COLICINS FOR TREATING BACTERIAL INFECTIONS

Abstract

The Invention relates to materials and methods for the treatment of conditions associated with bacterial biofilms, intracellular bacterial infections and/or adherent-invasive Escherichia coli infections, including Crohns' disease. In particular, the invention relates to the use of colicins and bacteria producing colicins, for the treatment of such conditions.

Claims

1-6. (canceled)

7. A method of treating Crohn's disease in a subject in need thereof, comprising administering to the subject an effective amount of a colicin.

8-9. (canceled)

10. The c method according to claim 1, wherein the method comprises treatment of an adherent-invasive Escherichia coli (AIEC) infection.

11. The method according to claim 10, wherein the AIEC infection is associated with a biofilm.

12. The method according to claim 10, wherein the AIEC infection an intracellular AIEC infection.

13. The method according to any claim 7, wherein the method of treating Crohn's disease is a prophylactic method of preventing Crohn's disease in a subject who has been determined to have a genetic predisposition to Crohn's disease.

14-19. (canceled)

20. A method of treating an AIEC infection in a subject in need thereof, comprising administering to the subject an effective amount of a colicin.

21-22. (canceled)

23. The method according to claim 20, wherein the AIEC of the infection are associated with a biofilm.

24. The method according to claim 20, wherein the AIEC of the infection are intracellular.

25. The method according to claim 24, wherein the intracellular AIEC are in macrophages.

26-44. (canceled)

45. The method according claim 7, wherein the subject is a human.

46-49. (canceled)

50. The method according to claim 7, wherein the colicin is a Group A colicin.

51. The method according to claim 7, wherein the colicin binds to an E.coli surface receptor selected from BtuB, FepA and Cir.

52. The method according to claim 51, wherein the colicin binds to BtuB.

53. The method according to claim 7, wherein the colicin is a membrane-depolarising or pore-forming colicin.

54. The method according to claim 53, wherein the colicin is colicin E1 or colicin IA.

55. The method according to claim 54, wherein the colicin is colicin E1.

56. The method according to claim 7, wherein the colicin is a nuclease.

57. The method according to claim 56 wherein the colicin is a DNase.

58. The method according to claim 57, wherein the colicin is colicin E9.

59. The method according to claim 56, wherein the colicin is an RNase.

60. The method according to claim 59, wherein the colicin is colicin E3.

61. The method according to claim 56, wherein the colicin is a tRNase.

62. The method according to claim 61, wherein the colicin is colicin D.

63-66. (canceled)

Description

DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1. Killing of AI E. coli LF82 growing in the biofilm state by colicins and small molecule antibiotics. Biofilms of E. coli Crohn's isolate LF82 treated with ampicillin, ciprofloxacin, metronidazole, rifaximin and colicins E1, E3, E9 and D for 1 h.

[0052] FIG. 2. Survival (%) of LF82 biofilms following treatment with the colicin E1 producing W3110 pColE1-K53 strain and the non-producing W3110 strain.

[0053] FIG. 3. Killing of intracellular bacteria by colicin E1.

[0054] FIG. 4. Killing of intracellular bacteria by colicin E9.

DETAILED DESCRIPTION OF THE INVENTION

[0055] Colicins

[0056] Colicins can be obtained directly from the bacterial supernatants of colicin producing bacterial strains. Typically, protein is precipitated from the supernatant by ammonium sulphate precipitation and protein purified by ion exchange chromatography and gel filtration. Alternatively, recombinant protein could be produced with an engineered affinity tag, such as a His-tag and the protein purified by affinity chromatography (metal affinity chromatography in the case of His-tagged protein). In some cases in accordance with the invention, the colicin is in a heterodimer with a corresponding immunity protein. Nuclease-type colicin-immunity protein complexes are suitable. In this case, an affinity tag can be engineered onto the immunity protein and the colicin-immunity protein complex isolated by affinity chromatography.

[0057] Bacteria that Produce a Colicin

[0058] Bacterial strains that produce (i.e. express and secrete) a colicin can be isolated from nature. In such cases, the colicin can be regarded as being endogenous to that strain. Alternatively, bacteria that produce (i.e. express and secrete) one or more colicins can be engineered using standard techniques that are well known in the art. Such engineering may be performed to enhance production (e.g. expression and/or secretion) of a colicin already naturally produced by the bacteria, i.e. to enhance production of an endogenous colicin. Alternatively, bacteria may be engineered to produce (i.e. express and secrete) a heterologous colicin, i.e. a colicin which is not naturally produced by those bacteria.

[0059] Colicins are typically encoded on plasmids. Thus, bacteria that produce a colicin may be engineered by introducing a plasmid carrying a gene for a colicin, although other expression vectors or constructs may be employed, including chromosomally-integrated expression constructs. Thus the bacterium may comprise an expression vector or expression construct comprising a nucleic acid sequence encoding a colicin, whereby the bacterium is able to express and secrete the colicin. The colicin may be endogenous or heterologous to the bacterium. In some cases the expression vector or construct (e.g. plasmid) additionally encodes (i.e. comprises a nucleic acid sequence encoding) an immunity protein and/or a colicin release protein, whereby the bacterium is able to express said immunity protein and/or release protein, and secrete them if appropriate. An example of a suitable plasmid is pColE1-K53.

[0060] Immunity proteins protect the cell from the activity of colicins produced either by the cell itself or by neighbouring cells. Each immunity protein is a specific antagonist of the activity of a corresponding colicin. For nuclease-type colicins, the immunity protein binds strongly to the colicin and prevents it from degrading nucleic acid in the cytoplasm. Typically a colicin-immunity protein complex is released from the producing cell, but the colicin dissociates from the immunity complex before or on entry to a target sensitive cell. Thus, in some cases in accordance with the invention, the bacterium produces both a colicin, for example a nuclease-type colicin, and a corresponding immunity protein. The bacterium may release a colicin-immunity protein complex.

[0061] Bacteria that produce a pore forming colicin may also produce a corresponding immunity protein. For pore-forming colicins, the immunity protein is typically an inner membrane protein that prevents exogenous colicin from depolarising the membrane. Cytoplasmic inophoric colicin is not active on the producing cell. Thus, in some cases in accordance with the invention, the bacterium produces both a pore-forming colicin and a corresponding immunity protein. In other cases, the bacterium that produces the pore-forming colicin does not express the receptor that is recognised by the colicin, so the producing cell is not sensitive to exogenous colicin made by neighbouring cells of the same strain. Example immunity proteins are disclosed in Cascales et al. (2007) and Papadakos et al (2011).

[0062] The colicin may be released from the bacterium by any suitable means. For example, a suitable targeting sequence may be engineered onto the colicin to direct its secretion from the cell. Alternatively, the colicin producing bacterium may co-express a colicin release protein. Colicin release proteins, also known as colicin lysis proteins, are small lipoproteins having a common consensus sequence that are sometimes co-expressed with a colicin. The release of colicins from a cell that co-expresses a colicin release protein occurs through a unique mechanism. Expression of the colicin release protein is lethal to the producing cell. The amino acid sequences of example lysis release proteins are disclosed in FIG. 2 of Cascales et al. (2007).

[0063] Probiotics

[0064] A living microorganism that confers a health benefit when administered or consumed in adequate amounts is referred to as a probiotic, or a probiotic organism. A probiotic organism can be used as an ingredient in a food product or in a diet supplement. In some cases, the bacterium in accordance with the invention is a probiotic bacterium.

[0065] Subjects for Treatment

[0066] Preferred subjects for treatment by the methods of the invention are mammals. Preferred subjects are primates (including humans), rodents (including mice and rats), and other common laboratory, domestic and agricultural animals, including but not limited to rabbits, dogs, cats, horses, cows, sheep, goats, etc.

[0067] Pharmaceutical Compositions and Methods of Treatment

[0068] The colicins and bacteria described herein can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular and intraperitoneal routes. Examples of suitable compositions and methods of administration are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). Preferably the route of administration is oral. Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

[0069] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. Whatever the nature of the active agent that is to be given to an individual (e.g. a cell, polypeptide, nucleic acid molecule, other pharmaceutically useful agent according to the present invention), administration is preferably in a prophylactically effective amount or a therapeutically effective amount (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0070] A composition may be administered alone or in combination with other treatments, including other colicins or colicin producing bacteria, either simultaneously or sequentially dependent upon the condition to be treated.

TABLE-US-00002 Sequences SEQIDNO:1 ColicinE1(UniProt:P02978,LastmodifiedJul.21,1986.Version 1.Checksum:A77C351BBC1AB7C1) Domainstructure:T-domain1-200,R-domain201-333,C-domain334- 522 METAVAYYKDGVPYDDKGQVIITLLNGTPDGSGSGGGGGKGGSKSESSAAIHATAKWSTAQLKKT QAEQAARAKAAAEAQAKAKANRDALTQRLKDIVNEALRHNASRTPSATELAHANNAAMQAEDERL RLAKAEEKARKEAEAAEKAFQEAEQRRKEIEREKAETERQLKLAEAEEKRLAALSEEAKAVEIAQ KKLSAAQSEVVKMDGEIKTLNSRLSSSIHARDAEMKTLAGKRNELAQASAKYKELDELVKKLSPR ANDPLQNRPFFEATRRRVGAGKIREEKQKQVTASETRINRINADITQIQKAISQVSNNRNAGIAR VHEAEENLKKAQNNLLNSQIKDAVDATVSFYQTLTEKYGEKYSKMAQELADKSKGKKIGNVNEAL AAFEKYKDVLNKKFSKADRDAIFNALASVKYDDWAKHLDQFAKYLKITGHVSFGYDVVSDILKIK DTGDWKPLFLTLEKKAADAGVSYVVALLFSLLAGTTLGIWGIAIVTGILCSYIDKNKLNTINEVL GI SEQIDNO:2 ColicinE3(Uniprot:P00646,LastmodifiedApr.1,1988.Version 1.Checksum:E444CE918D89ECD6) Domainstructure:T-domain1-315,R-domain316-450,C-domain 451-551 MSGGDGRGHNTGAHSTSGNINGGPTGLGVGGGASDGSGWSSENNPWGGGSGSGIHWGGGSGHGNG GGNGNSGGGSGTGGNLSAVAAPVAFGFPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFG LWGVALYGVLPSQIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRVVDDVKDERQN ISVVSGVPMSVPVVDAKPTERPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAG FTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHPVEAAERNYER ARAELNQANEDVARNQERQAKAVQVYNSRKSELDAANKTLADAIAEIKQFNRFAHDPMAGGHRMW QMAGLKAQRAQTDVNNKQAAFDAAAKEKSDADAALSSAMESRKKKEDKKRSAENNLNDEKNKPRK GFKDYGHDYHPAPKTENIKGLGDLKPGIPKTPKQNGGGKRKRWTGDKGRKIYEWDSQHGELEGYR ASDGQHLGSFDPKTGNQLKGPDPKRNIKKYL SEQIDNO:3 ColicinE9(Uniprot:P09883,LastmodifiedOct.1,1996. Version4.Checksum:47A71B57B45AFDD9) Domainstructure:T-domain1-315,R-domain316-450,C-domain 451-582 MSGGDGRGHNTGAHSTSGNINGGPTGIGVSGGASDGSGWSSENNPWGGGSGSGIHWGGGSGRGNG GGNGNSGGGSGTGGNLSAVAAPVAFGFPALSTPGAGGLAVSISASELSAAIAGIIAKLKKVNLKF TPFGVVLSSLIPSEIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRVVDDVKDERQ NISVVSGVPMSVPVVDAKPTERPGVETASIPGAPVLNISVNDSTPAVQTLSPGVTNNTDKDVRPA GFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHPVEAAERNYE RARAELNQANEDVARNQERQAKAVQVYNSRKSELDAANKTLADAIAEIKQFNRFAHDPMAGGHRM WQMAGLKAQRAQTDVNNKQAAFDAAAKEKSDADAALSAAQERRKQKENKEKDAKDKLDKESKRNK PGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKSFDDFRKAVWEEVSKDPELSKNLN PSNKSSVSKGYSPFTPKNQQVGGRKVYELHHDKPISQGGEVYDMDNIRVTTPKRHIDIHRGK SEQIDNO:4 ColicinD(Uniprot:P17998,LastmodifiedNov.1,1990. Version1.Checksum:F468B4A0172ABBB1)Domainstructure:C-domain 591-697 MSDYEGSGPTEGIDYGHSMVVWPSTGLISGGDVKPGGSSGIAPSMPPGWGDYSPQGIALVQSVLF PGIIRRIILDKELEEGDWSGWSVSVHSPWGNEKVSAARTVLENGLRGGLPEPSRPAAVSFARLEP ASGNEQKIIRLMVTQQLEQVTDIPASQLPAAGNNVPVKYRLMDLMQNGTQYMAIIGGIPMTVPVV DAVPVPDRSRPGTNIKDVYSAPVSPNLPDLVLSVGQMNTPVLSNPEIQEEGVIAETGNYVEAGYT MSSNNHDVIVRFPEGSDVSPLYISTVEILDSNGLSQRQEAENKAKDDFRVKKEEAVARAEAEKAK AELFSKAGVNQPPVYTQEMMERANSVMNEQGALVLNNTASSVQLAMTGTGVWTAAGDIAGNISKF FSNALEKVTIPEVSPLLMRISLGALWFHSEEAGAGSDIVPGRNLEAMFSLSAQMLAGQGVVIEPG ATSVNLPVRGQLINSNGQLALDLLKTGNESIPAAVPVLNAVRDTATGLDKITLPAVVGAPSRTIL VNPVPQPSVPTDTGNHQPVPVTPVHTGTEVKSVEMPVTTITPVSDVGGLRDFIYWRPDAAGTGVE AVYVMLNDPLDSGRFSRKQLDKKYKHAGDFGISDTKKNRETLTKFRDAIEEHLSDKDTVEKGTYR REKGSKVYFNPNTMNVVIIKSNGEFLSGWKINPDADNGRIYLETGEL SEQIDNO:5 ColicinIa(UniProt:P06716,LastmodifiedOct.24,2003. Version2.Checksum:3DC0DF322F405D39) Domainstructure:Tdomain1-298,Rdomain299-402,Cdomain403- 626 MSDPVRITNPGAESLGYDSDGHEIMAVDIYVNPPRVDVFHGTPPAWSSFGNKTIWGGNEWVDDSp TRSDIEKRDKEITAYKNTLSAQQKENENKRTEAGKRLSAAIAAREKDENTLKTLRAGNADAADIT RQEFRLLQAELREYGFRTEIAGYDALRLHTESRMLFADADSLRISPREARSLIEQAEKRQKDAQN ADKKAADMLAEYERRKGILDTRLSELEKNGGAALAVLDAQQARLLGQQTRNDRAISEARNKLSSV TESLNTARNALTRAEQQLTQQKNTPDGKTIVSPEKFPGRSSTNHSIVVSGDPRFAGTIKITTSAV IDNRANLNYLLSHSGLDYKRNILNDRNPVVTEDVEGDKKIYNAEVAEWDKLRQRLLDARNKITSA ESAVNSARNNLSARTNEQKHANDALNALLKEKENIRNQLSGINQKIAEEKRKQDELKATKDAINF TTEFLKSVSEKYGAKAEQLAREMAGQAKGKKIRNVEEALKTYEKYRADINKKINAKDRAAIAAAL ESVKLSDISSNLNRFSRGLGYAGKFTSLADWITEFGKAVRTENWRPLFVKTETIIAGNAATALVA LVFSILTGSALGIIGYGLLMAVTGALIDESLVEKANKFWGI SEQIDNO:6 ColicinE2(UniProt:B5TQU9,LastmodifiedNov.4,2008. Version1. Checksum:B046C6FC7BF0FE2C) Domainstructure:T-domain1-315,R-domain316-450,C-domain451- 581 MSGGDGRGHNTGAHSTSGNINGGPTGLGVGGGASDGSGWSSENNPWGGGSGSGIHWGGGSGHGNG GGNSNSGGGSGTGGNLSAVAAPVAFGFPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFG LWGVALYGVLPSQIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRVVDDVKDERQN ISVVSGVPMSVPVVDAKPTERPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAG FTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHPVEAAERNYER ARAELNQANEDVARNQERQAKAVQVYNSRKSELDAANKTLADAIAEIKQFDRFAHDPMAGGHRMW QMAGLKAQRAQTDVNNKQAAFDAAAKEKSDADAALSAAQERRKQKENKEKDAKDKLDKESKRNKP GKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKNFDDFRKKFWEEVSKDPDLSKQFKG SNKTNIQKGKAPFARKKDQVGGRERFELHHDKPISQDGGVYDMNNIRVTTPKRHIDIHRGK SEQIDNO:7 ColicinE7(Uniprot:Q47112,LastmodifiedMay30,2000.Version 2.Checksum:E5B05E73B2E17249) Domainstructure:T-domain1-315,R-domain316-450,C-domain451- 576 MSGGDGRGHNSGAHNTGGNINGGPTGLGGNGGASDGSGWSSENNPWGGGSGSGVHWGGGSGHGNG GGNSNSGGGSNSSVAAPMAFGFPALAAPGAGTLGISVSGEALSAAIADIFAALKGPFKFSAWGIA LYGILPSEIAKDDPNMMSKIVTSLPAETVTNVQVSTLPLDQATVSVTKRVTDVVKDTRQHIAVVA GVPMSVPVVNAKPTRTPGVFHASFPGVPSLTVSTVKGLPVSTTLPRGITEDKGRTAVPAGFTFGG GSHEAVIRFPKESGQKPVYVSVTDVLTPAQVKQRQDEEKRLQQEWNDAHPVEVAERNYEQARAEL NQANKDVARNQERQAKAVQVYNSRKSELDAANKTLADAKAEIKQFERFAREPMAAGHRMWQMAGL KAQRAQTDVNNKKAAFDAAAKEKSDADVALSSALERRKQKENKEKDAKAKLDKESKRNKPGKATG KGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFKSFDDFRKKFWEEVSKDPELSKQFSRNNNDR MKVGKAPKTRTQDVSGKRTSFELHHEKPISQNGGVYDMDNISVVTPKRHIDIHRGK SEQIDNO:8 ColicinE8(UniProt:C6GJY4,LastmodifiedSep.1,2009. Version1.Checksum:42ECBBDB92E0DB52) Domainstructure:Tdomain1-315,Rdomain316-450,Cdomain451- 573 MSGGDGRGHNTGAHSTSGNINGGPTGIGVSGGASDGSGWSSENNPWGGGSGSGIHWGGGSGRGNG GGNGNSGGGSGTGGNLSAVAAPVAFGFPALSTPGAGGLAVSISASELSAAIAGIIAKLKKVNLKF TPFGVVLSSLIPSEIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRVVDDVKDERQ NISVVSGVPMSVPVVDAKPTERPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPA GFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHPVEAAERNYE RARAELNQANEDVARNQERQAKAVQVYNSRKSELDAANKTLADAIAEIKQFNRFAHDPMAGGHRM WQMAGLKAQRAQTDVNNKQAADADAALSAAQERRKQKENKEKDAKDKLDKESKRNKPGKATGKQK PVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKNFDDFRRKFWEEVSKDPELSKQFNPGNKKRLSQ GLAPRARNKDTVGGRRSFELHHDKPISQDGGVYDMDNLRITTPKRHIDIHRGQ SEQIDNO:9 ColicinE4Cytotoxicdomain(Uniprot:Q47109,Lastmodified Nov.1,1996.Version1.Checksum:6F997EED8BF568D1) ERFAREPMAAGHRMWQMAGLKAQRAQTDVNNKKAAFDAAAKEKSDADAALSSAMESRKKKEDKKR SAENKLNEEKNKPRKGVKDYGHDYHPAPKTEEIKGLGELKKAPKKTPKQGGGGRRDRWIGDKGRK IYEWDSQHGELEGYRASDGEHIGAFDPKTGKQIKGPDPKGRNIKKYL SEQIDNO:10 ColicinE6(UniProt:P17999,LastmodifiedNov.1,1990. Version1.Checksum:D223D7F0770392E0) Domainstructure:T-domain1-315,R-domain316-450,C-domain451- 551 MSGGDGRGHNTGAHSTSGNINGGPTGLGVGGGASDGSGWSSENNPWGGGSGSGIHWGGGSGHGNG GGNGNSGGGSGTGGNLSAVAAPVAFGFPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFG LWGVALYGVLPSQIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRVVDDVKDERQN ISVVSGVPMSVPVVDAKPTERPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAG FTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHPVEAAERNYER ARAELNQANEDVARNQERQAKAVQVYNSRKSELDAANKTLADAIAEIKQFNRFAHDPMAGGHRMW QMAGLKAQRAQTDVNNKQAAFDAAAKEKSDADAALSSAMESRKKKEDKKRSAENKLNEEKNKPRK GVKDYGHDYHPDPKTEDIKGLGELKEGKPKTPKQGGGGKRARWYGDKGRKIYEWDSQHGELEGYR ASDGQHLGSFEPKTGNQLKGPDPKRNIKKYL SEQIDNO:11 ColicinE5cytotoxicdomain(UniProt:P18000,Lastmodified Nov.1,1990.Version1.Checksum:9DE1713F474B2BA4) RFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAFDAAAKEKADADAALSTAMESRKKKEDNKRD AEGKLNDELAKNKGKIPGLKIDQKIRGQMPERGWTEDDIKNTVSNGATGTSFDKRSPKKTPPDYL GRNDPATVYGSPGKYVVVNDRTGEVTQISDKTDPGWVDDSRIQWGNKNDQ SEQIDNO:12 ColicinA(UniProt:P04480,LastmodifiedAug.13,1987. Version1.Checksum:B80FA1F52A8CFC5D) Domainstructure:Tdomain1-172,Rdomain173-391,Cdomain392- 592 MPGFNYGGKGDGTGWSSERGSGPEPGGGSHGNSGGHDRGDSSNVGNESVTVMKPGDSYNTPWGKV IINAAGQPTMNGTVMTADNSSMVPYGRGFTRVLNSLVNNPVSPAGQNGGKSPVQTAVENYLMVQS GNLPPGYWLSNGKVMTEVREERTSGGGGKNGNERTWTVKVPREVPQLTASYNEGMRIRQEAADRA RAEANARALAEEEARAIASGKSKAEFDAGKRVEAAQAAINTAQLNVNNLSGAVSAANQVITQKQA EMTPLKNELAAANQRVQETLKFINDPIRSRIHFNMRSGLIRAQHNVDTKQNEINAAVANRDALNS QLSQANNILQNARNEKSAADAALSAATAQRLQAEAALRAAAEAAEKARQRQAEEAERQRQAMEVA EKAKDERELLEKTSELIAGMGDKIGEHLGDKYKAIAKDIADNIKNFQGKTIRSFDDAMASLNKIT ANPAMKINKADRDALVNAWKHVDAQDMANKLGNLSKAFKVADVVMKVEKVREKSIEGYETGNWGP LMLEVESWVLSGIASSVALGIFSATLGAYALSLGVPAIAVGIAGILLAAVVGALIDDKFADALNN EIIRPAH SEQIDNO:13 ColicinB(UniProt:P05819,LastmodifiedJan.23,2007. Version3.Checksum:8ABB972CF1925964) Domainstructure:TandRdomains1-291,Cdomain292-511 MSDNEGSVPTEGIDYGDTMVVWPSTGRIPGGDVKPGGSSGLAPSMPPGWGDYSPQGIALVQSVLF PGIIRRIILDKELEEGDWSGWSVSVHSPWGNEKVSAARTVLENGLRGGLPEPSRPAAVSFARLEP ASGNEQKIIRLMVTQQLEQVTDIPASQLPAAGNNVPVKYRLTDLMQNGTQYMAIIGGIPMTVPVV DAVPVPDRSRPGTNIKDVYSAPVSPNLPDLVLSVGQMNTPVRSNPEIQEDGVISETGNYVEAGYT MSSNNHDVIVRFPEGSGVSPLYISAVEILDSNSLSQRQEAENNAKDDFRVKKEQENDEKTVLTKT SEVIISVGDKVGEYLGDKYKALSREIAENINNFQGKTIRSYDDAMSSINKLMANPSLKINATDKE AIVNAWKAFNAEDMGNKFAALGKTFKAADYAIKANNIREKSIEGYQTGNWGPLMLEVESWVISGM ASAVALSLFSLTLGSALIAFGLSATVVGFVGVVIAGAIGAFIDDKFVDELNHKIIK SEQIDNO:14 ColicinN(UniProt:P08083,LastmodifiedAug.1,1988.Version 1.Checksum:1C4342E222F8CECD) Domainstructure:TandRdomains1-186,Cdomain187-387 MGSNGADNAHNNAFGGGKNPGIGNTSGAGSNGSASSNRGNSNGWSWSNKPHKNDGFHSDGSYHIT FHGDNNSKPKPGGNSGNRGNNGDGASAKVGEITITPDNSKPGRYISSNPEYSLLAKLIDAESIKG TEVYTFHTRKGQYVKVTVPDSNIDKMRVDYVNWKGPKYNNKLVKRFVSQFLLFRKEEKEKNEKEA LLKASELVSGMGDKLGEYLGVKYKNVAKEVANDIKNFHGRNIRSYNEAMASLNKVLANPKMKVNK SDKDAIVNAWKQVNAKDMANKIGNLGKAFKVADLAIKVEKIREKSIEGYNTGNWGPLLLEVESWI IGGVVAGVAISLFGAVLSFLPISGLAVTALGVIGIMTISYLSSFIDANRVSNINNIISSVIR SEQIDNO:15 ColicinM(UniprotP05820,LastmodifiedNov.1,1988. Version1.Checksum:B41B7BE107EC1DBA) Domainstructure:Tdomain1-35,Rdomain36-139,Cdomain140-271 METLTVHAPSPSTNLPSYGNGAFSLSAPHVPGAGPLLVQVVYSFFQSPNMCLQALTQLEDYIKKH GASNPLTLQIISTNIGYFCNADRNLVLHPGISVYDAYHFAKPAPSQYDYRSMNMKQMSGNVTTPI VALAHYLWGNGAERSVNIANIGLKISPMKINQIKDIIKSGVVGTFPVSTKFTHATGDYNVITGAY LGNITLKTEGTLTISANGSWTYNGVVRSYDDKYDFNASTHRGIIGESLTRLGAMFSGKEYQILLP GEIHIKESGKR SEQIDNO:16 ColicinS4(UniProt:Q9XB47,LastmodifiedNov.1,1999. Version1.Checksum:3E36C7271BF1D293) Domainstructure:Tdomain1-118,Rdomain119-299,Cdomain299- 499 MAKELSVYGPTAGESMGGTGANLNQQGGNNNSNSGVHWGGGSGSGNGGREHGSQTGWGWSKTNNP DVPPYVDDNGQVRITITNGLVKTPVYGVPGAGGNSDVQGGYIPENPNDEVARKWDKNNLPREIDV SIDGFKYRVTLNDNGRAIGILRIGVRPYVGSEKAKAGIMEKINHKTPEEIYEALGFNKDESQRQE KAKQQAEDAWDRLPPNVRKFDVDVEQFHYLVVLDDYGNVLSVTRTGVRPYVGSEKAKAGIMDKVD HKTPEEIYEALGFNNEEPQRQNQAKKAAYDVFYSFSMNRDRIQSDVLNKAAEVISDIGNKVGDYL GDAYKSLAREIADDVKNFQGKTIRSYDDAMASLNKVLSNPGFKFNRADSDALANVWRSIDAQDMA NKLGNISKAFKFADVVMKVEKVREKSIEGYETGNWGPLMLEVESWVLSGIASAVALGVFSATLGA YALSLGAPAIAVGIVGILLAAVVGALLDDKFADALNKEIIKPAH

EXAMPLES

Example 1

[0071] Activity of colicins against AI E. coli

[0072] Four adherent invasive E. coli isolates (AIEC) recovered from patients with Crohns' disease (isolates 95, 419, 615 and the AIEC type strain LF82) were shown to be sensitive to colicins E1, E3, E9 and D in spot tests, where purified colicin is spotted onto a growing lawn of cells.

[0073] Briefly, 25 l of a log phase bacterial culture (OD.sub.600=0.6) was added to 5 ml of molten 0.8% agarose and poured on top of an LB agar plate. A 2 l drop of each colicin (0.2 mg/ml) was spotted onto the overlay plate. The plates were incubated for 18 h and examined for zones of inhibition. All E. coli isolates tested were sensitive to the cytotoxic activity of the four colicin proteins, as indicated by the presence of clear zones representing cell killing (not shown).

Example 2

[0074] The Activity of a Colicin E1-Producing Strain Against AIEC Biofilms

[0075] The clinically relevant state for AI E. coli in the infected gut mucosa is the biofilm state in which bacteria show enhanced tolerance to antibiotics. We therefore tested the ability of colicins to kill AI E. coli in the biofilm state using the MBEC Physiology and Genetics Assay (Innovotech). In this assay, biofilm growth occurs on 96 identical pegs protruding from the lid of a 96-well microtitre plate. Biofilm formation was demonstrated using electron microscopy (not shown).

[0076] To determine the cytotoxicity of colicins against LF82 biofilms we compared the % cell survival of AIEC in biofilms treated with colicins with those exposed to antibiotics which are frequently prescribed in the treatment of Crohn's disease (FIG. 1). Biofilms were formed on the MBEC 96-peg plate platform for 24 h, then challenged for 1 h with 150 l dilutions of the antibiotics (ampicillin, ciprofloxacin, metronidazole and rifaximin) and colicins (E1, E3, E9 and D) in sterile PBS in the concentration range 0.002 g/ml-200 g/ml. All dilutions of antimicrobials were done in triplicate and control antimicrobial-free biofilms were treated with 150 l of sterile PBS. Following treatment, the viability of biofilm-associated cells was tested using the metabolic dye XTT. The XTT salt changes colour when it is metabolised by viable cells. This colour change can be compared to untreated antimicrobial-free biofilms, giving an indication of the percentage survival in biofilms exposed to the antimicrobials.

[0077] We further tested the ability of colicins E1 (a pore-forming colicin) E3 (a rRNase), E9 (a DNase), D (a tRNase) and IA (a pore-forming colicin) to kill 6 additional strains of AIEC in the biofilm state. All showed good killing activity against the multiple strains. Colicins E1 (a membrane depolarising colicin) and E9 (a DNase colicin) were the most effective in killing AIEC in the biofilm state. For all isolates, colicin E1 and E9 displayed superior antimicrobial activity against the biofilm-associated cells than the antibiotics ampicillin, metronidazole and rifaximin.

Example 3

[0078] The Activity of a Colicin E1 Producing Strain Against AIEC Biofilms

[0079] We envisage that colicins could be delivered in the form of a colicin producing bacterial strain. To determine if the addition of a colicin producing E. coli strain to LF82 resulted in killing of biofilm associated bacteria we added E.coli K-12 W3110 carrying the colicin El plasmid pColEl-K53 to 24 hour LF82 biofilms.

[0080] Biofilms of the LF82 strain were formed for 24 h on the MBEC 96-peg plate platform. A 150 l culture volume of W3110 pColE1-K53 was also grown in the wells of a 96-well flat-bottom plate in LB broth supplemented with a sub-inhibitory concentration of the antibiotic ciprofloxacin (0.001 g/ml). Ciprofloxicin induces colicin production through activation of the SOS response to DNA damage. The antibiotic induces the expression of the colicin E1 protein in this E. coli K12 strain. The 24 h LF82 biofilms were exposed to the W3110 pColE1-K53 strain for 1, 2, 4, 6, and 24 h. The pegs with the treated LF82 biofilms were then removed from the plate, placed in 1 ml of sterile PBS, sonicated and vortexed to remove the bacteria from the surface. The sonicate was plated out on LB agar containing 50g/ml ampicillin to select for colonies of LF82, which are resistant to ampicillin. The plates were incubated for 18 h and colonies were counted to determine the survival of LF82 biofilm-associated cells following treatment with the K-12 colicin E1-producing strain. As a control, biofilms of LF82 were also exposed to the non-colicin-producing K-12 strain W3110 to ensure that the presence of the K-12 strain did not affect LF82 cell viability.

[0081] Addition of the colicin producing strain W3110 pColE1-K53 greatly reduced survival of LF82, whereas addition of W3110 lacking the colicin E1 plasmid had no effect on survival of the AI E. coli strain (FIG. 2).

Example 4

[0082] The Activity of Colicins E1 and E9 in a Macrophage Model of AIEC Infection

[0083] AI E. coli are able to infect and replicate within macrophages and epithelial cells. To determine if colicin E1 is able to kill intracellular bacteria raw murine macrophages (J774) were infected with AI E. coli LF82 and treated with colicin E1 (FIG. 3).

[0084] Raw murine macrophages (J774) were grown in 24-well tissue culture plates in RPMI tissue culture media (containing 3% fetal calf serum, 1% L-glutamate, 1% pen/strep) at 37 C. in 5% CO.sub.2 until the cells reached 70-80% confluency. The macrophages were then infected with E. coli LF82 at a multiplicity of infection of 50 (MOI 50). After 2 h of infection, the macrophages were exposed to the antibiotic gentamicin (100 g/ml) to kill any bacteria outside the macrophage which hadn't successfully invaded the cell. The LF82-infected macrophages were then treated with colicin E1 (10, 0.1, 0.001 g/ml) for 3 h at 37 C. The macrophages were then scraped from the surface of the plate, lysed with Triton X and the intra-cellular bacteria were plated on LB agar containing 50 g/ml ampicillin for selection of LF82. The plates were incubated overnight at 37 C. and colony counts were performed. Treatment with colicin E1 (10 g/ml) resulted in approximately an 80% reduction in the number of bacteria recovered from the macrophages showing that this colicin can kill intracellular bacteria.

[0085] We also tested the ability of colicin E9 and a catalytically inactive colicin E9 variant (colicin E9 H575A) for the ability to kill intracellular E. coli LF82 in infected macrophages. Experiments were performed as described for colicin E1 except that infected macrophages were treated with colicin for 4 and 24 hours. Like colicin E1, wild-type colicin E9 was able to kill intracellular LF82 but in infected macrophages treated with the catalytically inactive mutant colicin E9 H575A no killing of intracellular bacteria was observed. See FIG. 4. These data indicate that killing is a direct consequence of the cytotoxic activity of the colicin and not through activation of host (macrophage) cell killing pathways.

[0086] Fluorescence microscopy has been used to visualise infected macrophages treated with colicin E9 tagged with red fluorescent protein. These studies clearly show internalisation of colicin E9 into the macrophages, further reinforcing our findings that colicins are capable of entering macrophage cells and acting on intracellular bacteria. (Data not shown.)

EQUIVALENTS

[0087] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

[0088] All references, including patent documents, disclosed herein are incorporated by reference in their entirety for all purposes, particularly for the disclosure referenced herein.

REFERENCES

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