: 49) of diffocin 4 was recombined into a heterologous ORF 1374 (SEQ ID NO: 87) from strain M68, and the 3 'position of BPAR (SEQ ID NO: 78) of diffocin 4 was replaced by ATCC 43593 (SEQ ID NOs: 89 and 90). Recombined into a cognate BPAR (SEQ ID NO: 88) from strain M68 with a chaperone and expressed in Bacillus subtilis as described in Example 6. The resulting diffocin, Diff4_M68-1374, had a narrower bactericidal spectrum than the diffocin preparation isolated directly from the mitomycin C-derived M68 strain. While no other diffocin gene cluster was found in the M68 gene sequence, a plurality of putative prophage gene insertions having homology to known myoviruses were found. Several genes in the diffocin gene cluster have homology to genes in the contractile tail of Clostridium difficile myovirus phage, and the genes of Clostridium difficile and Clostridium difficile genomes are: It could be hypothesized that it contains a non-limiting prophage sequence and may function as a heterologous RBD source to alter the diffocin target. However, no RBD gene, prophage or other ORF in the gene sequence corresponding to the spectral determinant or any Clostridium difficile phage has been identified so far.

  By comparing the ORF derived from the diffocin gene cluster with the ORF derived from the putative prophage inserted into M68, a large number of similar genes and similar gene cluster structures were confirmed. However, downstream of the ORF with homology to the putative substrate J-aggregated protein and its BPAR, it contains up to 1,700 amino acid residues for a typical diffocin RBD (eg, SEQ ID NOs: 17, 49-53). A shorter ORF that encodes a protein of 400-560 amino acid residues where the ORF encoding the group is present but does not share homology with the typical diffocin RBD (Table III and SEQ ID NOs: 92, 94, 96, 98).

  Sequence analysis of these ORFs located downstream of the putative prophage BPAR revealed that they are homologous to each other in at least one of the three domains (N-terminal, middle, and C-terminal), and It is homologous to the ORF (SEQ ID NO: 92) downstream of the annotated BPAR in phiC2 phage, SEQ ID NO: 54. Interestingly, the DNA sequence encoding SEQ ID NO: 92 in phiC2 was not annotated with the expected protein in phiC2 phage. This is because the ORF immediately downstream of BPAR in the phage and prophage sequences was found to be homologous to the domain-like format and all downstream of the cognate BPAR and hence the ORF1374 gene. These ORFs were predicted to potentially encode RBD. As provided in this disclosure, rather than encode a potential RBD located in the genome of another Clostridium difficile isolate or located downstream of a putative BPAR in a phage that infects Clostridium difficile. A number of DNA sequences have been identified. It is the result of an original observation from Clostridium difficile M68. The RBD sequence numbers used to obtain functional heterologous diffocins are listed in the table (Table III).

  In order to determine whether these newly identified ORFs with previously unknown functions are direct diffocins that are bactericidal against the target strain of the novel Clostridium difficile, Four ORF 1374s (SEQ ID NO: 49) were replaced by ORF encoding the putative RBD by gene recombination to create a new heterologous diffocin. The amino acid sequence encoding the putative RBD and the adjacent 3 'position of the ORF encoding its cognate BPAR were cloned into a vector (described in Example 2) containing the Diff4 gene cluster (SEQ ID NO: 61) and ORF1374 of diffocin 4 ( SEQ ID NO: 49) and the CPAR of BPAR of the diffocin 4 (SEQ ID NO: 78) were replaced. SEQ ID NOs of amino acid residues of the fusion BPAR are listed in Table III. We observed the putative RBD of interest incorporated into heterologous diffocin 4 without the C-terminus of at least half of the cognate BPAR that is minimally bactericidal or inactive. Since the insert did not have a compatible cloning site within the vector backbone, three cloning strategies were designed to overcome this barrier.

  For the construct made with Strategy I, three double-stranded DNA segments with overlapping ends (referred to as upstream, intermediate, and downstream segments) were generated by PCR (Table IV, panels A, B, and C). ). The upstream segment (Table IV, panel A) consists of the unique BstBI of SEQ ID NO: 61, including up to the 5 'end of the BPAR gene encoding SEQ ID NO: 78. The middle segment (Table IV, panel B) consists of the total ORF of the putative RBD and the 3 ′ half of the upstream cognate BPAR. The downstream segment (Table IV, panel C) encodes SEQ ID NOs 79 and 80 that extend immediately downstream of SEQ ID NO 49 and into the unique AscI restriction site in the vector polylinker described in Example 2 above. Consists of regions. All three segments use oligonucleotides complementary to the template for proper assembly and contain overhangs complementary to adjacent fragments. Oligonucleotides with coding and non-coding helical structures used to amplify each construct are listed in Table IV by SEQ ID NO. The DNA template for each PCR reaction is listed in Table IV. Each of the three segment constructs was then combined into a single fragment by PCR using oligonucleotides complementary to the upstream segment coding helix and the downstream segment non-coding helix and amplified (SEQ ID NO: Table IV). reference). As a result, the combined single-stranded PCR fragments were cleaved with BstBI and AscI and ligated into a Diff4 vector backbone that had been previously digested with BstBI and AscI to produce the constructs listed in the table. Each other construct was then transformed into Bacillus subtilis and expressed using 0.5 mM hydrogen peroxide as a small molecule inducer as in Example 3 above.

  For the constructs created in Strategy II, use the overlapping PCR products complementary to the upstream segment, the intermediate segment containing the RBD region, and the downstream segment using the oligonucleotides listed in Table IV and the template for each construct. And as described in Strategy I. The overlapping PCR product was then pre-degraded with BstBI and AscI, and a vector backbone comprising SEQ ID NO: 61 (Example 4) assembled into a single stranded construct by the Gibson method (Gibson et al., 2009). Mixed). Each resulting construct was transformed into Bacillus subtilis separately and expressed in the manner described in Example 3 above, except that 0.5 mM hydrogen peroxide was used as the low molecular inducer. I let you.

  The Strategy III construct was created as specified in Strategy II except that it did not create a downstream segment. Instead, the intermediate segment containing the RBD region is expanded to include the ORF immediately downstream of the putative RBD (SEQ ID NO listed for each construct in Table IV) and to overlap the vector backbone AscI site. did. As a result, DNA encoding SEQ ID NOs: 79-80, a DNA that is a putative chaperone for tail structure assembly of diffocin 4, is a DNA sequence encoding an ORF found immediately downstream in the cognate putative RBD, and It was replaced with a DNA sequence that appeared to be a chaperone that assembles the cognate putative RBD. Each resulting construct is then transformed into Bacillus subtilis and expressed as described in Example 3 above, except that 0.5 mM hydrogen peroxide is used as the small molecule inducer. It was.

  A diffocin preparation from a heterologous diffocin DNA construct expressed in Strategy I-III was made and tested for bactericidal activity against an isolated Clostridium difficile panel. The sensitivity of the isolate to the bactericidal activity of each heterologous diffocin construct is listed in Table II. In comparison to the Diff4_R20291-RBD1 construct containing the native diffocin 4 chaperone (SEQ ID NO: 79-80) relative to R20291-RBD1, the construct comprising the Diff4_R20291-RBD1 + cognate chaperone (SEQ ID NO: 113, 114), A more stable bactericidal activity was observed.

10. Enhanced stability of heterologous diffocins including new, non-1374 based RBDs In order for diffocins to be effectively produced and delivered to animals in need, they need to be active in a variety of different physical environments. For this purpose, the physical properties of naturally occurring and heterogeneous diffocins were examined. The diffocin was made as described in Example 9, except that the prepared diffocin centrifuge pellet was resuspended in the appropriate buffer in each study.

  For temperature stability studies (Table V), diffocin was resuspended in HN50 (pH 7.4) and incubated at the indicated temperature and time.

  In pH sensitivity studies (Table VI), in 5 mM sodium citrate solution acidified with citric acid to the specified acidic pH, or 12.5 mM sodium bicarbonate / HN50 (or specified alkali) The diffocin was resuspended in TN50 for a diffocin 4 solution alkalized with sodium hydroxide to pH. Samples were incubated for 30 minutes at room temperature at the specified pH.

  In both studies, samples were serially diluted (5-fold) in HN50 and tested for susceptibility to Clostridium difficile isolates by bactericidal activity by spot assay (used to test the bactericidal activity of each diffocin. Sensitive strains are listed in Tables V and VI). The diffocins studied include: That is, natural diffocin 4 (encoded by SEQ ID NO: 61), heterologous Diff4_43593-1374 (RBD encoded by SEQ ID NO: 53), heterologous Diff4_M68-RBD1 (BPAR and RBD encoded by SEQ ID NOs: 91 and 92), heterologous Diff4_M68- RBD4 (BPAR and RBD encoded by SEQ ID NOs: 95 and 96), heterologous Diff4_M68-RBD5 (BPAR and RBD encoded by SEQ ID NOs: 97 and 98).

  The heterologous diffocin containing phage RBD was stabilized at a higher temperature for a longer time than the native diffocin or heterologous diffocin containing ORF1374 protein as RBD (Table V). A heterologous diffocin preparation containing the novel RBDsM68-RBD1 (SEQ ID NO: 91, 92), M68-RBD4 (SEQ ID NO: 95, 96) and M68_RBD5 (SEQ ID NO: 97, 98) was compared to a culture at 37 ° C. for 15 minutes. Incubated at 45 ° C. for 1 hour to correct or enhance activity (Table V). Interestingly, the native diffocin 4 RBD (SEQ ID NO: 49) and the heterologous Diff4_43593-1374 RBD compared to a novel, small molecule, non-1374 based RBD heterologous diffocin that retained bactericidal activity under the same temperature conditions. A diffocin containing a CD1374-based RBD, such as (SEQ ID NO: 53) did not retain it. The former diffocins containing these two 1374 base RBDs lost 80% and over 95% bactericidal activity when heated at 45 ° C for 1 hour, respectively.

  In addition to the development of enhanced thermal stability, the heterologous diffocin containing RBD M68-RBD4 (SEQ ID NO: 96) was stable over a wider pH range compared to native diffocin 4 containing ORF1374 (Table VI). The results of the bactericidal activity test by spot assay showed that Diff4_M68-RBD4 retained bactericidal activity at pH 3.4 to pH 9 and also retained detectable residual activity even in the range up to pH 2.5 and pH 10.6. Diffocin 4 remained active only in the pH 5.5 to pH 10 range. Diffocin, which has the ability to cope with expanded pH, is suitable for in vivo functions in places where an acidic environment such as the stomach may occur. These results show that heterologous diffocins containing RBD, which is a new class of non-1374, low molecular RBDs, are more stable and more efficient in producing and treating natural diffocins or macromolecules, diffocins containing heterologous 1374-based RBDs, etc. It shows that it possesses advantages for application.

11. Bactericidal activity of heterologous diffocin containing novel RBD remaining in the gastrointestinal tract of the mouse The ability of oral administration of diffocin to remain in the body was evaluated in the mouse. As a positive control, a mixture consisting of natural diffocin 4 (encoded by SEQ ID NO: 61), heterologous Diff4_M68-RBD4, and R-type bacteriocin obtained by genetic manipulation not related to the present disclosure, AvR2-V10 (Scholl et al., 2009) was prepared in 12.5 mM sodium bicarbonate and administered orally to normal healthy mice (n = 3). Two hours prior to administration, an H ₂ antagonist, ranitidine (100 mg / kg), was injected to prevent or minimize gastric acidification. Feces were collected every hour for 8 hours, homogenized in HN50 containing protease inhibitors, and centrifuged to remove tissue debris. The supernatant was filtered through a 0.45 micron filter, diluted 5-fold with HN50, and tested for bactericidal activity by spot assay on the bacterial flora of susceptible C. difficile isolates. Clostridium difficile 19137 is inherently sensitive to bactericidal properties of diffocin 4 and Clostridium difficile CF5 is inherently sensitive to bactericidal properties of diffocin 4_M68-RBD4, and E. coli EDL933 is an AvR2-V10 There was an inherent susceptibility to bactericidal properties and susceptibility to each other's inherent bactericidal components was observed in the oral mixture. The results of the bactericidal test in each susceptible strain were in contrast to the test mouse feces. An aliquot of the oral cocktail was stored and spotted in parallel with each flora as a positive control.

  Bactericidal activity in mouse fecal samples was demonstrated in heterologous Diff4_M68-RBD4 and not in native diffocin 4 (Table VII).

  In the activity comparison between mice, Diff4_M68-RBD4 activity was observed as early as 2 hours and as late as 8 hours after oral gavage. In the bactericidal activity observed after 25-fold dilution, the peak of activity restoration was observed 3 and 4 hours after oral gavage. Native diffocin 4 was not active in fecal samples at any time. The activity against a certain amount of strain 19137 of the cocktail solution stored separately was confirmed, and it was confirmed that natural diffocin 4 in the oral cocktail was active at the time of oral administration. These results and the facts of Example 10 showed that the heterogeneous diffocin, including the novel, a heterologous diffocin including the non-1374 base RBD, was stable at high temperatures and in more acidic environments when compared to the native diffocin. . Furthermore, it was shown that these bactericidal activities remained after oral administration even after exposure to the gastrointestinal tract of animals, which are sites of Clostridium difficile growth and pathogenesis.

12 Heterologous diffocin that reduces in vivo CDI The effect of diffocin on Clostridium difficile infection (CDI) was examined using mice exposed to Clostridium difficile spores. As a pre-treatment, 2 groups of mice (6 mice each) were given 5 days of water containing an appropriate amount of cefoperazone (0.5 mg / mL), destroyed the gastrointestinal flora and mice were orally administered with spores. Was susceptible to infection with Clostridium difficile. Both groups of mice were given a 36-hour recovery period prior to ranitidine administration (dose 100 mg / kg / day) in drinking water. Twelve hours after the start of ranitidine administration, the treatment group was given a heterologous Diff4_M68-RBD4 in a 12.5 mM sodium bicarbonate solution by gavage (dose 10 ¹¹ bactericidal unit-Gebhart et al.,). 2012, Ritchie et al., 2011, Scholl et al., 2009, described and defined). Two hours later, both groups of mice were exposed to 2 × 10 ⁵ CFU of Clostridium difficile spores prepared from CD630. Forced administration of diffocin to the treatment group was started 4 hours after spore contact and continued every 6 hours. 24 hours after spore contact, feces from both groups of mice are collected, weighed, homogenized, and placed on a Clostridium difficile selective plate containing 0.05% sodium taurocholate to promote germination. Then, it was spread as a 10-fold diluted solution. Total CFU per sample was counted and converted to CFU / g feces (FIG. 10). The geometric mean CFU / g feces for each group was calculated and compared with the Student t test.

Data analysis showed that the heterologous diffocin, Diff4_M68-RBD4, was active in vivo and reduced in vitro elimination and colonization of Clostridium difficile strain CD630. Mice that were challenged with and before Clostridium difficile excreted Clostridium difficile at a rate of 3.7 × 10 ⁴ CFU / g feces (geometric mean), whereas mice without diffocin administration Clostridium difficile was discharged at a rate of 6.9 × 10 ⁵ CFU / g feces (geometric mean). This meant a 18.6-fold dilution of Clostridium difficile. Comparison of efflux results by Student's t test gave a null hypothesis <0.05, and the difference in efflux caused by diffocin was shown to be statistically significant. This experiment demonstrated that a novel, non-1374 based RBD-containing heterologous diffocin is active in vivo and reduces Clostridium difficile efflux and colonization.

  The term “comprising” is used interchangeably with “including”, “containing” or “characterized by”, but is an inclusive or unconstrained term And does not preclude additional, non-repeated elements and method steps. The phrase “consisting of” does not exclude any element, procedure, or composition not specified in the claims. The phrase “consisting essentially of” limits the claim to those specific substances or procedures that do not materially adversely affect the specific substance or procedure and the fundamental and novel features of the claimed invention. To do. This disclosure contemplates embodiments of inventive compositions and methods that correspond to the scope of each sentence. Thus, a composition or method consisting of the elements or procedures to be repeated contemplates certain embodiments in a composition or method that essentially comprises or includes those elements or procedures.

  All references, including patents, patent proposals, and publications cited in this disclosure, are hereby incorporated by reference in their entirety, whether or not specifically referred to before.

  Throughout the description of the invention, the same can be done within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation. Those skilled in the art will understand.

  However, while the invention has been described with reference to particular embodiments, it will be understood that further modifications are possible. This application generally includes departures from the present disclosure within the principles of the invention and within the well-known scope or practice routinely practiced by those skilled in the art and applies to the essential qualities specified above. It is intended to include any modifications, uses, and adaptations of the present invention that are possible.

  While the invention has been described with reference to the above examples, it will be understood that it encompasses modifications and variations within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

  This application includes a sequence listing that electronically corresponds to the ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on March 6, 2014 and has the name CF210341_SL. At txt, the size is 476,921 bytes.

Claims (30)

An isolated nucleic acid molecule encoding an R-type high molecular weight (hmw) bacteriocin, wherein the nucleic acid molecule is derived from the genome of a first strain of Clostridium difficile and consists of:
A first polynucleotide sequence that is at least 80% identical to a second polynucleotide encoding SEQ ID NOs: 66-77, wherein said nucleic acid molecule is further a prophage derived from the genome of a second strain of Clostridium difficile, or Consisting of a heterologous sequence encoding the receptor-binding domain (RBD) of the prophage residue or the RBD of a bacteriophage that infects Clostridium difficile, and
Wherein the R-type hmw bacteriocin consists of at least 50 contiguous amino acids at the amino terminal end of the first substrate junction region (BPAR) polypeptide that is at least 80% identical to the polypeptide SEQ ID NO: 78; and
Here, the R-type hmw bacteriocin has bactericidal activity on at least one of Clostridium difficile, and
Here, it is the third polynucleotide encoding at least the C-terminal part of the second BPAR homologous to the RBD.   2. The RBD of claim 1, wherein the RBD is at least 80% identical to an RBD selected from the gene group consisting of SEQ ID NOs: 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 116. The described nucleic acid molecule.   2. The nucleic acid molecule of claim 1, wherein the third polynucleotide encodes all portions of the cognate BPAR of the RBD.   2. The third polynucleotide encoding all portions of a cognate BPAR of RBD is inserted into a nucleic acid molecule, so that the C-terminus of the cognate BPAR of RBD is fused to the amino terminus of the first BPAR. A nucleic acid molecule according to 1.   The group of genes wherein the third polynucleotide encoding the C-terminal part of the cognate BPAR of RBD is SEQ ID NO: 88, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 115 2. The nucleic acid molecule according to claim 1, comprising a sequence encoding BPAR that is at least 80% identical to a sequence selected from:   2. The nucleic acid molecule of claim 1, further comprising a fourth polynucleotide encoding a cognate chaperone of RBD.   The nucleic acid molecule according to claim 6, wherein the cognate chaperone is selected from the group of genes consisting of SEQ ID NOs: 89, 90, 113, 114, and 117.   An expression cassette comprising the nucleic acid molecule according to claim 1.   9. The expression cassette of claim 8, wherein the expression cassette of R-type high molecular weight (hmw) bacteriocin is inducible or repressive.   10. An expression cassette according to claim 9, wherein expression is induced by a small molecule inducer (inducer) or derepressor (delipressor).   The expression cassette according to claim 10, wherein the small molecule inducer or depressor is a generator of reactive oxygen species (ROS) or ROS.   12. The expression cassette of claim 11, wherein the ROS is a peroxide that is non-toxic to humans or other animals.   13. The expression cassette of claim 12, wherein the peroxide is hydrogen peroxide.   R-type hmw bacteriocin encoded by claim 1.   15. R-type hmw bacteriocin according to claim 14, wherein the bacteriocin can be administered orally to an animal and can be excreted in the feces in a form where bactericidal activity is still present.   15. The R-type hmw bacteriocin according to claim 14, wherein the bacteriocin still has a bactericidal activity after culturing at 25 ° C. for 30 minutes between pH 2.5 and pH 10.   15. The R-type hmw bacteriocin according to claim 14, wherein the bacteriocin remains a bactericidal activity even after incubation at 45 ° C. for 30 minutes.   A method for producing R-type hmw bacteriocin comprising exposure of a producer cell comprising a nucleic acid molecule according to claim 1 and binding to an inducible promoter by genetic recombination and being effective in inducing expression of R-type hmw bacteriocin A method of binding to an inducing agent at a suitable concentration, thereby producing R-type hmw bacteriocin.   The nucleic acid molecule encoding R-type hmw bacteriocin is heterologous to the genome of the producer cell, and the nucleic acid molecule is contained in the producer cell's chromosome or is contained in the producer cell's extrachromosomal expression vector. The method of claim 18.   19. The method of claim 18, wherein the producer cell is a non-pathogenic and not an obligate anaerobic bacterium.   21. The method of claim 20, wherein the non-pathogenic and non-obligate anaerobic bacterium is a species derived from a bacterial gene selected from the group consisting of Bacillus subtilis, lactic acid bacteria, lactic acid cocci, and listeria.   The method of claim 21, wherein the bacterial species is Bacillus subtilis.   23. The method of claim 22, wherein the Bacillus subtilis does not dissolve when induced for production of R-type hmw bacteriocin.   A Clostridium difficile comprising contacting the pathogenic Clostridium difficile with an effective amount of the R-type hmw bacteriocin of claim 14, wherein the R-type hmw bacteriocin binds and kills the pathogenic Clostridium difficile. Disinfection method of difficile bacteria.   25. The method of claim 24, wherein Clostridium difficile is in the body of the animal and a bactericidal amount of the R-type hmw bacteriocin is administered to the animal.   26. The method of claim 25, wherein the animal is a mammal.   27. The method of claim 26, wherein the mammal is a human.   A method of treating Clostridium difficile infection in an animal, comprising administering to the animal a producer cell comprising a nucleic acid molecule according to claim 1 in an amount necessary to produce a bactericidal amount of said bacteriocin, thereby treating said infection .   A composition comprising the R-type bacteriocin of claim 14 and a pharmaceutically acceptable carrier.   2. The nucleic acid molecule of claim 1, wherein the RBD is from a bacteriophage that infects a second strain of Clostridium difficile.