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WO2014143033A1 - Identification de clostridium difficile cspc comme récepteur de germination de l'acide biliaire - Google Patents

Identification de clostridium difficile cspc comme récepteur de germination de l'acide biliaire Download PDF

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WO2014143033A1
WO2014143033A1 PCT/US2013/032464 US2013032464W WO2014143033A1 WO 2014143033 A1 WO2014143033 A1 WO 2014143033A1 US 2013032464 W US2013032464 W US 2013032464W WO 2014143033 A1 WO2014143033 A1 WO 2014143033A1
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cspc
bile acid
spore
germination
clostridium
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Joseph A. SORG
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • Clostridium difficile infections are steadily increasing in the United States and other countries [1,2].
  • C. difficile spores In a host, C. difficile spores must germinate to form the actively growing, anaerobic bacteria that produce the two toxins that are necessary for disease (TcdA and TcdB) [4,5,6]. These two toxins are secreted by the bacterium where they then enter host epithelial cells by receptor- mediated endocytosis and, upon escape into the cytosol, glucosylate members of the Rho-family of GTPases [7].
  • Embodiments of the present invention relate to methods of identifying compounds that modulate germination of a spore of a Clostridium species.
  • the invention provides methods of identifying a test compound that binds a germination-related protease-like protein (CspC) by providing a bile acid-binding fragment of a CspC in a mixture with the test compound and determining the presence of a complex between the test compound and the bile acid-binding fragment of CspC, where the presence of the complex identifies the test compound as a compound that binds CspC.
  • CspC germination-related protease-like protein
  • the bile acid-binding fragment of CspC comprises a sequence at least 60% identical to SEQ ID NO: 1. In certain embodiments, the bile acid-binding fragment of CspC is on the surface of a bacterial spore or is embedded in the spore cortex.
  • the bile acid-binding fragment of CspC is in a cell- free system.
  • the test compound is detectably labeled.
  • the bile acid-binding fragment of CspC is detectably labeled.
  • the detectable label is a fluorescent label, dye label, isotopic label, radio label, or a combination thereof, e.g a fluorescent label.
  • the mixture is deposited onto nitrocellulose and allowed to dry to determine the presence of a complex between the test compound and the bile acid-binding fragment of CspC.
  • the test compound is a small molecule or biological macromolecule.
  • the test compound is a small molecule and the small molecule is a bile acid.
  • the mixture further comprises a bile acid.
  • the bile acid is taurocholic acid, or a salt or an ester thereof.
  • the bile acid is chenodeoxycholic acid, or a salt or an ester thereof.
  • the bile acid may detectably labeled, e.g. , a fluorescent label, dye label, isotopic label, radio label, or a combination thereof.
  • the bile acid is provided to the mixture with the bile acid-binding fragment of CspC before adding the test compound to the mixture.
  • the methods further include detecting displacement of the bile acid from a complex of the bile acid with the bile acid-binding fragment of CspC.
  • the test agent is a modulator of germination of a spore of a Clostridium species, e.g. an agonist of germination of a spore of a Clostridium species or an antagonist of germination of a spore of a Clostridium species.
  • the Clostridium species is a Clostridium difficile, e.g., is an epidemic strain, e.g., the B I NAP 1/027 strain.
  • the invention provides an isolated antibody, or antigen- binding fragment thereof, that specifically binds a CspC or a bile acid-binding fragment of CspC.
  • the invention provides an isolated bile acid-binding fragment of CspC.
  • the invention provides methods of inhibiting germination of a spore of a Clostridium species or treating and/or preventing a Clostridium infection in a mammalian subject by contacting the spore with a test molecule identified by any one of the methods provided by the invention, or any of the antibodies or bile acid-binding fragments of CspC provided by the invention.
  • FIGs. 1 A-1B show the strategy to identify C. difficile ger phenotypes.
  • Spores were generated (1) and purified (2). After purification, spores were germinated in BHIS medium supplemented with TA (3) and germinated spores heat- killed at 65 °C (4). Spores that survived (4) were artificially germinated (5) before plating on BHIS medium (6).
  • C. difficile UKl spores or C. difficile gerl spores were serially diluted and spotted on BHIS medium supplemented with 0.1% TA or germinated by thioglycollate / lysozyme and were serially diluted and spotted on BHIS medium
  • FIGs. 2A-2C show purified C. difficile UKl spores (A) or C. difficile gerl spores (B) were suspended in BHIS medium ( ⁇ ) or BHIS medium supplemented with 5 mM TA ( ⁇ ) or 50 mM TA (A) and the initiation of germination was followed at A 6 oo- (C) Ca ++ -DPA release from spores suspended in germination buffer supplemented with TA and glycine was analyzed at A270.
  • FIGs. 3A-3D show purified C. difficile UKl spores (A) or C. difficile JSC 10 ⁇ cspCr. ermB) spores (B) or C. difficile JSC10 ⁇ cspCr. ermB) pJS123 (pcspBA (C) were suspended in BHIS medium ( ⁇ ) or BHIS medium supplemented with 5 mM TA ( ⁇ ) or 50 mM TA ( A) and the initiation of germination was followed at A 6 oo- (D) Ca ++ -DPA release from spores suspended in germination buffer supplemented with TA and glycine was analyzed at A270.
  • FIG. 4 shows purified C. difficile JSC10 ⁇ cspCr. ermB) pJS144
  • spores were suspended in BHIS medium ( ⁇ ) or BHIS medium supplemented with 1 mM chenodeoxycholic acid (A) or 5 mM chenodeoxycholic acid (T) or 10 mM TA ( ⁇ ) and the initiation of germination was followed at ⁇ 0 ⁇
  • FIG. 5 shows the Kaplan-Meier survival curve of clindamycin-treated Syrian hamsters inoculated with 1,000 spores of C. difficile UKl or C. difficile JSC 10 ⁇ cspCr. ermB) or C. difficile JSC10 ⁇ cspCr. ermB) pJS123 ⁇ pcspBAQ. Animals showing signs of C. difficile infection (wet tail, poor fur coat, lethargy) were euthanized.
  • FIG. 6 shows the C. difficile CspC and C. perfringens CspC protein sequence alignments were performed with the Interactive Structure based Sequences
  • STRAP Alignment Program using the ClustalW method.
  • FIG. 7 illustrates the Differential Radial Capillary Action of Ligand Assay (DRaCALA) methodology.
  • FIG. 8 is the amino acid sequence of SEQ ID NO: 1, the CspC sequence for Clostridium difficile R20291 under Entrez Accession Number YP_003218633.
  • CspC is a bacterial germination-related protease-like protein and is exemplified by SEQ ID NO: 1, as well as variants that are at least 60% identical to this sequence. This protein also has an Entrez Accession number of YP_003218633 in the National Center for Biotechnology Information database. CspC refers to protease inactive and catalytic inactive variants, and CspC variants described herein can be characterized by one or more point mutations at or within 30 residues of one or more residues of the catalytic triad at D109, T170, and G485 of SEQ ID NO: 1. As used herein, CspC does not refer to major cold shock protein. In particular embodiments, the catalytic triad of a CspC is impaired for, or lacks completely, catalytic activity. In some embodiments, a CspC is protease inactive.
  • a "bile acid-binding fragment" of CspC is a fragment of CspC that retains specific binding of one or more bile acids.
  • a bile-acid binding fragment comprises the CspC protein sequence corresponding to the proximate (N-terminal and/or C-terminal) 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or more amino acids of one or more (i.e. 1, 2, or all 3) of each of the three residues of the catalytic triad of YP_003218633, as identified above or, in some embodiments, as identified by alignment to Entrez conserved domain cd07478.
  • Bile acid is a steroid acid and encompasses primary bile acids (cholic acid or chenodeoxycholic acid), secondary bile acids (deoxycholic acid or lithocholic acid), and conjugates (e.g. glycine or taurine conjugates) of primary or secondary bile acids, as well as salts, esters, derivatives, and conjugates of any of these. Additional bile acids are described in WO 2010/062369, U.S. Patent Application Publication No. US 20110280847, and Howerton, A. et al. [38] (including, in particular embodiments, compound T15 therein) and Howerton et ah, J. Inf. Dis.
  • the "bile acids” encompass both free carboxylic acids and the corresponding carboxylic acid salts, and vice versa.
  • cholic acid can refer to the free acid (cholic acid) as well as the corresponding carboxylic acid salt (cholate).
  • a “complex" between, for example, a compound and a bile acid-binding fragment of CspC is an intermolecular association that can be characterized by, e.g., binding affinity, dissociation constant K ⁇ , n , ⁇ ⁇ 13 ⁇ 4 et cetera, and is a specific association where the molecules in the complex are in an at least semi-stable association that can be differentiated from non-specific associations by virtue of, for example, the binding affinity.
  • Clostridium species refers to bacteria of the genus Clostridium such as
  • Clostridium difficile or C. difficile Clostridium difficile or C. difficile.
  • An "epidemic strain” refers to bacteria isolated during a widespread occurrence of an infectious disease in a community at a particular time.
  • epidemic strains of C. difficile include BI/NAP 1/027, UK1, and R20291.
  • C. difficile UK1 refers to an epidemic strain (REA type BI23) was isolated during a 2006 outbreak at Stoke-Mandeville Hosptial in the United Kingdom [19].
  • bile acids and/or polypeptide e.g. bile-acid binding fragments of CspC or antibodies to CspC
  • pharmaceutically acceptable salts of the bile acids and/or polypeptide ⁇ e.g. bile-acid binding fragments of CspC or antibodies to CspC) compounds disclosed herein are included in the present invention.
  • an acid salt of a compound containing an amine or other basic group can be obtained by reacting the compound with a suitable organic or inorganic acid, resulting in pharmaceutically acceptable anionic salt forms.
  • anionic salts include the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate,
  • pantothenate phosphate/diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, triethiodide, and trifluoroacetate salts.
  • Salts of the compounds containing an acidic functional group can be prepared by reacting with a suitable base.
  • a suitable base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as
  • modulator refers to agents which interact with the target receptor or the spore and affect biological function.
  • modulators include full agonists, partial agonists, neutral antagonists, and inverse agonists.
  • the term "agonist" refers to any agent, either naturally occurring or synthetic, that, upon interacting with (e.g. , binding to) its target, here, a spore, raises the germination activity above its basal level.
  • An agonist can also refer to any agent, either naturally occurring or synthetic, that, upon interacting with (e.g. , binding to) its target, here, a CspC or a bile acid-binding fragment of CspC, raises the signaling activity of a CspC or a bile acid-binding fragment of CspC above its basal level.
  • An agonist can be a superagonist (i.e.
  • a full agonist i. e. a compound that elicits a maximal response following receptor occupation and activation
  • a partial agonist i.e. a compounds that can activate receptors but are unable to elicit the maximal response of the receptor system.
  • the term "antagonist” refers to any chemical compound, that, upon interacting with (e.g. , binding to) its target, here, a spore, blocks, in a dose dependent manner, the germination activity of an agonist compound with the spore.
  • the term “antagonist” can also refer to any chemical compound, that, upon interacting with (e.g., binding to) its target, here, a CspC or a bile acid-binding fragment of CspC, blocks, in a dose dependent manner, the signaling activity of an agonist compound with the CspC or the bile acid-binding fragment of CspC.
  • amino acid includes both a naturally occurring amino acid and a non-natural amino acid.
  • a CspC can be made by any means known in the art.
  • a CspC, or bile acid- binding fragment thereof can be isolated from a natural source such as C. difficile spores.
  • a CspC, or bile-acid binding fragment thereof can be produced by recombinant techniques.
  • a consensus sequence for CspC is provided by SEQ ID NO: 1, which is shown in FIG. 8.
  • a CspC is a bacterial germination-related protease- like protein.
  • a CspC contains point mutations, for example, in one or more residues of the catalytic triad at D109, T170, and G485 or within 30 residues of these sites.
  • the catalytic triad of a CspC is impaired for, or lacks completely, catalytic activity.
  • a CspC germination-related protein is protease inactive.
  • a CspC germination-related protein is found in a Clostridium species such as Clostridium difficile.
  • bile acids are primary bile acids (cholic acid or chenodeoxycholic acid), secondary bile acids (deoxycholic acid or lithocholic acid), and conjugates (e.g. glycine or taurine conjugates) of primary or secondary bile acids, as well as salts and esters of any of these.
  • the bile acids and pharmaceutically acceptable salts thereof that target the CspC germinant receptor in C. difficile are represented by structural formula:
  • Ri is selected from the group consisting of -C0 2 H, -C0 2 (R 2 ),
  • each of R and R 5 is independently selected from the group consisting of -H, -N3 ⁇ 4, -NH(R 2 ), -N(R 2 ) 2 , -OH, -0(R 2 ), and -OAcyl, wherein:
  • each R 2 is independently a straight or branched chain CI -CIO alkyl
  • R 3 is selected from the group consisting of -C0 2 H, -SO 3 H, -CONH 2 , - S0 2 N3 ⁇ 4, -OC 2 (R 2 ), and -S0 3 (R 2 );
  • aliphatic includes both saturated and branched
  • unsaturated,straight chain i. e., unbranched
  • branched i.e., branched
  • acyclic i.e., carbocyclic
  • cyclic i.e., carbocyclichydrocarbons, which are optionally substituted with one or more functional groups.
  • aliphatic is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.
  • alkyl includes straight, branched and cyclic alkyl groups.
  • An analogous convention applies to other generic terms such as “alkenyl", "alkynyl", and the like.
  • alkyl encompass both substituted and unsubstituted groups.
  • aliphatic is used to indicate those aliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-20 carbon atoms.
  • Aliphatic group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy,
  • the bile acids and pharmaceutically acceptable salts thereof that bind the CspC germinant receptor in C. difficile include, for example, cholic acid, lithocholic acid, chenodeoxycholic acid, deoxycholic acid, cholanic acid, dehydrocholic acid, ursodeoxycholic acid, and hyodeoxycholic acid.
  • the bile acids and pharmaceutically acceptable salts thereof that target the CspC germinant receptor are represented by any one of the following structural formulas:
  • dehydrocholic acid ursodeoxycholic acid glycocholic acid
  • a bile acid is a taurocholate analog of structural formula:
  • R-6 is selected from the group consisting of -H, -NH 2 , -NH(R 2 ), -N(R 2 ) 2 , - OH, -0(R 2 ), and -OAcyl [38]. Values and preferred values of the remainder of the variables are as defined above with respect to Formula (I).
  • a bile acid is a labeled variant of any of the foregoing, including radio labeled bile acids, fluorescently labeled bile acids, et cetera.
  • a bile acid-binding fragment of CspC is a monomer. In some embodiments, a bile acid-binding fragment of CspC is soluble and can be free in solution. In some embodiments, a bile acid-binding fragment of CspC is a soluble monomer.
  • CspC or a bile acid-binding fragment of CspC are combined with a bile acid and another compound such as an amino acid.
  • the additional compound is glycine.
  • Clostridium spores are suspended in a mixture of a bile acid with a co-germinant, such as an amino acid.
  • a co-germinant such as an amino acid.
  • C. difficile spores are suspended in buffered taurocholic acid with a co-germinant, such as glycine, is added [17].
  • Receptor binding assays for determining the presence of a complex between a test compound and a bile-acid binding fragment of CspC, and in some
  • determining a binding constant (Kd) or inhibition concentration (IC5 0 ) for displacing a radio-labeled test compound from a CspC or a bile acid-binding fragment of CspC may be conducted by any means known in the art.
  • Functional assays to determine agonist or antagonist status of a test compound interaction with a CspC or a bile acid-binding fragment of CspC may be conducted by any means known in the art.
  • the presence of a complex between a test compound and a bile-acid binding fragment of CspC can be evaluated by
  • DRaCALA Differential Radial Capillary Action of Ligand Assay
  • Clostridium difficile spores must germinate in vivo to become actively growing bacteria in order to produce the toxins that are necessary for disease.
  • C. difficile spores germinate in vitro in response to certain bile acids and glycine.
  • proteins embedded within the inner membrane of the spore sense the presence of germinants and trigger the release of Ca ++ -dipicolinic acid (Ca ++ -DPA) from the spore core and subsequent hydrolysis of the spore cortex, a specialized peptidoglycan.
  • Ca ++ -dipicolinic acid Ca ++ -dipicolinic acid
  • C. difficile Based upon homology searches of known germinant receptors from other spore-forming bacteria, C. difficile likely uses unique mechanisms to recognize germinants.
  • CspC the C. difficile bile acid germinant receptor
  • bile acid-mediated germination is important for establishing C. difficile disease in the hamster model of infection.
  • germinant receptors are embedded within the inner membrane of bacterial spores [10,32]. Germinants must pass through layers of coat proteins, an outer membrane, the cortex and germ cell wall before interacting with their respective receptors.
  • Ca ++ -DPA is released from the spore core in exchange for water. This exchange is essential to rehydrate the core and allow metabolism to begin.
  • the release of Ca -DPA triggers the activation of cortex hydrolases allowing a vegetative bacterium to grow from the germinating spore [33].
  • CspA, CspB and CspC are all members of the subtilisin family of serine proteases and have complete catalytic triads, suggesting that any one of these proteins can activate SleC-mediated cortex hydrolysis.
  • C. difficile the cspB and cspA coding sequences have been fused [34]. Only CspB contains a complete catalytic triad while CspA and CspC have lost their catalytic residues. Based on sequence analysis, one would predict that only CspB would have an active role in stimulating C. difficile cortex hydrolysis.
  • CspBA undergoes autoprocessing to generate CspB, which can cleave the cortex hydrolase pro-SleC to an active form [34].
  • C. difficile CspC plays an active and essential role during germination by functioning as the bile acid germinant receptor.
  • CspA The 12a-hydroxyl group that differentiates between cholic acid and chenodeoxycholic acid protrudes from the molecule. This hydroxyl, in wild-type CspC, may penetrate the hypothetical binding pocket, resulting in a conformational change that is transmitted to C. difficile CspB [34]. CspB would then cleave SleC, initiating cortex hydrolysis [34].
  • Two of the identified SNPs in the germination-null screen were nonsense mutations in cspBA (Q632stop and W359stop). In the CspBA hybrid protein, Q632 is located in CspA while W359 is in CspB. The generation of a premature stop codon in cspB would result in a truncated protein with an incomplete catalytic triad [34]. CspA may be important in C. difficile spore germination.
  • Lysozyme is able to induce germination of C. difficile spores in vitro [21].
  • recent evidence has suggested that lysozyme at physiological levels may not be able to stimulate C. difficile spore germination [35] and we observe most efficient lysozyme-mediated spore germination after spore coat removal.
  • this other receptor may be localized to the inner membrane to aid in the release of Ca ++ -DPA from the spore core during germination.
  • Metabolically dormant spores are formed by selected bacterial species in response to changes in environmental conditions, including nutrient availability [9]. During spore formation, the proteins required for germination are pre-packaged into the spore, priming the spore to germinate when conditions are appropriate [10]. In many spore-forming species, the interaction of the metabolically dormant spore with specific germination-inducing molecules (germinants) leads to the release of large amounts of Ca ++ -dipicolinic acid (DP A) from the dehydrated spore core in exchange for water.
  • DP A Ca ++ -dipicolinic acid
  • hydrolases embedded within the spore cortex a specialized peptidoglycan, become activated and begin cortex hydrolysis.
  • a vegetative cell begins to grow out from the germinated spore. This process is largely conserved among spore-forming bacteria, though the signals that initiate germination can vary.
  • L-alanine or a mixture of L-asparagine, glucose, fructose and potassium ions triggers gennination, while spores of certain strains of Clostridium perfringens initiate germination in response to inorganic phosphate and sodium ions [1 1].
  • Bile acids are small amphipathic, cholesterol-based molecules that aid in the absorption of fats and cholesterol during digestion.
  • the liver synthesizes two main bile acids, cholic acid (3a,7a,12a-trihydroxy-5p-cholanic acid) and chenodeoxycholic acid (3a,7a-dihydroxy-5P-cholanic acid), which are further modified with the addition of either a taurine or glycine amino acid [16].
  • cholic acid 3a,7a,12a-trihydroxy-5p-cholanic acid
  • chenodeoxycholic acid 3a,7a-dihydroxy-5P-cholanic acid
  • chenodeoxycholic acid was unable to stimulate colony formation or the initiation of spore germination [17]. Subsequent studies identified chenodeoxycholic acid as a competitive inhibitor of cholic acid- mediated germination [18,19]. While the chemical signals that promote the initiation of C. difficile spore germination are known, the proteins that respond to these germinants had not been identified.
  • Example 1 Identifying and characterizing germination-null C difficile strains Bacterial strains and growth conditions
  • C. difficile UK1 [19] was grown in a Model B, Coy Laboratory Chamber at 37°C under anaerobic conditions (85% nitrogen, 10% hydrogen, 5% carbon dioxide) in BHIS medium (Brain Heart Infusion supplemented with 5g / L yeast extract and 0.1% L-cysteine). Antibiotics were added as needed (20 ⁇ g/ml thiamphenicol, 10 ⁇ lincomycin, 5 ⁇ g/ml rifampin). E. coli DH5a [40] was routinely grown at 37°C in LB medium. Antibiotics were added as needed (50 ⁇ g/ml kanamycin or 20 ⁇ g/ml chloramphenicol). Bacillus subtilis was grown at 37°C in LB medium and antibiotics were added as needed (2.5 ⁇ g/ml chloramphenicol, 5 ⁇ g/ml tetracycline). EMS mutagenesis
  • spores were suspended in 40 ml BHIS + 10% w/v taurocholic acid (TA) and incubated overnight at 37°C to germinate those spores that recognized TA as a germinant. Spores were collected and heated to 65°C for 1 hour to inactivate germinated spores (dormant spores are resistant to 65°C).
  • TA w/v taurocholic acid
  • spores were again collected and treated with 250 mM thioglycollate for 30 min at 50°C followed by incubation with 4 mg / ml lysozyme for 15 min at 37°C [21] and 25- ⁇ aliquots were spread on BHIS agar plates to allow spore formation.
  • spores were again collected and germinated as described above.
  • C. difficile UKl was mutated as described above with the following modification.
  • spores generated from mutated bacteria were spread on BHIS medium supplemented with 0.5 mM chenodeoxycholic acid. Colonies from spores that germinated on this medium were purified and the germination phenotype of their spores was confirmed using standard germination techniques.
  • the ⁇ 916 oriT from Bacillus subtilis Bs49 was amplified using
  • oligonucleotides 5 n916SLIC and 3'Tn916SLIC (Table 1) and introduced into the BstAPI restriction site of pBLlOO [43] using Sequence and Ligation Independent Cloning (SLIC), generating pJS107.
  • the pJS107 plasmid was used as a TargeTron vector to introduce mutations in to C. difficile.
  • the group II intron insertion sites for C, difficile cspC were identified using an algorithm, the Intron Site Finder maintained by the Egyptian Bioinformatics Consortium and entering the cspC gene DNA sequence.
  • the intron fragment was generated as described previously using oligonucleotides cspC (115) EBS2, cspC (115) IBS, cspC (115) EBS1 and EBSU, cloned into pCR2.1-TOPO and then sub-cloned at the Hindlll and BsrGI sites of pJS107, yielding pJS130.
  • the B. subtilis - C. difficile shuttle vector, pJSl 16 was generated through the introduction of the ⁇ 916 oriT into the Apal restriction site of the E. coli - C.
  • C. difficile shuttle vector pMTL84151 [44] using oligonucleotides 5'Tn916ApaI and 3 n916ApaI which amplify the Tn i 6 oriT.
  • the C. difficile cspBAC loci were amplified with Phusion polymerase using 5'cspBA_CXbaI and 3'cspBA_CXhoI oligonucleotides and cloned into the B. subtilis - C. difficile shuttle vector, pJS 1 16. The nucleotide sequences for all constructs were confirmed before use.
  • B. subtilis BS49 was used as a donor for conjugation with C, difficile.
  • Plasmids were introduced into B. subtilis BS49 using standard techniques. Conjugation experiments were carried out as described previously [28]. C. difficile transconjugants were screened for the presence of ⁇ 916 using tetracycline resistance. Thiamphenicol-resistant, tetracycline-sensitive (plasmid-containing, transposon negative) transconjugants were selected for further use. Potential TargeTron mutants were generated by screening lincomycin-resistant C. difficile for the insertion of the intron into C. difficile cspC using primers specific for full-length C. difficile cspC, the 5' intron insertion site and the 3' intron insertion site and a positive clone was identified, C. difficile JSC10 (Table 2). Table 2. Strains and plasmids used in this study
  • FIG. 1A We mutagenized C. difficile strain UK1 [19] using the DNA alkylating agent ethyl methanesulfonate (EMS). The EMS-mutagenized bacteria were allowed to recover during overnight incubation in fresh medium and spread on solid medium to allow efficient spore formation.
  • EMS DNA alkylating agent ethyl methanesulfonate
  • CspA, CspB and CspC are germination-specific proteases that cleave the spore cortex lytic enzyme, SleC, to the active form [22,23,24]. This allows precise control of the timing of cortex hydrolysis during germination.
  • CspA, CspB and CspC all members of the subtilisin-family of proteases, have identifiable catalytic triads, while, in C. difficile, only CspB has obvious catalytic residues.
  • CspA and CspC catalytic triads appear to have been lost (FIG. 6).
  • Eight of the 10 mutant strains had mutations in cspC (Table 4), suggesting that, despite the apparent absence of catalytic activity, wild-type CspC may still have a role in C. difficile spore germination.
  • C. difficile JSC 10 cspC::ermB
  • TA TA
  • FIG. 3C wild-type C. difficile UKl initiates germination in response to TA
  • FIG. 3A wild-type C. difficile UKl initiates germination in response to TA
  • Spores were purified from BHIS agar medium as described previously [19] with the following modification. Spores from antibiotic-resistant strains ⁇ i.e.
  • plasmid-containing or mutant strains were generated on SMC medium [45] supplemented with appropriate antibiotics and purified as described previously.
  • the initiation of spore germination was analyzed in a Lambda 25 Perkin Elmer spectrophotometer at Agoo every 18 seconds, as described previously [17,18,19].
  • Ca ++ -DPA release was measured by incubating purified spores at 37°C in germination salts (0.3mM (NH 4 ) 2 S0 4 , 6.6mM KH 2 P0 4 , 15mM NaCl, 59.5mM NaHC0 3 and 35.2mM Na 2 HP0 4 ) supplemented with 10% TA and 1 mM glycine for 1 hour.
  • Bile acid-mediated germination is important for C. difficile infection in hamsters
  • Protein or whole-cell lysates in 1 x cdiGMP binding buffer (20 iL) were mixed with 4 nM radiolabeled nucleotide and allowed to incubate for 10 min at room temperature. Radiolabeled nucleotide was competed away by cold nucleotides in concentrations and for times indicated.
  • Purified proteins were tested in technical replicates. Whole-cell lysates in were tested in biological triplicates. Whole-cell lysates in were tested in technical replicates. These mixtures were pipetted (2.5-5 ⁇ ,) onto dry untreated nitrocellulose (GE Healthcare) in triplicate and allowed to dry completely before quantification.
  • An FLA7100 Fujifilm Life Science An FLA7100 Fujifilm Life Science
  • Phosphorlmager was used to detect luminescence following a 5-min exposure of blotted nitrocellulose to phosphorimager film. Data were quantified using Fujifilm Multi Gauge software v3.0. Roelofs, K.G. et al. [49] which is incorporated by reference in its entirety.
  • the protein is a CspC or a bile acid-binding fragment of CspC.
  • the DRaCALA system involves a C. difficile spore.
  • the test compound is detectably labeled, for example, with a radiolabel.
  • A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated.
  • each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A,
  • Pruitt RN Lacy DB (2012) Toward a structural understanding of Clostridium difficile toxins A and B. Front Cell Infect Microbiol 2: 28.
  • Clostridium difficile spore germination J Bacteriol 191 : 11 15-11 17.
  • protease CspB is essential for initiation of cortex hydrolysis and dipicolinic acid (DPA) release during germination of spores of Clostridium perfringens type A food poisoning isolates. Microbiology 155: 3464-3472.
  • ClosTron A universal gene knock-out system for the genus Clostridium. J Microbiol Methods 79: 452-464.

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Abstract

L'invention concerne, entre autres, un procédé d'identification d'un composé test qui se lie à une protéine de type protéase associée à la germination (CspC), par exemple des composés qui sont des agonistes ou des antagonistes de la germination d'une spore d'une espèce de Clostridium, telle que Clostridium difficile. L'invention concerne également des méthodes de traitement et/ou de prévention d'une infection par Clostridium chez un sujet mammifère en ayant besoin.
PCT/US2013/032464 2013-03-15 2013-03-15 Identification de clostridium difficile cspc comme récepteur de germination de l'acide biliaire Ceased WO2014143033A1 (fr)

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ADAMS ET AL.: "`Structural and functional analysis of the CspB protease required for Clostridium spore germination`", PLOS PATHOGENS, vol. 9, no. ISSUE, 7 February 2013 (2013-02-07), pages 1 - 17 *
BURNS ET AL.: "`SleC is essential for germination of Clostridium difficile spores in nutrient-rich medium supplemented with the bile salt taurocholate`", JOURNAL OF BACTERIOLOGY, vol. 192, no. 3, 2010, pages 657 - 664 *
DATABASE GENBANK 23 December 2012 (2012-12-23), accession no. P_003215124.1 *
FRANCIS ET AL.: "`Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection`", PLOS PATHOGENS, vol. 9, no. ISSUE, May 2013 (2013-05-01), pages 1 - 9 *
HEEG ET AL.: "`Spores of Clostridium difficile clinical isolates dis play a diverse germination response to bile salts`", PLOS ONE, vol. 7, no. ISSUE, 2012, pages 1 - 9 *
PAREDES-SABJA ET AL.: "`Germination of spores of Bacillales and Clostridiales species: mechanisms and proteins involved`", TRENDS IN MICROBIOLOGY, vol. 19, no. 2, 2011, pages 85 - 94 *

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