Topical and orally administered protease inhibitors and bacterial vectors for the treatment of disorders and methods of treatment

Abstract

The present invention provides purified protease inhibitors derived from microorganisms alone or in combination with bacteriocins and/or antibodies. The protease inhibitors may also be expressed by microbiome or probiotic microorganisms alone or in combination with bacteriocins and/or antibodies. The invention also provides methods and compositions for improving the expression of endogenous or heterologous protease inhibitors alone or in combination with bacteriocins and/or antibodies. The invention is useful for treating a variety of inflammatory disorders including acne, psoriasis, eczema, atopic dermatitis and inflammatory bowel disease.

Claims

1. A method of screening microorganism from a mixed population of microorganisms for the production of secreted protease inhibitors, comprising: growing the mixed population of microorganisms on protease-detection media comprising a protease substrate; adding a protease followed by at least one antibiotic to the mixed population of microorganisms grown on the protease-detection media; and detecting inhibition of the added protease.

2. The method according to claim 1, further comprising selecting from the mixed population of microorganisms grown on the protease-detection media, a subpopulation displaying production of protease inhibitors.

3. The method according to claim 1, further comprising mutating at least one microorganism from the mixed population of microorganisms to form at least one mutant.

4. The method according to claim 3, further comprising selecting at least one mutant based on the detected inhibition of the added protease.

5. The method according to claim 1, further comprising selecting at least one colony from the mixed population of microorganisms grown on the protease-detection media for enhanced production of at least one protease inhibitor.

6. The method according to claim 5, further comprising isolating at least one protease inhibitor produced by the selected at least one colony.

7. The method according to claim 6, further comprising administering the isolated at least one protease inhibitor in a pharmaceutically acceptable dosage form to a human, for treatment of at least one inflammation associated from a disorder selected from the group acne, psoriasis, atopic dermatitis, eczema and inflammatory bowel disease.

8. The method according to claim 1, wherein the protease-detection media selects for inhibition of at least one protease selected from the group consisting of chymase, calpain, Casp-1, and neutrophil serine protease elastase.

9. The method according to claim 2, wherein the subpopulation is selected from the group consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus caprae, Staphylococcus epidermidis, Streptococcus pyogenes, Candida albicans, Proteus sp., Bacillus sp., Clostridium sp., Serratia sp., Campylobacter, Streptomyces sp., Porphromonas gingivalis, Lactococcus lactus, Lactococcus casei, Lactobacillus acidophilus, Streptococcus salivarus, Propionibacterium sp, Corynebacterium sp., E. coli, S. agalaciae, and Listeria monocytogenes.

10. The method according to claim 1, further comprising: selecting at least one colony of the mixed population of microorganisms based on the detected protease inhibition by the colony; and subjecting the selected at least one colony to at least one of guided evolution, mutagenesis, and genetic engineering to produce altered microorganisms having a change a respective genome of members of the colony with respect to the selected at least one colony.

11. The method according to claim 10, wherein the altered microorganism is further selected based on concurrent expression of a combination of a protease inhibitor and a bacterocin that facilitates colonization of a target tissue of an animal by the altered microorganisms.

12. The method according to claim 11, wherein the target tissue is selected from the group consisting of: skin exhibiting symptoms of atopic dermatitis, skin exhibiting symptoms of eczema, skin exhibiting symptoms of acne, and skin exhibiting symptoms of psoriasis.

13. The method according to claim 10, wherein the selected microorganism produces a protease inhibitor selected from the group consisting of an inhibitor of at least one of chymase, calpain, Casp-1, proteases from Staphylococcus aureus, and neutrophil serine protease elastase.

14. The method according to claim 2, wherein the subpopulation produces a protease inhibitor that reduces inflammation of at least one of skin and mucus membranes in an animal.

15. The method according to claim 1, wherein the mixed population of microorganisms comprises a probiotic bacteria which colonies at least one of human skin, human mucous membranes, and human gut substantially without causing disease in health adult humans, and which produces a protease inhibitor in situ.

16. The method according to claim 10, wherein said subjecting comprises genetically engineering the selected at least one colony to include synthetic DNA.

17. The method according to claim 10, further comprising: growing the altered microorganisms on protease-detection media; adding a protease followed by at least one antibiotic to the altered microorganisms grown on the protease-detection media; and detecting inhibition of the added protease by the altered microorganisms.

18. A method of screening microorganisms for the production of secreted protease inhibitors, comprising: culturing colonies of microorganisms on protease-detection media comprising a protease substrate; preserving a replica of the cultured colonies; adding a protease to the cultured microorganisms, detectable by the protease-detection media; adding at least one antibiotic to cultured microorganisms cultured on the protease-detection media; detecting inhibition of the added protease by a protease inhibitor produced by respective cultured colonies; and selecting at least one colony from the preserved replica of the cultured colonies based on said detecting.

19. The method according to claim 18, further comprising identification of at least one protease inhibitor from the selected at least one colony by: collecting a supernatant from growth of the selected at least one colony; separating components of the collected supernatant in a gel comprising a protease-sensitive protein; treating the gel having the separated components comprising a protease-sensitive protein with a protease; determining regions of the gel in which the protease is inhibited; and identifying the at least one protease inhibitor based on at least a region of the gel in which the protease is inhibited.

20. A method of screening microorganism from a mixed population of microorganisms for the production of secreted inhibitors of a protease from proteolytic bacterium of a human microbiome, comprising: growing separated colonies of the mixed population of microorganisms on protease-detection media comprising a protease substrate; duplicating the separated colonies of the mixed population of microorganisms on growth media; adding the protease from proteolytic bacterium of the human microbiome and at least one antibiotic the protease-detection media; determining regions of the protease-detection media corresponding to respective individual colonies, which show inhibition of the protease from the proteolytic bacterium of the human microbiome; and selecting duplicated separated colonies of the mixed population of microorganisms which correspond to the determined regions of the protease-detection media.

Description

4. BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a stepwise procedure for detecting protease inhibitors.

(2) FIGS. 2A and 2B show protease inhibitor zones of protease protection.

(3) FIGS. 3A and 3B show protease inhibitor precipitation zone.

(4) FIG. 4 shows correlation of the protease inhibitor molecular mass with the diffusion zone that creates the zone of protease protection.

(5) FIGS. 5 and 5B show bacterial protease inhibitor zones of protease protection.

(6) FIGS. 6A and 6B show a pathogenic Propionibacterium acnes and a non-pathogenic bacterium resistant to phage that produces a microcin that kills Propionibacterium acnes, respectively.

5. DETAILED DESCRIPTION OF THE INVENTION

(7) The present invention provides, according to one embodiment, live attenuated bacterial strains that co-express protease inhibitors together with one or more plasmids, phage, phagemids or viroids that carry peptides, antibodies, DNA or RNA based therapeutics. The plasmids, phage, phagemids, or viroids may be carried by either gram negative bacteria, wherein the phage is based on M13, or gram positive bacteria, wherein the phage is based on B5; the viroids which can be carried in either gram positive or gram negative are based on plant viroids or mammalian hepatitis D (Rocheleau L, Pelchat M (2006). “The Subviral RNA Database: a toolbox for viroids, the hepatitis delta virus and satellite RNAs research”. BMC Microbiol. 6: 24. doi:10.1186/1471-2180-6-24). The phage may be particularly effective in suppressing inflammatory responses through a combination of the effects of the protease inhibitor together with either an externally displayed anti-inflammatory peptide, an externally displayed anti-inflammatory antibody, a DNA encoded anti-inflammatory molecule or a therapeutic RNA, including miRNAs, antisense miRNAs and siRNAs. Certain modifications of the phage, phagemids or viroids may also be useful in treating certain virally infected cells, cancer or parasitic diseases such as worms.

(8) The present invention provides, according to various embodiments, improved live attenuated therapeutic bacterial strains that express one or more therapeutic molecules. The primary characteristic of the bacteria of certain embodiments of the invention is the enhanced effect in treatment of inflammatory disease of the skin and other locations. In one embodiment, the percent increase in effect is approximately 2% to approximately 95%, approximately 2% to approximately 75%, approximately 2% to approximately 50%, approximately 2% to about 40%, approximately 2% to about 30%, approximately 2% to about 25%, approximately 2% to about 20% or about 2% to approximately 10% greater than the parental strain of bacteria without expressing one or more invasion mutations or cell wall defects under the same conditions.

(9) For reasons of clarity, the detailed description is divided into the following subsections: mixed microorganism protease inhibitor assay, protease inhibitors, bacteriocins, antibody producing bacteria and determination of synergy.

(10) 5.1 Mixed Microorganism Protease Inhibitor Assay.

(11) Protease inhibitor producing microbiota of the invention are novel protease inhibitors isolated from the human microbiome; methods and examples for their isolation are described herein. The assay is directed toward the identification of microorganisms secreting protease inhibitors in order for the inhibitor to be identified and facilitate its use as a purified protease inhibitor such as a topical formulation. The assay also allows identification of microorganisms secreting protease inhibitors in order for the microorganism to be used as a probiotic. The same microorganism can be phenotypically selected or genetically modified to produce a greater quantity of the protease inhibitor using methods known to those skilled in the arts. The microorganisms can also be further modified to express one or more bacteriocins and/or anti-inflammatory antibodies or peptides using methods known to those skilled in the arts.

(12) Microbiome samples are taken using standard techniques including skin swabs and Bioré Deep Cleansing Pore Strips, (Kao Brands Company, Cincinnati, Ohio) which have been used for human skin microbiome studies (e.g., Fitz-Gibbon et al., 2013 Propionibacterium acnes Strain Populations in the Human Skin Microbiome Associated with Acne, Journal of Investigative Dermatology 133:2152-60). Standard microbiological media described by Kreig, 1981 (Chapter 8, Enrichment and Isolation, p. 112-142 in Manual of Methods for General Bacteriology, Gerhardt et al., eds, American Society for Microbiology, Washington, D.C.) such as Mueller Hinton and chocolate agar under aerobic, microaerophilic and anaerobic conditions. These organisms can then be screened as described in Example 1.

(13) Petri plate modifications. Typical petri plates are contain 1.5% agar in order to generate a solid support. Modified supports may consist of higher or lower agar concentrations, and/or varying concentrations of starch, silica, acrylamide, gelatin, casein, cellulose, clay, montmorillonites, or other solidifying supports.

(14) Petri plates may be further modified with porous membranes on the surface that they allow protein diffusion and facilitate the microbial colonies to be transferred to another growth medium or to a biochemical assay while preserving their spatial organization which then allows retracing a position on a plate to a particular organism on a master plate. Porous membranes include cellulose, cellulose esters, cellophane, polyestersulfone (PES), nylon, polytetrafluoroethylene (PTFE, a.k.a. Teflon) including hydrophobic and hydrophilic forms, as well as membranes with protein binding ability that allow small molecule diffusion including polyvinylidene fluoride, or polyvinylidene difluoride (PVDF) and nitrocellulose. The purpose of these supports is that they allow transfer of the metabolically active microorganism from a type of petri plate that may not be fully compatible with the assay and allows the bacteria secrete the protease inhibitor(s) during the period the microorganism remain metabolically active.

(15) The composition of the petri plates is matched to the protease for which the specific protease inhibitor is being screened for. For isolation of general protease inhibitors, the compositions of Table 1 may be used. Variations, such as alternate proteins as indicators, such the dye-bound protein azocoll (Chavira et al., 1984, Assaying proteases with azocoll, Anal Biochem 136: 446-450), can also be used.

(16) TABLE-US-00001 TABLE 1 Classes of proteases, substrates, proteases for substrate clearance, and cognate inhibitor controls that can be used for a petri plate assay to isolate classes of protease inhibitors. Protease Cognate Class of for substrate inhibitor Protease Substrate clearance control Serine Gelatin/casein A) Trypsin, Aprotinin B) Thymostrypsin, C) Elastase Cysteine Gelatin/casine Papain Leupeptin Aspartic acid Gelatin/casein Pepsin Pepstatin Metalloprotease Gelatin/casein Thermolysin α-2- macroglobulin

(17) The general formula outlined above in Table 1 can be further modified to screen for specific inhibitors (Table 2).

(18) TABLE-US-00002 TABLE 2 Representative petri plate components for isolation of protease enzyme specific inhibitors. Cognate Protease Protease for substrate inhibitor Target Substrate clearance control Propionibacterium Gelatin/casein Purified I proteases from acnes Propionibacterium acnes (Ingram et al., 1983 J. Appl. Bacteriol 54: 263- 271; Lee et al., 2010 Arch Dermatol Res. 302: 745-756) Propionibacterium Gelatin/casein Purified II proteases from acnes Propionibacterium acnes acnes (Ingram et al., 1983 J. Appl. Bacteriol 54: 263-271; Lee et al., 2010 Arch Dermatol Res. 302: 745-756) Propionibacterium Gelatin/casein Purified III proteases acnes from Propionibacterium acnes acnes (Ingram et al., 1983 J. Appl. Bacteriol 54: 263-271; Lee et al., 2010 Arch Dermatol Res. 302: 745- 756) Chymase Gelatin/casein Chymase Z-Arg-Glu-Thr- N-Succinyl-Ala-Ala- Phep(OPh).sub.2 Pro-Phe p-nitroanilide (MP Biomedical) (Sigma Aldrich) Calpain Gelatin/casein Calpain Calpastatin, Proluminescent calpain N-Acetyl-L- substrate, Suc-LLVY- leucyl-L-leucyl- aminoluciferin L-norleucinal (Promega, Madison, (Sigma Aldrich) WI) Furin Gelatin/Casein Furin Furin Inhibitor I Boc-Arg-Val-Arg-Arg- N.sup.2-(Decanoyl- AMC (AMC = 7- RVKR-CMK) Amino-4- (EMD Millipore) methylcoumarin) (Enzo Life Science) Casp-1 Gelatin/casein Casp-1 Caspase-1 YVAD-AFC emits Inhibitor VI (Z- blue light (400 nm); YVAD-FMK) upon cleavage of the (Calbiochem) substrate by caspase-1 or Neutrophil serine Gelatin/Casein Neutrophil serine Elafin protease elastase protease elastase inhibitor inhibitor MMP2/9 Gelatin/casein MMP2/9 MMP-2/MMP-9 MMP-2/MMP-9 Inhibitor II; CAS Substrate II Ac-Pro- 193807-60-2 Leu-Gly-(2-mercapto- (Calbiochem) 4-methylpentanoyl)- Leu-Gly-OEt (Millipore EMD 444224)

(19) The general formulas outlined above in Tables 1 and 2 can be further modified to screen for species specific inhibitors (Table 3) against purified enzymes using published methods.

(20) TABLE-US-00003 TABLE 3 Representative petri plate components for isolation of species-specific protease enzyme inhibitors for purified proteases. Protease Protease for producing species substrate target Substrate clearance Cognate inhibitor control Propionibacterium Gelatin/casein Protease acnes containing culture supernatant from Propionibacterium acnes (Ingram et al., 1983 J. Appl. Bacteriol 54: 263- 271; Lee et al., 2010 Arch Dermatol Res. 302: 745-756) Staphylococcus Gelatin/casein SspB Cysteine E-64 aureus protease from Staphylococcus aureus. Pseudomonas Gelatin/casein Zinc HSCH2 aeruginosa metalloprotease (DL)CH[CH2CH(CH3)2]CO-Phe- from Ala-NH2 Pseudomonas aeruginosa Pseudomonas Gelatin/casein Elastase 2-mercaptoacetyl-L-phenylalanyl-L- aeruginosa Pseudomonas leucine aeruginosa

(21) The general formulas outlined above in Tables 1, 2 and 3 can be further modified to screen for inhibitors culture supernatants (Table 4). The culture supernatants can contain proteases that have not been characterized, or they may contain mixtures of proteases.

(22) TABLE-US-00004 TABLE 4 Representative petri plate components for isolation of species-specific protease enzyme inhibitors. Protease producing Protease for substrate species target Substrate clearance Staphylococcus Gelatin/casein Protease containing culture aureus supernatant of Staphylococcus aureus. Staphylococcus Gelatin/casein Protease containing culture epidermidis supernatant of Staphylococcus epidermidis. Clostridium Gelatin/casein Protease containing culture difficile supernatant of Clostridium difficile Pseudomonas Gelatin/casein Protease containing culture aeruginosa supernatant of Pseudomonas aeruginosa

(23) The bacteria may be further selected for enhanced protease inhibitor production. The methods described herein include a culture-based method for the isolation of microorganisms producing protease inhibitors. The production of the protease inhibitors is visualized on a petri dish (FIGS. 5A and 5B). Using methods known to those skilled in the arts which include various mutagenesis methods such as exposure to ultraviolet light, chemical mutagens such as nitrosoguanidine, or genetic methods such as transposon mutagenesis, organisms with improved production of protease inhibitors are visualized as producing more intensely staining zone of protease protection.

(24) 5.2 Protease Inhibitors.

(25) Optionally, the protease inhibitors may be known polypeptide inhibitors engineered to be expressed by the bacteria. The inhibitors include both synthetic peptides and naturally occurring, endogenous peptides and metabolites. To result in the desired activity, the peptides or metabolites should be surface displayed, released or secreted outside of the microorganism. Accordingly, the peptides are modified by fusing them to secretion signals. The secretion signals may be: N-terminal (LPP:OmpA, M13pIII, M13pIII with a signal recognition particle site, M13pVIII, zirS (Finlay et al., 2008, PLoS Pathogens 4 (4), e100003); heat-stable (ST; thermostable) toxins from Escherichia and Vibrio (U.S. Pat. No. 5,399,490, expressly incorporated herein by reference); E. coli enterotoxin II (Kwon et al., U.S. Pat. No. 6,605,697, expressly incorporated herein by reference); by colicin fusions together with colicin lysis proteins, or using autotransporter fusions; fusion to the M13 pIX may also be used (WO 2009/086116, expressly incorporated herein by reference); hlyA C-terminal signal sequence last 60 amino acids of the E. coli HlyA hemolysin, together with the required HlyBD supplied in trans and endogenous tolC as shown in FIGS. 6A and 6B.

(26) The N-terminal signal sequences are well known and characterized by the presence of a protease cleavage site for an endogenous bacterial protease. Thus, N-terminal signal sequences provide free protease inhibitors, free from the signal sequence. The C-terminal signal sequence may be further engineered to have a protease cleavage site in between the protease inhibitory peptide and the signal sequence. The cleavage site may be for the same protease that the peptide inactivates. Thus, the protease activates its own inhibitor. The protease cleavage site may also be for a protease other than for the protease inhibitor, thus deactivating another protease. Multiple protease inhibitor peptides may be used in-frame with multiple protease cleavage signals (polymeric protease activated protease inhibitors), where the inhibitors alternate with cleavage sites.

(27) The polymeric protease activated protease inhibitors can be homo- or hetero-inhibitor polymers (i.e., have inhibitors for the same or different proteases, respectively), and/or homo- or hetero-protease cleavage polymers (i.e., have the same or different protease cleavage sites). Proteases upregulated within tumors for which protease cleavage sites may be engineered include: tissue plasminogen activator, activated protein C, factor Xa, granzyme (A, B, M), cathepsin, thrombin, plasmin, urokinase, matrix metaloproteaes, prostate specific antigen (PSA) and kallikrein 2 (e.g., Edwards et al. (eds) 2008, The Cancer Degradome: Proteases and Cancer Biology, Springer, 926 pp), as well as proteases of lysosomes and the gut.

(28) Protease inhibitors have been reviewed by Laskowski and Kato, 1980, (Annual Review of Biochemistry 49: 593-626), expressly incorporated by reference herein. Serine proteases inhibitors, the largest group, include 1) bovine pancreatic trypsin inhibitor (Kunitz) family, 2) pancreatic secretory trypsin inhibitor (Kazal) family, 3) Streptomyces subtilisin inhibitor family, 4) soybean trypsin inhibitor (Kunitz) family, 5) soybean proteinase inhibitor (Bowman-Birk) family 6) potato I inhibitor family, 7) potato II inhibitor family, 8) Ascaris trypsin inhibitor family, and 9) others. Protease inhibitors have also been grouped within the MEROPS peptidase database (Rawlings et al., 2008 Nucleic Acids Res. 36 Database issue, D320-325). Specific examples of protease inhibitors that may be expressed as complete proteins or peptide fragments corresponding to the active inhibitory site include but are not limited to aprotinin, autodisplay aprotinin (Jose J, Zangen D (2005) Autodisplay of the protease inhibitor aprotinin in Escherichia coli. Biochem Biophys Res Commun 333:1218-1226; Jose, 2006, Autodisplay: efficient bacterial surface display of reombinant proteins, Appl Microbiol Biotechnol 69: 607-614), cathepsin inhibitor peptide sc-3130, lympocyte protease inhibitor, maspin, matrix metalloprotease inhibitors, macroglobulins, antithrombin, equistatin, Bowman-Birk inhbitor family, ovomucoid, ovoinhibitor-proteinase inhibitors from avian serum, dog submandibular inhibitors, inter-a-trypsin inhibitors from mammalian serum, chelonianin from turtle egg white, soybean trypsin inhibitor (Kunitz), secretory trypsin inhibitors (Kazal) a.sub.i-proteinase inhibitor, Streptomyces subtilisin inhibitor, plasminostreptin, plasmin inhibitor, factor Xa inhibitor, coelenterate protease inhibitors, protease inhibitor anticoagulants, ixolaris, human Serpins (SerpinA1(alpha 1-antitrypsin), SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinA10, SerpinA11, SerpinA12, SerpinA13, SerpinB1, SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinC1 (antithrombin), SerpinD1, SerpinE1, SerpinE2, SerpinF1, SerpinF2, SerpinG1, SerpinNI1, SerpinNI2), cowpea trypsin inhibitor, onion trypsin inhibitor, alpha 1-antitrypsin, Ascaris trypsin and pepsin inhibitors, lipocalins, CI inhibiotor, plasminogen-activator inhibitor, collegenase inhibitor, Acp62F from Drosophila, bombina trypsin inhibitor, bombyx subtilisin inhibitor, von Willebrand factor, leukocyte secretory protease inhibitor. Short peptide inhibitors of protease are preferred. Many protease inhibitors have one or more disulfide bonds. Fusion to thioredoxin (trxA) is known to improve protease inhibitor activity (e.g., Furuki et al., 2007, Fukuoka University Science Reports 37: 37-44). Fusion to glutathione-S transferase (GST) and co-expression with disulfide bond isomerase (DsbA) or nusA (Harrison 2000, Expression of soluble heterologous proteins via fusion with NusA protein. inNovations 11: 4-7) are also known to improve solubility. Methods to isolate novel protease inhibitors using M13 phage display have been described by Roberts et al., 1992 (Gene 121: 9-15). Neutrophil serine protease inhibitors derived from elafin (also known as trappin-2 or SKALP (skin-derived anti-leukoproteinase) which targets elastase and proteinase 3) and SLPI (which targets elastase and cathepsin G) have been described as polyvalent inhibitors of neutrophil serine proteases (Zani et al., 2009 Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases, Protein Science 2009 18: 579-594). Koivunen et al., (1999 Tumor targeting with a selective gelatinase inhibitor, Nature Biotechnology 17: 768-774) have described a short peptide (CTTHWGFTLC SEQ ID: 003) inhibitory to MMP2 and MMP9 and Bjorklund et al. have described the leukocyte specific β-2 integrin binding partner for pro-MMP-9 “DDGW” SEQ ID: 004 (Bjorklund et al., 2004 Peptide Inhibition of catalytic and noncatalytic activities of matrix metalloproteinase-9 blocks tumor cell migration and invasion, J. Biol. Chem. 279: 29589-29597). Other peptides include DX-88 which contains the kunitz domain from human liopoprotein-associated coagulation inhibitor domain 1 (LACI-D1) or the variant DX-1000. Calpastatin and novel secreted derivatives including transmembrane transport (i.e., cell penetrating peptides or ferry peptides such as TAT (Heitz et al., 2009, Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics Br J Pharmacol. 2009 May; 157(2): 195-206) described herein are also encompassed.

(29) The peptide inhibitors are engineered to be secreted from the gram negative bacteria secretion signals known to those skilled in the arts and may optionally be engineered to contain a signal recognition particle translocation sequence Steiner et al., 2006, signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display, Nature Biotechnol 24: 823-831), including E. coli cytolethal distending toxin, Shiga toxin, LPP:OmpA, M13pIII, M13pVIII, zirS (Finlay et al., 2008, PLoS Pathogens 4 (4), e100003), heat-stable (ST; thermostable) toxins from Escherichia and Vibrio (U.S. Pat. No. 5,399,490), E. coli enterotoxin II (Kwon et al., U.S. Pat. No. 6,605,697) N-terminal signal sequences, or hlyA C-terminal signal sequence (requires addition of hlyBD and TolC), or by colicin fusions together with colicin lysis proteins, or using autotransporter (autodisplay) fusions. The autotransporter surface display has been described by Berthet et al., WO/2002/070645, expressly incorporated by reference herein. Other heterologous protein secretion systems utilizing the autotransporter family can be modulated to result in either surface display or complete release into the medium (see Henderson et al., 2004, Type V secretion pathway: the autotransporter story, Microbiology and Molecular Biology Reviews 68: 692-744; Jose, 2006 Applied Microbiol. Biotechnol. 69: 607-614; Jose J, Zangen D (2005) Autodisplay of the protease inhibitor aprotinin in Escherichia coli. Biochem Biophys Res Commun 333:1218-1226 and Rutherford and Mourez 2006 Microbial Cell Factories 5: 22). For example, Veiga et al. (2003 Journal of Bacteriology 185: 5585-5590 and Klauser et al., 1990 EMBO Journal 9: 1991-1999) demonstrated hybrid proteins containing the β-autotransporter domain of the immunoglogulin A (IgA) protease of Nisseria gonorrhea. Fusion to the M13 pIX may also be used (WO 2009/086116) or fusions to type III secretion system of Salmonella or other bacteria (Wilmaier et al., 2009 Mol Sys Biol 5: 309. The inhibitors can be further modified to have the protease cleavage signal of the protease that they inhibit or for a different protease. Secretion signal from gram positive bacteria include that from listerialysin O (LLO), alkaline phosphatase (phoZ) (Lee et al., 1999, J Bacteriol. 181: 5790-5799), CITase gene (Shiroza and Kuramitsu 1998, Methods in Cell Science, 20: 127-136) or the twin arginine translocation system (Berks et al., 2005, Protein targeting by the bacterial twin-arginine translocation (Tat) pathway, Current Opinion in Microbiology 8: 174-181). Enhanced secretion may be achieved as described in U.S. Pat. No. 7,358,084, WO/2009/139985 Methods and materials for gastrointestinal delivery of a pathogen toxin binding agent; van Asseldonk, M et al. 1990, Cloning of usp45, a gene encoding a secreted protein from Lacotococcs lactis subsp. lactis MG1363 Gene 95, 15-160; Kim et al., Display of heterologous proteins on the surface of Lactococcus lactis using the H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix J Appl Microbiol. 2008 June; 104(6):1636-43. Epub 2008 Feb. 19).

(30) The proteins may have one or more additional features or protein domains known to those skilled in the art which are designed to be active or catalytic domains that facilitate them being secreted or released by autolytic peptides such as those associated with colicins or bacteriophage release peptides or thoredoxin or glutation S-transferase (GST) fusions that improve solubility.

(31) 5.3 Bacteriocins, Production and Resistance, and Resistance to Phage

(32) In one embodiment, the probiotic bacteria express one or more bacteriocins and one or more bacteriocin immunity proteins. In another embodiment, the bacteria co-expresses a protease inhibitor and bacteriocin/bacteriocin immunity proteins. Bacteriocins (bacterially produced antibacterial agents that inhibit other strains of bacteria but not the host strain that produces them), such as lactococcins, microcins or colicins (Riley and Chavan 2006, Bacteriocins: Ecology and Evolution, Springer; de Vuyst and Vandamme 2012, Bacteriocins of lactic acid bacteria; Microbiology, genetics and applications, Blackie Academic & Professional Press). In a more preferred embodiment, the bacteriocin is the acnecin from Propionibacterium acnes (Fujimura and Nakamura 1978) or the bacteriocin from Propionibacterium shermanii (Ayers et al., Propionibacteria peptide microcin U.S. Pat. No. 5,635,484 A), the bacteriocin from Streptococcus salivarius (Bowe et al., 2006, J. Drugs Dermatol 5: 868-870), the bacteriocin from Lactococcus sp. HY 449 (Oh et al., 2006. Effect of bacteriocin produced by Lactococcus sp HY 449 on skin inflammatory bacteria, Food Chem Toxicol 44: 1184-1190) or the bacteriocin from Lactococcus sp. HY 49 or Lactobacillus casei HY 2782 described by Kim et al., (U.S. Pat. No. 6,329,002 Food for inhibiting infection and treating gastritis, gastric and duodenal ulcers). In most preferred embodiment, the probiotic bacterium is a Propionibacteria acnes of ribotype 6 (RT6) associated with normal skin (Fitz-Gibbon, S. et al., 2013 Propionibacterium acnes Strain Populations in the Human Skin Microbiome Associated with Acne, Journal of Investigative Dermatology 133:2152-60) that expresses an anti-Propionibacterium acnes bacteriocin effective against ribotypes associated with acne such as RT4,5,7, 8, 9 & 10 (Fitz-Gibbon et al., 2013), such that the bacteriocin allows the normal skin P. acnes to penetrate into the skin and overtake the population of P. acnes associated with acne due to the ability of the bacteria of the invention to be immune to acne-associated strain's bacteriocin, and thus can kill and occupy the acne-producing strains location, thereby reducing or eliminating the symptoms of acne.

(33) The bacteria may be further selected for enhanced bacteriocin production using standard methods for visualizing production of bacteriocins which uses an indicator strain usually embedded in a soft agar overlay, and a test strain, or library or mixed population of strains, applied to the surface. The production of the bacteriocin is then visualized as an increased zone of inhibition of the indicator strain. Using methods known to those skilled in the arts which include various mutagenesis methods such as exposure to ultraviolet light, chemical mutagens such as nitrosoguanidine, or genetic methods such as over expression on plasmids, insertion of strong promoters, transposon mutagenesis, organisms with improved production of bacteriocins are visualized as producing wider zones of bacterial inhibition.

(34) Resistance to phage by the Propionibacteria acnes RT6, and many other bacteria species, is already understood to occur, at least in part, by the CRISPER (Clustered Regularly Interspaced Short Palindromic Repeats) systems, but the “immunity” may be incomplete, and could allow phage from resident pathogenic bacteria to kill the probiotic bacterium, preventing it from having as fully effective therapeutic action. The bacteria of the invention may be further engineered to have phage resistance proteins, such as phage repressor proteins related to lambda phage c1 repressor, including those identified by Marinelli, et al., 2012 (Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates, mBio 3(5) doi:10.1128/mBio.00279-1). These authors suggest the possible use of the phage as a form of “phage therapy”, i.e., to kill Propionibacterium acnes, but do not propose or suggest the use of the Propionibacterium acnes, or phage-resistant bacteria, or bacteria with bacteriocins, or bacteria with bacteriocins and protease inhibitors as an effective form of therapy for acne. Use of standard methods of isolating and/or identifying phage resistant strains is also encompassed. It is of importance that the therapeutic bacterial strain, such as the RT6 strain of Propionibacterium acnes, be resistant to the resident, disease-associated organisms such as pathogenic ribotypes RT4,5,7, 8, 9 & 10 of Propionibacterium acnes (Fitz-Gibbon et al., 2013) or bacteria such as Staphylococcus aureus or Streptomyces pyogenes. Methods for selecting resistant strains selection for spontaneous resistance by exposure of the strain such as RT6 to the phage such as those described by Marinelli, et al., 2012 (Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates, mBio 3(5) doi:10.1128/mBio.00279-1), and recovery of the survivors, or the strain can be initially modified by chemical, ultraviolet or transposon mutagenesis, to create a mixed genetic population followed by exposure to the phage, and selection of survivors (Levin, 1994, Isolating multiple strains of Escherichia coli for coliphage isolation, phage typing, and mutant recovery, Chapter 4 pages 63-72, in Tested studies for laboratory teaching, Volume 15 (C. A. Goldman, Editor). Proceedings of the 15th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 390 pages; Exploitation of a new flagellatropic phage of Erwinia

(35) For positive selection of bacterial mutants attenuated in plant virulence: towards phage therapy T. J. Evans, J Appl Microbiol 108 (2010) 676-685). The surviving strains may contain genetically integrated phage, i.e., lysogens. Preferred strains are those that do not contain the phage, which is readily determinable by genetic techniques such as PCR. Similar techniques are used to select therapeutic strains that are resistant to the bacteriocins of pathogenic strains.

(36) The resulting bacteria are both capable of resisting attack by the virulent bacterial bacteriocins and/or their phage, are able to persist, colonize and kill pathogenic ribotypes by expressing bacteriocins the pathogens are sensitive to, and secrete protease inhibitors that suppress inflammation.

(37) 5.4 Antibody Expressing Bacteria

(38) In another embodiment, the probiotic bacteria displays an anti-inflammatory antibody, such as anti-TNF-alpha antibody, TNF-beta antibody, anti-IL-12, IL-17 or IL-23 antibody, either by surface display (Nhan et al., 2011 Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosis, Microbial Cell Factories 2011, 10:22; Lee et al., 2003, Microbial Surface Display, Trends in Biotechnology 21: 45-52; Kramer et al., 2003, Autodisplay: Development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains, Infec. Immun. 71: 1944-1952) or by carrying a phage that displays the antibody when secreted. The microbiome, probiotic, commensal or attenuated pathogenic bacterium may be either gram negative, such as E. coli, or gram positive, such as Lactococcus sp. or Lactobacillus sp. isolated from the human microbiome. The gram negative bacteria may express and secrete an anti-TNF antibody as an autotransporter display protein or a pIII fusion on a phage such as those derived from M13, fd and other filimentous phage. The Gram positive bacteria will express and secrete an anti-TNF antibody such as an M13 pIII homolog fusion (p6 on a phage such as that derived from B5; Chopin et al. 2005). The antiTNF single chain antibody can be one such as described by Mukai et al., 2006; Yang et al., 2010) and may be fully humanized (United States Patent Application 2012/0308575, expressly incorporated herein by reference).

(39) 5.5 Determination of Synergy

(40) Overall improvement is defined as an increase in effect, such as the ability to inhibit inflammatory disease symptoms. The contribution of the enhanced protease inhibitor production with bacteriocin production is determined individually and in combination. Additivity, synergy or antagonism may determined using the median effect analysis (Chou and Talaly 1981 Eur. J. Biochem. 115: 207-216) or other standard methods and used to select improved bacteria with optimal combinations in their ability to suppress inflammation.

6. FIGURE LEGEND

(41) FIG. 1 shows a stepwise procedure for detecting protease inhibitors.

(42) FIGS. 2A and 2B show protease inhibitor zones of protease protection. Protease inhibitors were pipetted onto the surface of a casein plate containing BCG. After 1 hour the plate was flooded with 1.0 ml of sterile-filtered 0.625 mg/ml trypsin and allowed to absorb, followed by addition of 1 ml carbenicillin/streptomycin and incubation at 37° C. overnight. FIG. 2A shows the BCG containing plate prior to PS staining and FIG. 2B shows the same plate following PS staining and destaining. 1) α-2-macroglubulin (2 ml of 7.8 mg/ml in dH.sub.2O), 2) aprotinin (2 ml of 0.3 mM in sterile dH.sub.2O), 3) leupeptin (2 ml of 10 mM in sterile dH.sub.2O), and 4) bestatin (2 ml of 1 mM in methanol).

(43) FIGS. 3A and 3B show protease inhibitor precipitation zones. The aprotinin protease protection zones were visibly distinct under conditions of increased amounts of trypsin on the different protein containing plates. FIG. 3A) Aprotinin (2 ml of 0.3 mM in sterile dH.sub.2O) with 2.5 mg/ml trypsin on a BCG gelatin plate and FIG. 3B) aprotinin (2 ml of 0.3 mM in sterile dH.sub.2O) with 2.5 mg/ml trypsin on a BCG casein plate.

(44) FIG. 4 shows correlation of the protease inhibitor molecular mass with the diffusion zone that creates the zone of protease protection. The inhibition zones created by the three trypsin inhibitors were plotted as the radius against the inverse molecular mass of the inhibitors (α-2-macroglobulin, ˜720 kDa; aprotinin, 6511 Da; and leupeptin 427 Da). R.sup.2 linear regression was inserted using Microsoft Excel. Dotted line, BCG gelatin; solid line, GCG casein.

(45) FIGS. 5A and 5B show bacterial protease inhibitor zones of protease protection. The bacteria were grown for 2 days at 30° C. on casein plates with BCG. The bacteria were gently washed off and the plate flooded with 1.0 ml of sterile-filtered 0.625 mg/ml trypsin and allowed to absorb, followed by addition of 1 ml carbenicillin/streptomycin and incubation at 37° C. overnight. The plate was further stained with Ponseau S and then destained in water overnight. FIG. 5A shows the Growth pattern of the colonies on the BCG plate containing casein following incubation at 30° C. FIG. 5B shows PS staining pattern of the same plate. 1) Photorhabdus luminescens 1° form, 2) Photorhabdus luminescens 2° form, 3) Citrobacter freundii B, 4) S. epidermidis, 5) S. aureus, 6) Enterococcus fecalis, α2m) α-2-macroglubulin.

(46) FIG. 6A shows a pathogenic Propionibacterium acnes carrying phage and a virulence associated plasmid that secretes one or more proteases.

(47) FIG. 6B shows a non-pathogenic genetically engineered probiotic Propionibacterium, or any other nonpathogenic bacterium of the microbiome engineered to have the properties that it is resistant to the phage from the pathogenic form, can kill the pathogenic form with a bacteriocin/microcin and optionally, secretes a protease inhibitor to reduce inflammation.

7. EXAMPLES

(48) In order to more fully illustrate the invention, the following examples are provided.

Example 1: A Novel Technique for Detection of Secreted Microbial Protease Inhibitors

(49) The overall procedure is shown in FIG. 1. Because the protocol is destructive to the plate assayed, a duplicate or replica plate is generated and retained for identification and further analysis of the strains using methods known to those skilled in the arts. The initial step is the exposure of the protease detection plate to a protease inhibitor or bacterial growth. Either commercially available protease inhibitor peptides or growth of bacterial strains on the protease detection plates can be detected. Protease inhibitors include α-2-macroglobulin (7.8 mg/ml in sterile water; Thermo Fisher), aprotinin (0.3 mM in sterile dH.sub.2O; Thermo Fisher), leupeptin (10 mM in sterile dH.sub.2O; Thermo Fisher), and bestatin (1 mM in methanol; Thermo Fisher) that are pipeted as 2.0 ml drops onto the surface of the protein containing plates and allowed to be absorbed into the plate and diffuse for 1 hour. The bacterial strains Photorabdus (Xenorhabdus) luminescens strain Hm primary and secondary forms where the secondary and not the primary form is known to secrete a protease inhibitor and the other strains are negative controls (Wee et al. 2000, A new broad-spectrum protease inhibitor from the entomopathogenic bacterium Photorhabdus luminescens. Microbiology 146: 3141-3147), Citrobacter freundii (ATCC #33128), Staphylococcus aureus, S. epidermidis and Enterococcus faecalis are allowed to grow two days at 30° C. in air.

(50) The base media can be any microbiological media designed to facilitate the growth of cultivable microorganism; different media can be used to favor different strains as appropriate (Kreig, 1981, Chapter 8, Enrichment and Isolation, p. 112-142 in Manual of Methods for General Bacteriology, Gerhardt et al., eds, American Society for Microbiology, Washington, D.C.). Variations on the type of protease being targeted, the protease substrate and cognate inhibitors are shown in Table 1 and used as a guide in the modification of media. A casein base media based on that of Vijayaraghavan and Vincent (2013, A simple method for the detection of protease activity on agar plates using bomochresolgreen dye, J. Biochem. Tech. 4: 628-630), which contained 5 grams of peptone, 1.5 grams of yeast extract, 1.5 grams of sodium chloride per liter with, except that the casein 0.5% is used (instead of 1.0%) and dissolved as described by Montville (1983, Dual-substrate plate diffusion assay for proteases, Appl Environ Microbiol 45: 200-204) using 0.02 N NaOH. When bromochresol green (BCG) is added directly to the media, 0.0015% is used. BCG preincorporated into the media as well as post incubation addition of a BCG dye reagent containing 0.028% BCG dissolved in 0.56% (w/v) succinic acid, 0.1% (w/v) NaOH (Vijayaraghavan and Vincent; 2013) with 0.6% Brij-35 and then acidified to pH 4.2 can also be used following exposure to a protease inhibitor or bacterial growth and subsequent exposure to a protease. After staining and then destaining in water the by completely removing the agar from the lower plastic, the plates are observed for the presence and absence of protein. Ponseau S staining by flooding the plate with 5 ml of the stain containing 0.1% Ponseau S (PS; Sigma, St. Louis, Mo.) and 5% acetic acid in water for 1-2 hrs and then marking the agar with a 23 ga syringe needle dipped in India ink at a corresponding register marked on the plastic petri plate, removing the agar from the plate and destaining overnight in water before observing them and is highly sensitive.

(51) A gelatin based media (Medina and Baresi 2007, Rapid identification of gelatin and casein hydrolysis using TCA, J Microbiol Meth 69: 391-393) which uses 40 grams per liter of tryptic soy agar powder without glucose (Becton Dickinson, Sparks, Md.) modified to contain 8 g of gelatin (instead of 16 g) per liter may be preferable for certain organisms depending upon their growth requirements and is used as is appropriate. Approximately 5 ml of 20% trichloroacetic acid (TCA; Thermo Fisher, Waltham, Mass.) was used for protein precipitation per petri plate. This media can also be combined with preincorporated BCG, or post-incubation BCG dye reagent or Ponseau S, each as described above for the casein plates.

(52) Following incubation, the bacterial strains are washed off the plate using a gentle stream of water in order to remove the colonies and eliminate their potential for surface inhibition effects on diffusion of the protease and/or dyes. The next step is to expose the plate to a protease-containing solution to cause clearing of the substrate (Table 1; FIG. 1). Casein-containing plates required a higher concentration of trypsin to achieve clearing, using trypsin concentrations as high as 2.5 mg/ml. The plates are flooded with either 1.0 ml of sterile-filtered 0.625 mg/ml trypsin or 0.0625 mg/ml trypsin in 10 mM Tris 1 mM EDTA pH 8.0 for casein and gelatin respectively, and allowed to incubate at 37° C. overnight. After the trypsin is absorbed into the plate (20-60 min.), 1 ml of sterile filtered water containing 2.5 mg carbenicillin and 750 mg streptomycin is also allowed to absorb into the plate in order to stop any further bacterial growth.

(53) In order to detect the zones of protease inhibition, the plates are observed for the presence of opaque zones surrounded by clearing that are detected with TCA precipitation as per Medina and Baresi 2007 (Rapid identification of gelatin and casein hydrolysis using TCA. J. Microbiol. Meth. 69: 391-393) or by bromochrosol green (BCG) previously incorporated into the media (Vijayaraghavan and Vincent. 2013, A simple method for the detection of protease activity on agar plates using bomochresolgreen dye, J. Biochem. Tech. 4: 628-630), or by the addition Ponseau S dye. Radial diffusion is measured in triplicate, entered into Microsoft Excel, plotted, and analyzed using linear regression.

(54) A comparison of the different variations on protein substrates, dyes and destains is shown in Table 1. Following the addition of a protease inhibitor and its diffusion into the plate, the majority of the protein contained within the plates is subsequently hydrolyzed by the addition of the protease trypsin. However, in the presence of the protease inhibitors, a zone of protection against proteolysis is created. When the plates are observed for the presence of protein either by TCA precipitation or the localized concentration of a dye such as BCG or PS, a zone of unhydrolyzed protein is observed in the location of the protease inhibitor. Using α-2-macroglobulin, aprotinin, leupeptin and bestatin we found that we were only able to faintly visualize the α-2-macroglobulin and aprotinin on the casein plates with preincorporated BCG (FIG. 2A). However, the combination of BCG-containing plates further stained with PS is the most sensitive when 0.625 mg/ml trypsin was used for casein and 0.0625 mg/ml trypsin was used for gelatin (Table 5; FIG. 2B). Using those plates and staining procedure we are able to visualize all three of the trypsin inhibitors α-2-macroglobulin, aprotinin, and leupeptin, with no staining for bestatin. Differences in a white ring of protein precipitation were observed that occurred strongly on the gelatin plates when higher concentrations of trypsin (2.5 mg/ml) were used, but only slightly on the casein plates when they were treated with the same trypsin concentration (FIGS. 3A and 3B). It was also observed on the casein plates, but to a lesser degree the gelatin plates, that the zone of inhibition for leupeptin (427 Da) was notably larger than the zone of inhibition for aprotinin (6511 Da), or that of the α-2-macroglobulin (˜720 kDa) which had the smallest diffusion zone (FIG. 2). When the radius was measured and plotted against the inverse of the molecular mass, a general relationship of size and diameter was also apparent for the casein plates and to a lesser degree for the gelatin plates (FIG. 4). An R.sup.2 regression analysis was performed using Microsoft Excel, but the results were not significant for either the casein plates (R.sup.2=0.73) or the gelatin plates (R.sup.2=0.47). The relative intensity of the zone of inhibition based on different amounts of protease inhibitor allows for observation and selection of strains producing the most inhibitor.

(55) Analysis of the six bacterial strains, Photorabdus luminescens Hm primary and secondary forms, Citrobacter freundii, Staphylococcus aureus, S. epidermidis and Enterococcus faecalis showed that the Photorabdus luminescens Hm secondary form, and not the primary form, was protease inhibitor positive (FIGS. 5A and 5B). These results were therefore consistent with previous results (Schmidt et al., 1988; Woo et al, 2000). The P. luminescens secondary form colony grew fairly large (FIG. 5A), and it was apparent that the inhibitor only diffused a short distance from the edge of the colony. Likewise, the α-2-macroglobulin also diffused only a short distance. These data establish the capability of this novel assay to detect protease inhibitors from large numbers of bacteria growing in mixed populations on petri plates.

(56) TABLE-US-00005 TABLE 5 Protease inhibitor detection efficiency, protein substrates, dyes and destaining. Dye or Dye Reagent Relative Detection Efficiency Protein incorporated added after α-2 Variation Substrate in plate growth Destain Macroglobulin Aprotonin Leupeptin 1 0.5% Casein None BCG 1 hour Water + +/− +/− 2 0.5% Casein None Ponseau S Overnight +++ +++ +++ Water 3 0.5% Casein None TCA 15 min + +/− +/− development 4 0.5% Casein BCG BCG 1 hour Water + + +/− 5 0.5% Casein BCG Ponseau S Overnight +++ +++ +++ Water 6 0.5% Casein BCG TCA 15 min +/− +/− +/− development 7 0.8% Gelatin None BCG 1 hour Water − − − 8 0.8% Gelatin None Ponseau S Overnight +++ +++ +++ Water 9 0.8% Gelatin None TCA 15 min +++ +++ +++ development 10 0.8% Gelatin BCG BCG 1 hour Water − − − 11 0.8% Gelatin BCG Ponseau S Overnight +++ +++ +++ Water 12 0.8% Gelatin BCG TCA 15 min +++ +++ +++ development

Example 2: Isolation of Microorganisms with Inhibitors of Chymase

(57) Mixed microbial organisms such as environmental samples, microbiome, mutant populations, or DNA libraries of microorganisms are plated to microbiological support media, with or without a membrane support which may be transferred from a media to an assay support at a microbial density that allows spatial distinction or localized enrichment of the bacteria on that support. Typically, an 85 mm Petri plate would display 300 to 3000 individual colonies; fewer or greater microbial colonies could be used.

(58) The media may also be modified to be selective for various groups of microorganisms, such as salt tolerance (e.g., Staphylococcus growth on mannitol salt agar), or the addition of antibiotics such as cyclohexamide and nystatin at various concentrations (e.g., for selective growth of Actinomycetes and Streptomyces by addition of various antibiotics known to those skilled in the arts; Williams and Davies 1965, Use of antibiotics for selective isolation and enumeration of Actinomycetes in soil, J. Gen. Microbiol. 38: 251-261; Zhang, J. 2011, Improvement of an isolation medium for Actinomycetes. Modern Applied Science. 5 (2) 124-127; Seong et al., 2001, An improved selective isolation of rare Actinomycetes from forest soil, The Journal of Microbiology 39: 17-23), or the isolation of Propionibacterium on lactate and other isolation techniques (Kreig, 1981, Chapter 8, Enrichment and Isolation, p. 112-142 in Manual of Methods for General Bacteriology, Gerhardt et al., eds, American Society for Microbiology, Washington, D.C.).

(59) Growth conditions may also be varied, including temperatures above the normal body temperature (e.g., 37 C for humans) such as 42 C, or below normal temperature such as 30 C. The atmospheric conditions of oxygen may be normal atmospheric, microaerophilic, hypoxic or anaerobic.

(60) One or more media may be employed in one or more environmental conditions for the same biological sample, subdivided for each of the media and conditions, and modified as per Table 2. By way of example, a mixed bacterial population of the human skin microbiome is obtained by a skin swab plated at a density of approximately 1000 bacterial colonies per 85 mm Petri plate with tryptic soy agar media incubated in a 5% CO2/air chamber at 35 C for 16 hours. The tryptic soy agar (Difco Manual Difco & BBL Manual: Manual of Microbiological Culture Media, Second Edition, M. J. Zimbro et al., (Eds), Becton Dickinson and Co., 2009) and either N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (3 μL/ml of Sigma Aldrich Cat. No. S0448) or 1 micromole of benzoyl-L-tyrosine ethyl ester (BTEE). The following day the bacteria are replica plated by methods known to those skilled in the arts (e.g., Lederberg, J and Lederberg, E M, 1952, Replica plating and indirect selection of bacterial mutants. J Bacteriol. 63: 399-406) to serve as a master plate from which the bacteria may be preserved as live and/or DNA samples such that those of interest can be individually identified and used for further study. The bacteria are then removed, either by removing their porous membrane support or by washing the bacteria off. The plate is then exposed to a chymase containing solution (0.5-1.0 U/ml) by addition to the surface of the plate, which is then allowed to adsorb into the petri plate and incubate.

(61) N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide is a colorimetric substrate yielding a product with absorbance at 405 nm, a blue/violet color. After the purified chymase is added to the entire plate and allowed to cleave the the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide, the plate is then observed for zones of decreased blue/violet absorption (lack of the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide activation) compared to the surrounding areas where the chymase has activated the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide and increased the absorbance. The lighter areas associated with particular bacterial colonies correspond to ones producing one or more chymase inhibitors.

(62) The chymase enzyme also has the ability to degrade BTEE to N-Benzoyl-L-Tyrosine+Ethanol, which increases the absorption at 256. When BTEE is used, the plate is then observed at or near 256 nm ultraviolet light for zones of decreased absorption compared to the surrounding areas where the chymase has degraded the BTEE and increased the absorbance. The lighter areas associated with particular bacterial colonies correspond to ones producing one or more chymase inhibitors.

Example 3: Isolation of Microorganisms with Inhibitors of Calpain

(63) Mixed microbial organisms such as environmental samples, microbiome, mutant populations, or DNA libraries of microorganisms are plated to microbiological support media, with or without a membrane support which may be transferred from a media to an assay support at a microbial density that allows spatial distinction or localized enrichment of the bacteria on that support. Typically, an 85 mm Petri plate would display 300 to 3000 individual colonies; fewer or greater microbial colonies could be used.

(64) The media may also be modified to be selective for various groups of microorganisms, such as salt tolerance (e.g., Staphylococcus growth on mannitol salt agar), or the addition of antibiotics such as cyclohexamide and nystatin at various concentrations (e.g., for selective growth of Actinomycetes and Streptomyces by addition of various antibiotics known to those skilled in the arts; Williams and Davies 1965, Use of antibiotics for selective isolation and enumeration of Actinomycetes in soil, J. Gen. Microbiol. 38: 251-261; Zhang, J. 2011, Improvement of an isolation medium for Actinomycetes. Modern Applied Science. 5 (2) 124-127; Seong et al., 2001, An improved selective isolation of rare Actinomycetes from forest soil, The Journal of Microbiology 39: 17-23), or the isolation of Propionibacterium on lactate and other isolation techniques (Kreig, 1981, Chapter 8, Enrichment and Isolation, p. 112-142 in Manual of Methods for General Bacteriology, Gerhardt et al., eds, American Society for Microbiology, Washington, D.C.).

(65) Growth conditions may also be varied, including temperatures above the normal body temperature (e.g., 37 C for humans) such as 42 C, or below normal temperature such as 30 C. The atmospheric conditions of oxygen may be normal atmospheric, microaerophilic, hypoxic or anaerobic.

(66) One or more media may be employed in one or more environmental conditions for the same biological sample, subdivided for each of the media and conditions. By way of example, a mixed bacterial population of the human skin microbiome is obtained by a skin swab plated at a density of approximately 1000 bacterial colonies per 85 mm Petri plate with tryptic soy agar media incubated in a 5% CO2/air chamber at 35 C for 16 hours. The tryptic soy agar (Difco Manual Difco& BBL Manual: Manual of Microbiological Culture Media, Second Edition, M. J. Zimbro et al., (Eds), Becton Dickinson and Co., 2009).

(67) The procedure of Example 2 is modified for calpain as indicated in Table 1.

Example 4: Isolation of Microorganisms with Inhibitors of Caspase-1

(68) Mixed microbial organisms such as environmental samples, microbiome, mutant populations, or DNA libraries of microorganisms are plated to microbiological support media, with or without a membrane support which may be transferred from a media to an assay support at a microbial density that allows spatial distinction or localized enrichment of the bacteria on that support. Typically, an 85 mm Petri plate would display 300 to 3000 individual colonies; fewer or greater microbial colonies could be used.

(69) The media may also be modified to be selective for various groups of microorganisms, such as salt tolerance (e.g., Staphylococcus growth on mannitol salt agar), or the addition of antibiotics such as cyclohexamide and nystatin at various concentrations (e.g., for selective growth of Actinomycetes and Streptomyces by addition of various antibiotics known to those skilled in the arts; Williams and Davies 1965, Use of antibiotics for selective isolation and enumeration of Actinomycetes in soil, J. Gen. Microbiol. 38: 251-261; Zhang, J. 2011, Improvement of an isolation medium for Actinomycetes. Modern Applied Science. 5 (2) 124-127; Seong et al., 2001, An improved selective isolation of rare Actinomycetes from forest soil, The Journal of Microbiology 39: 17-23), or the isolation of Propionibacterium on lactate and other isolation techniques (Kreig, 1981, Chapter 8, Enrichment and Isolation, p. 112-142 in Manual of Methods for General Bacteriology, Gerhardt et al., eds, American Society for Microbiology, Washington, D.C.)

(70) Growth conditions may also be varied, including temperatures above the normal body temperature (e.g., 37 C for humans) such as 42 C, or below normal temperature such as 30 C. The atmospheric conditions of oxygen may be normal atmospheric, microaerophilic, hypoxic or anaerobic.

(71) One or more media may be employed in one or more environmental conditions for the same biological sample, subdivided for each of the media and conditions. By way of example, a mixed bacterial population of the human skin microbiome is obtained by a skin swab plated at a density of approximately 1000 bacterial colonies per 85 mm Petri plate with tryptic soy agar media incubated in a 5% CO2/air chamber at 35 C for 16 hours. The tryptic soy agar (Difco Manual Difco& BBL Manual: Manual of Microbiological Culture Media, Second Edition, M. J. Zimbro et al., (Eds), Becton Dickinson and Co., 2009).

(72) The procedure of Example 1 is modified for Caspase-1 as indicated in Table 2. The YVAD-AFC emits blue light (400 nm) upon cleavage of the substrate by caspase-1; thus the petri plates are observed for areas that lack blue light emission.

Example 5: Isolation of Microorganisms with Inhibitors Neutrophil Serine Protease Elastase Inhibitors

(73) Mixed microbial organisms such as environmental samples, microbiome, mutant populations, or DNA libraries of microorganisms are plated to microbiological support media, with or without a membrane support which may be transferred from a media to an assay support at a microbial density that allows spatial distinction or localized enrichment of the bacteria on that support. Typically, an 85 mm Petri plate would display 300 to 3000 individual colonies; fewer or greater microbial colonies could be used.

(74) The media may also be modified to be selective for various groups of microorganisms, such as salt tolerance (e.g., Staphylococcus growth on mannitol salt agar), or the addition of antibiotics such as cyclohexamide and nystatin at various concentrations (e.g., for selective growth of Actinomycetes and Streptomyces by addition of various antibiotics known to those skilled in the arts; Williams and Davies 1965, Use of antibiotics for selective isolation and enumeration of Actinomycetes in soil, J. Gen. Microbiol. 38: 251-261; Zhang, J. 2011, Improvement of an isolation medium for Actinomycetes. Modern Applied Science. 5 (2) 124-127; Seong et al., 2001, An improved selective isolation of rare Actinomycetes from forest soil, The Journal of Microbiology 39: 17-23), or the isolation of Propionibacterium on lactate and other isolation techniques (Kreig, 1981, Chapter 8, Enrichment and Isolation, p. 112-142 in Manual of Methods for General Bacteriology, Gerhardt et al., eds, American Society for Microbiology, Washington, D.C.).

(75) Growth conditions may also be varied, including temperatures above the normal body temperature (e.g., 37 C for humans) such as 42 C, or below normal temperature such as 30 C. The atmospheric conditions of oxygen may be normal atmospheric, microaerophilic, hypoxic or anaerobic.

(76) One or more media may be employed in one or more environmental conditions for the same biological sample, subdivided for each of the media and conditions. By way of example, a mixed bacterial population of the human skin microbiome is obtained by a skin swab plated at a density of approximately 1000 bacterial colonies per 85 mm Petri plate with tryptic soy agar media incubated in a 5% CO2/air chamber at 35 C for 16 hours. The tryptic soy agar (Difco Manual Difco & BBL Manual: Manual of Microbiological Culture Media, Second Edition, M. J. Zimbro et al., (Eds), Becton Dickinson and Co., 2009).

(77) The procedure of Example 1 is modified for neutrophil serine protease elastase inhibitors as indicated in Table 2.

Example 6: Enhanced Production of Protease Inhibitors

(78) The bacteria may be further selected for enhanced protease inhibitor production. The methods described herein include a culture-based method for the isolation of microorganisms producing protease inhibitors. The production of the protease inhibitors is visualized on a petri dish (FIGS. 5A and 5B). Using methods known to those skilled in the arts which include various mutagenesis methods such as exposure to ultraviolet light, chemical mutagens such as nitrosoguanidine, or genetic methods such as overexpression by plasmids or introduction of strong promoters, transposon mutagenesis, organisms with improved production of protease inhibitors are visualized as producing more intensely staining zone of protease protection. In order to develop strains with advantageous properties, biological, chemical or physical mutagens may be provided to generate new strains not preexisting in the source sample. The microbes may be recognized, for example, by their enhanced ability to secrete protease inhibitors. For example, a sample of purified or mixed bacteria may be subjected to radiation, such as ultraviolet light or gamma radiation, or chemical mutagens (Kodym, A. and Afzar, R. 2003, Physical and chemical mutagenesis. Methods in Molecular Biology 236: 189-204), to produce mutants, which can then be cultured and subcultured to select mutants with desired properties. Alternatively, the microbes may be altered by directed evolution (Lutz, S. 2010. Beyond directed evolution—semi-rational protein engineering and design. Curr. Opin. Biotechnol. 21: 734-743; Cai, W. et al., 2014. Directed evolution of three-finger toxin to produce serine protease inhibitors. J. Recept. Signal Transduct. Res. 34: 154-161). Likewise, the bacteria may be subject to the methods described by Pawelek et al., WO 96/40238, expressly incorporated herein by reference. The bacteria may be subject to genetic exchange technologies with natural or synthetic DNA from various sources (Vergunst, A and O'Callaghan, D. (eds) 2014, Host-Bacteria Interactions: Methods and Protocols. Methods in Molecular Biology 1197: 366 p.; Chaparro-Riggers, J. F. et al., 2007, Better library design: data driven protein engineering. Biotechnol. J. 2: 180-191), such as plasmids, transposons (Pajunen, M. I. et al., 2005. Generation of transposing insertion mutant libraries for gram-positive bacteria by electroporation of phage Mu DNA transposition complexes. Microbiology 151: 1209-1218), co-cultured strains, and the like. The result is various populations of bacteria that are distinct from their parent strains, some small portion of which may have properties which are considered advantageous by the selection process.

Example 7: Identification of Novel Secreted Protease Inhibitors

(79) The secreted protease inhibitors as derived in the Examples identified above are inherently capable of secreting a protease inhibitor into the media. Supernatants of the media containing the protease are collected by centrifuging the bacteria and passing the supernatant through a 0.22 μm filter. Then, in a novel modification of protease zymography (Lantz and Ciborowski 1994, Zymographic techniques for detection and characterization of microbial proteases. Methods Enzymol. 1994; 235:563-594), a native, non-denaturing gel containing the cognate protein gelatin is run in duplicate, one with embedded gelatin and one without embedded gelatin. Rather than running a protease in the gel, the protease inhibitor supernatant is run. For the gelatin-embedded gel, the gel is then incubated in the exoenzyme protease supernatant which then digests all of the gelatin protein, except at the location of the protein band of the peptide protease inhibitor, which is determined by developing in 15% TCA (Hanspal et al., 1983, Detection of protease inhibitors using substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Anal Biochem. 132(2): 288-293). The duplicate gel is stained, the appropriate corresponding gel band is excised from the gel. The protein is identified using MALD-TOF.

(80) Other protease inhibition assays based on individual clones or strains are already known to those skilled in the arts, and generally couple a biochemical test for proteolysis with purified fractions that potentially contain protease inhibitors. The protease inhibition assays as conducted by Wee et al. (2000) for identification of the P. luminescens protease inhibitor utilizes ammonium sulfate precipitation of the culture supernatant, redisolution of the precipitate in phosphate buffer, dialysis, separation by isoelectric focusing, and then testing of fractions for inhibitory activity using azocoll (Chavira et al., 1984 Assaying proteases with azocoll, Analytical Biochem. 136: 446-450). Inhibitory activity can also be monitored by determining hydrolysis of the chromogenic peptide N-a-benzoyl-DL-arginine-p-nitroanilide (BAPNA) or by following the change in absorbance at 275 nm of the protease substrate N.sup.a-p-tosyl-L-argininemethylester. The purified protease inhibitor is then identified by MALDI-TOF (Webster and Oxley 2012, Protein identification by MALDI-TOF mass spectrometry, Methods in Molecular Biology 2012; 800:227-40. doi: 10.1007/978-1-61779-349-3_15; Yukihira et al., 2010 MALDI-MS-based high-throughput metabolite analysis for intracellular metabolic dynamics, Analytical Chemistry, 15; 82(10):4278-82. doi: 10.1021/ac100024w).

Example 8: Genetically Engineering Bacteria to Produce Bacteriocins

(81) Methods known to those skilled in the arts are used, including those described by Bermudes et al. U.S. Pat. No. 7,452,531 Compositions and Methods for Tumor-Targeted Delivery of Effector Molecules, expressly incorporated herein by reference in its entirety.

Example 9: Selection of Bacterial Strains with Enhanced Production of Bacteriocins

(82) The bacteria may be further selected for enhanced bacteriocin production using standard methods for visualizing production of bacteriocins which uses an indicator strain usually embedded in a soft agar overlay, and a test strain, or library of strains, applied to the surface. The production of the bacteriocin is then visualized as an increased zone of inhibition of the indicator strain. Using methods known to those skilled in the arts which include various mutagenesis methods such as exposure to ultraviolet light, chemical mutagens such as nitrosoguanidine, or genetic methods such as introduction of strong promoters, overexpression on plasmids, transposon mutagenesis, organisms with improved production of bacteriocins are visualized as producing wider zones of bacterial inhibition.

Example 10: Construction of Propionibacterium acnes Strains for Treatment of Acne Vulgaris

(83) Propionibacterium acnes, such as RT6, is genetically engineered to express one or more bacteriocins that kill pathogenic Propionibacterium acnes using methods known to those skilled in the arts. The bacteria may be further selected for enhanced bacteriocin production using standard methods for visualizing production of bacteriocins which uses an indicator strain usually embedded in a soft agar overlay, and a test strain, or library or mixed population of strains, applied to the surface. The indicator strains are pathogenic bacteria, such as Staphylococcus aureus, Streptococcus pyogenes, or Propionibacterium acnes pathogenic ribotypes. The production of the bacteriocin is then visualized as an increased zone of inhibition of the indicator strain. Using methods known to those skilled in the arts which include various mutagenesis methods such as exposure to ultraviolet light, chemical mutagens such as nitrosoguanidine, or genetic methods such as over expression on plasmids, insertion of strong promoters, transposon mutagenesis, organisms with improved production of bacteriocins are visualized as producing wider zones of bacterial inhibition. Using similar techniques, the therapeutic Propionibacterium is selected for resistance to the pathogen bacteriocins, such as those of pathogenic Propionibacterium ribotypes.

(84) The bacteria are further engineered to have phage resistance proteins, such as phage repressor proteins related to lambda phage c1 repressor, including those identified by Marinelli, et al., 2012 (Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates, mBio 3(5) doi:10.1128/mBio.00279-1). Use of standard methods of isolating and/or identifying phage resistant strains is also encompassed. It is of importance that the therapeutic bacterial strain, such as the RT6 strain of Propionibacterium acnes, be resistant to the resident, disease-associated organisms such as pathogenic ribotypes RT4, 5, 7, 8, 9 and 10 of Propionibacterium acnes (Fitz-Gibbon et al., 2013). Methods for selecting resistant strains selection for spontaneous resistance by exposure of the strain such as RT6 to the phage such as those described by Marinelli, et al., 2012 (Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates, mBio 3(5) doi:10.1128/mBio.00279-1), and recovery of the survivors, or the strain can be initially modified by chemical, ultraviolet or transposon mutagenesis, to create a mixed genetic population followed by exposure to the phage, and selection of survivors (Levin, 1994, Isolating multiple strains of Escherichia coli for coliphage isolation, phage typing, and mutant recovery, Chapter 4 pages 63-72, in Tested studies for laboratory teaching, Volume 15 (C. A. Goldman, Editor). Proceedings of the 15th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 390 pages; Exploitation of a new flagellatropic phage of Erwinia for positive selection of bacterial mutants attenuated in plant virulence: towards phage therapy T. J. Evans J Appl Microbiol 108 (2010) 676-685). The surviving strains may contain genetically integrated phage, i.e., lysogens. Preferred strains are those that do not contain the phage, which is readily determinable by genetic techniques such as PCR.

(85) The resulting bacteria are both capable of resisting attack by the virulent bacterial bacteriocins and/or their phage, are able to persist, colonize and kill pathogenic strains by expressing bacteriocins the pathogenic strains are sensitive to, and secrete protease inhibitors that suppress inflammation.

Example 11: Treatment of Disease Using Purified Protease Inhibitors

(86) Following identification and purification protease inhibitors, the inhibitors may be used for the treatment of protease-mediated diseases. By way of example, the purified protease inhibitor such as a chymase inhibitor or pathogenic Propionibacterium inhibitor is formulated in to an acceptable pharmaceutical carrier known to those skilled in the arts such as a cream, ointment or gel. The inhibitor is applied in sufficient frequency to eliminate the symptoms to a patient with dermatitis.

Example 12. Use of Microbiome Bacteria for the Treatment of Psoriasis, Acne Vulgaris, or Other Inflammatory Skin Diseases

(87) The purified protease inhibitor bacteria are used for treatment of psoriasis or acne vulgaris, for example. A sufficient amount of the bacteria are applied to the affected sites in a saline, gel cream or ointment formulation to result in colonization and inhibition of the inflammatory response, resulting in decrease in the size and/or number of inflammatory lesions.

(88) A sufficient amount of the bacteria are applied to the affected sites in a pharmaceutically acceptable formulation such as saline, gel, cream or ointment to result in colonization and inhibition of the inflammatory response, resulting in decrease in the size and/or number of inflammatory lesions.

(89) In general, the dosage ranges from about 1.0 cfu/kg to about 1×10.sup.10 cfu/kg; optionally from about 1.0 cfu/kg to about 1×10.sup.8 cfu/kg; optionally from about 1×10.sup.2 cfu/kg to about 1×10.sup.8 cfu/kg; optionally from about 1×10.sup.4 cfu/kg to about 1×10.sup.8 cfu/kg.

Example 13. Use of Protease Inhibitor for the Treatment of Psoriasis or Acne Vulgaris

(90) The purified protease inhibitor proteins are used for treatment of psoriasis. A sufficient amount of the substantially purified protease inhibitor, obtained using standard protein purification procedures known to those skilled in the art, is applied to the affected sites in a pharmaceutically acceptable carrier such as saline, gel, cream or ointment formulation to result in inhibition of the inflammatory response, resulting in decrease in the size and/or number of inflammatory lesions.

Example 14: Identification of Microbiome Bacteria Secreting Protease Inhibitors

(91) Secreted protease inhibitors of the human microbiome are determined from individual bacteria or mixed colonies of bacteria collected from human body sites by culturing the bacteria and screening for zones of protease inhibition. First, the cognate protein, e.g., collagen, or collagen fragments (gelatin), is embedded into a nutrient agar using methods known to those skilled in the arts. Second, a proteolytic bacterium of the human microbiome is grown under conditions for which it produces an exoenzyme protease, such as that for collagen or gelatin, the secretion of such which can be determined using the said gelatin-containing agar plate (Vermelho et al., 1996, Detection of Extracellular Proteases from Microorganisms on Agar Plates Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(6): 755-760). Non-proteolytic bacteria are incubated on the gelatin agar plate, which may be a mixed culture including known or unknown organisms, and then replica plated to generate a master plate, to later recover bacteria of interest. The gelatin plate is then flooded with the exoenzyme protease supernatant and incubated for a sufficient time to degrade all of the gelatin embedded within the plate. The protease plate is then “developed” by precipitating undigested protein using 15% trichloroacetic acid (TCA). For microbiome bacteria secreting protease inhibitors, a halo of precipitated, undigested protein is observed due the presence of a protease inhibitor, and the corresponding bacterium selected from the master plate.

(92) It will be understood that the foregoing is only illustrative of the principles of the invention, and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the invention.