WO2014143033A1 - Identification of clostridium difficile cspc as a bile acid germinant receptor - Google Patents

Abstract

The invention provides, inter alia, method of identifying a test compound that binds a germination-related protease-like protein (CspC), e.g., compounds that are agonists or antagonists of germination of a spore of a Clostridium species, such as Clostridium difficile. The invention also provides methods of treating and/or preventing a Clostridium infection in a mammalian subject in need thereof.

Description

IDENTIFICATION OF CLOSTRIDIUM DIFFICILE CSPC AS A BILE ACID

GERMINANT RECEPTOR

BACKGROUND OF THE INVENTION

Clostridium difficile infections (CDI) are steadily increasing in the United States and other countries [1,2]. The use of broad-spectrum antibiotics, often unrelated to CDI, leads to alteration of the colonic microbiota that normally provides resistance to C. difficile colonization [3]. 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]. The action of these toxins lead to symptoms normally associated with CDI (e.g. diarrhea) and release of C. difficile spores into the environment [8]. Bile salts stimulate germination of Clostridium difficile spores, and so the cycle of infection persists.

In view of the increased incidence of CDI, the associated economic and personal costs of these infections, a need exists for methods of identifying agents that treat or prevent CDIs.

SUMMARY OF THE INVENTION Embodiments of the present invention relate to methods of identifying compounds that modulate germination of a spore of a Clostridium species.

In a first aspect, 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.

In some embodiments, 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.

In some embodiments, the bile acid-binding fragment of CspC is in a cell- free system.

In certain embodiments, the test compound is detectably labeled. In some embodiments, the bile acid-binding fragment of CspC is detectably labeled. In more particular embodiments, the detectable label is a fluorescent label, dye label, isotopic label, radio label, or a combination thereof, e.g a fluorescent label.

In some embodiments, 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.

In certain embodiments, the test compound is a small molecule or biological macromolecule. In more particular embodiments, the test compound is a small molecule and the small molecule is a bile acid. In some embodiments, the mixture further comprises a bile acid. In more particular embodiments the bile acid is taurocholic acid, or a salt or an ester thereof. In other in more particular embodiments the bile acid is chenodeoxycholic acid, or a salt or an ester thereof. In either of these particular embodiments, the bile acid may detectably labeled, e.g. , a fluorescent label, dye label, isotopic label, radio label, or a combination thereof. In more particular embodiments, the bile acid is provided to the mixture with the bile acid-binding fragment of CspC before adding the test compound to the mixture. In more particular embodiments 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.

In some embodiments, 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. In certain particular embodiments, the Clostridium species is a Clostridium difficile, e.g., is an epidemic strain, e.g., the B I NAP 1/027 strain.

In another aspect, the invention provides an isolated antibody, or antigen- binding fragment thereof, that specifically binds a CspC or a bile acid-binding fragment of CspC.

In another aspect, the invention provides an isolated bile acid-binding fragment of CspC.

In another aspect, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

FIGs. 1 A-1B show the strategy to identify C. difficile ger phenotypes. (A) 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). (B) 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 A₆oo- (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 A₆oo- (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

(pcspBACG457R) 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 Α ₀ο·

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

Alignment Program (STRAP) using the ClustalW method. The locations of the catalytic residues for C. perfringens CspC, a subtilisin-like protease, were identified using the MEROPS database, which is maintained by the Wellcome Trust Sanger Institute. Catalytic residues (highlighted and double underlined), SNPs identified in the germination-null screen (highlighted), SNP that alters germinant specificity (highlighted and underlined).

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.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

"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. In particular embodiments, 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. (2013; PMID 2342096) which are incorporated by reference in their entirety. As used herein, the "bile acids" encompass both free carboxylic acids and the corresponding carboxylic acid salts, and vice versa. For example, the term "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, Κ_ο1¾ 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.

A "Clostridium species" refers to bacteria of the genus Clostridium such as

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. For example, 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].

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.

For example, 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. Examples of 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. Such a pharmaceutically acceptable salt can be made with a 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

trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, Ν,Ν'-dibenzylethylenedi amine, 2-hydroxyethylamine, bis-(2- hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, Ν,Ν'-bisdehydroabietylamine, glucamine, N- methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine.

As used herein, the term "modulator" refers to agents which interact with the target receptor or the spore and affect biological function. Examples of modulators include full agonists, partial agonists, neutral antagonists, and inverse agonists.

As used herein, 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 compound that is capable of producing a greater maximal response than the endogenous agonist for the target receptor, and thus has an efficacy of more than 100%), a full agonist (i. e. a compound that elicits a maximal response following receptor occupation and activation) or a partial agonist (i.e. a compounds that can activate receptors but are unable to elicit the maximal response of the receptor system).

As used herein, 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. As used herein, 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.

As used herein, the term "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. Alternatively, 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.

In example embodiments, a CspC is a bacterial germination-related protease- like protein. In some embodiments, 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. In example embodiments, the catalytic triad of a CspC is impaired for, or lacks completely, catalytic activity. In some embodiments, a CspC germination-related protein is protease inactive. In some embodiments, a CspC germination-related protein is found in a Clostridium species such as Clostridium difficile.

In some embodiments, 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.

In some embodiments, the bile acids and pharmaceutically acceptable salts thereof that target the CspC germinant receptor in C. difficile are represented by structural formula:

wherein:

Ri is selected from the group consisting of -C0₂H, -C0₂(R₂),

-CONH₂, -CON(R₂)₂, -CONHCH₂CH₂(R₃), -CON(R₂)CH₂CH₂(R₃), -NH₂ , - NH(R₂), and -N(R₂)₂; and

each of R and R₅ is independently selected from the group consisting of -H, -N¾, -NH(R₂), -N(R₂)₂, -OH, -0(R₂), and -OAcyl, wherein:

each R₂ is independently a straight or branched chain CI -CIO alkyl; and

R₃ is selected from the group consisting of -C0₂H, -SO₃H, -CONH₂, - S0₂N¾, -OC₂(R₂), and -S0₃(R₂);

as shown in U.S. Patent Application Publication 201 1/0280847, which is incorporated herein in its entirety.

The term "aliphatic," as used herein, includes both saturated and

unsaturated,straight chain (i. e., unbranched), branched, acyclic, and cyclic (i.e., carbocyclic)hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl", and the like. Furthermore, as used herein, the terms "alkyl", "alkenyl", "alkynyl", and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "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, acyloxy, and the like, each of which may or may not be further substituted).

"Acyl" refers to a group -C(=0)R, where R is H or alkyl, as defined above.

In some embodiments, 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. In certain embodiments, the bile acids and pharmaceutically acceptable salts thereof that target the CspC germinant receptor are represented by any one of the following structural formulas:

chenodeoxycholic acid deoxycholic acid hyodeoxycholic acid

dehydrocholic acid ursodeoxycholic acid glycocholic acid

taurocholic acid

In some embodiments, a bile acid is a taurocholate analog of structural formula:

wherein:

R-6 is selected from the group consisting of -H, -NH₂, -NH(R₂), -N(R₂)₂, - OH, -0(R₂), and -OAcyl [38]. Values and preferred values of the remainder of the variables are as defined above with respect to Formula (I).

In some embodiments, a bile acid is a labeled variant of any of the foregoing, including radio labeled bile acids, fluorescently labeled bile acids, et cetera.

In some embodiments, 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.

In some embodiments, CspC or a bile acid-binding fragment of CspC are combined with a bile acid and another compound such as an amino acid. In example embodiments, the additional compound is glycine. In some embodiments,

Clostridium spores are suspended in a mixture of a bile acid with a co-germinant, such as an amino acid. In example embodiments, 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

embodiments, determining a binding constant (Kd) or inhibition concentration (IC5₀) 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. In particular embodiments, the presence of a complex between a test compound and a bile-acid binding fragment of CspC can be evaluated by

Differential Radial Capillary Action of Ligand Assay (DRaCALA), as decribed in, for example, Roelofs et ah, PNAS 107:15528-33(2011), which is incorporated by reference in its entirety.

EXEMPLIFICATION

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. In other sporulating bacteria, 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. Based upon homology searches of known germinant receptors from other spore-forming bacteria, C. difficile likely uses unique mechanisms to recognize germinants. Here, we identify the germination-specific protease, CspC, as the C. difficile bile acid germinant receptor and show that bile acid-mediated germination is important for establishing C. difficile disease in the hamster model of infection. These results highlight the importance of bile acids in triggering in vivo germination and provide the first description of a C. difficile spore germinant receptor. Blocking the interaction of bile acids with the C. difficile spore represents an attractive target for novel therapeutics.

Classically, 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. Upon interaction, 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. In some bacteria, the release of Ca -DPA triggers the activation of cortex hydrolases allowing a vegetative bacterium to grow from the germinating spore [33]. In C. perfringens the germination-specific proteases cleave the cortex hydrolase, SleC, to an active form [23,24]. The signals that stimulate this proteolysis in C. perfringens are not known. In C. perfringens, 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. In 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. Indeed, a recent study by Adams and colleagues has shown that 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.

In C. difficile CspC, two of the three catalytic residues have been lost, T170 (conserved HI 98 in C. perfringens CspC) and G485 (conserved S517 in C.

perfringens CspC) (FIG. 6 - highlighted and double underlined). Interestingly, several the SNPs identified in the germination-null screen lie near T170 or G485 (FIG. 6 - highlighted). When we screened for C. difficile mutants that germinated in response to an inhibitor of germination (chenodeoxycholic acid), we identified G457R (FIG. 6 - highlighted and underlined). This residue is approximately 30 amino acids removed from G485. G457R, being a fairly drastic substitution, may modify the bile acid binding pocket to allow for a less-stringent recognition of germination-inducing bile acids. 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.

Our data indicate that host-derived bile acids mediate C. difficile spore germination and that recognition of bile acids is required for infection in the hamster model to be maximally effective. Still, 50% of the animals succumbed to disease when infected with the cspC mutant, suggesting (i) that enough spores

spontaneously germinated in the GI tract of the animal to cause disease, or (ii) that other, as yet unidentified, host signals can stimulate spore germination. Lysozyme is able to induce germination of C. difficile spores in vitro [21]. However, 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.

Previously, we identified inhibitors of C. difficile spore germination that had increased potency when compared to chenodeoxycholic acid [19]. A recent study by Howerton and coworkers has shown dosing antibiotic-treated mice with an inhibitor of germination can reduce disease severity [36]. The identification of the molecular target of bile acids in the C. difficile spore may allow even more potent inhibitors to be rationally designed. Further, these inhibitors may aid in the identification in the bile acid-binding pocket in CspC by providing high-affinity interaction, instead of the relatively low affinity (in the mM range) for taurocholic acid [19,37]. The relative affinities of bile acids for the C. difficile spore were determined using kinetics of germination [18,19,37,38,39]. Also, it has been proposed that the bile acid germinant receptor binds taurocholic acid cooperatively [37]. The

identification of the bile acid germinant receptor now permits testing these interactions.

We hypothesize that 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. Subsequently, hydrolases embedded within the spore cortex, a specialized peptidoglycan, become activated and begin cortex hydrolysis. Once the core is rehydrated and the cortex is degraded, 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. In Bacillus subtilis, 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].

The proteins that respond to these signals, ger receptors, share homology among many spore-forming bacteria. However, based on homology searches, C. difficile is not among the spore-forming bacteria that have such canonical germinant receptors, suggesting that C. difficile responds to unique germinants or uses a novel mechanism for spore germination or both [12].

Approximately 30 years ago, Wilson and others showed that certain bile acids increased the frequency of C. difficile colony formation from environmental samples [13,14,15]. Bile acids are small amphipathic, cholesterol-based molecules that aid in the absorption of fats and cholesterol during digestion. Typically, 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]. We demonstrated that all cholic acid derivatives can stimulate C. difficile colony formation from spores with approximately equal efficiency [17]. Further, we showed that exposure to the combination of taurocholic acid and glycine were required to initiate C. difficile spore germination [17]. Interestingly,

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 μg/ml thiamphenicol, 10 μ^ηιΐ lincomycin, 5 μg/ml rifampin). E. coli DH5a [40] was routinely grown at 37°C in LB medium. Antibiotics were added as needed (50 μg/ml kanamycin or 20 μg/ml chloramphenicol). Bacillus subtilis was grown at 37°C in LB medium and antibiotics were added as needed (2.5 μg/ml chloramphenicol, 5 μg/ml tetracycline). EMS mutagenesis

One overnight culture of C. difficile UK1 was diluted 1 :100 in 5 ml fresh medium and grown to OD₆oo = 0.5 before adding ethyl methanesulfonate (EMS) to 1% final concentration. The culture was incubated for 3 hours, washed in BHIS medium and recovered overnight in 40-ml BHIS. A sample was taken to score mutation frequency on rifampin-containing BHIS agar medium and 50^L samples were spread on 20 BHIS plates to allow spore formation of the mutagenized bacteria. Plates were incubated for 4 days before spores were harvested and purified as describe previously [19]. Purified 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). To germinate the remaining dormant spores, 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-μί aliquots were spread on BHIS agar plates to allow spore formation. To enrich for germination null phenotypes, spores were again collected and germinated as described above. To select for mutations that change the affinity of the germinant receptor from TA to chenodeoxycholic acid, C. difficile UKl was mutated as described above with the following modification. Purified 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.

Illumina Sequencing

High-quality, high-molecular weight genomic DNA was extracted, as described previously [41 ,42], and submitted to Tufts University School of Medicine Genomics Core facility for Paired-End 50 Illumina re-sequencing. The samples were sonicated in a 4°C water bath with a Branson sonicator. Illumina libraries were then prepared using the Illumina TruSeq genomic DNA kit and tagged with individual Illumina barcodes. Final libraries were checked on said advanced analytical device, and then diluted to ΙΟηΜ prior to being loaded on a lane of an Illumina HiSeq2000. Illumina single-end sequencing was carried out for 50 cycles. The resulting sequence data in fastq format was aligned against the C. difficile R20291 genome using CLC Genomics Workbench, and SNPs were called at any position where more than 66% of the reads had an alternate base from the reference

Molecular Biology

The Ύη916 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 Victorian 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 Ίη916 oriT into the Apal restriction site of the E. coli - 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.

Table 1. Oligonucleotides used in this study.

Conjugation and mutant selection

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 Ύη916 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

Strain Description / Phenotype Reference

E. coli DH5a F^" endAl glnV44 thi-1 [40]

recAl relAl gyrA96 deoR

nupG <D80d/acZAM15

MlacZYA-argF)\ \69,

hsdRl 7(Γ ^{" η}ΐκ⁺), λ-

B. subtilis Bs49 Tn916 donor strain, Tet^R [48]

C. difficile UK1 Wild type, PCR ribotype [19]

027

C. difficile JSC 10 cspC TargeTron mutant, This study

germination null

Plasmids

pMTL84151 E. coli-C. difficile shuttle [44]

vector (pCD6 ColEl traJ

Cm^R)

pBLlOO TargeTron vector [43]

pJS107 TnPi 6 or iT in pBLlOO This study

pJS116 B. subtilis-C. difficile This study

shuttle vector (pCD6

ColEl TnP 16 oriT Cm^R) pJS123 cspBAC locus cloned in This study

pJSl 16, complements cspC

mutation

pJS130 a¾?C-targeted TargeTron This study

in pJS107

pJS144 cspBACG457R locus cloned This study

in pJS116

Identifying germination-null phenotypes

Previously, we demonstrated that the cholic acid family of bile acids causes spores to initiate germination in rich medium [17]. To identify the C. difficile bile acid germinant receptor, we employed a strategy schematized in 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. Spores were purified and incubated overnight at 37°C in rich medium + 10% w/v taurocholic acid [(TA); 185 mM] to germinate those spores that were still able to respond to TA as a germinant. The spore suspension

(containing both germinated and non-germinated spores) was incubated at 65°C for 30 minutes to heat-kill the germinated spores; dormant, non-germinated spores are resistant to 65°C. The surviving spores were artificially germinated using thioglycollate and lysozyme [21] and then plated on rich medium to recover, as colonies, the artificially germinated spores. Mutants that failed to germinate under these conditions were enriched and 10 colonies, among thousands, were isolated and tested for the ability of their spores to germinate in response to TA.

Spores from all 10 isolates (gerl - gerlO) were unable to form colonies on rich medium + TA, but did form colonies after artificial germination [21] (FIG. IB; only wild-type C. difficile UK1 and ger 1 are shown). This suggests that the ger mutants are either blocked at the outgrowth stage of germination (inability to grow as a vegetative cell from the germinated spore) or blocked in the initiation of germination (inability to respond to TA as a germinant). Characterizing germination-null C. difficile strains

To determine at what stage the mutants are blocked, we analyzed the initiation of germination as measured by a decrease in A₆₀o over time. Wild-type C. difficile UK1 initiated germination in the presence of 5 mM TA and 50 mM TA but not in the absence of TA (FIG. 2A). However, spores derived from C. difficile ger I (FIG. 2B) did not initiate germination even at the highest TA concentration used (50 mM). Also, while wild-type C. difficile released Ca⁺⁺-DPA in response to TA and glycine, C. difficile gerl - ger 10 spores were unable to release the majority of the stored Ca⁺⁺-DPA (FIG. 2C); Ca⁺⁺-DPA release from the spore core is one of the first measurable events in bacterial spore germination [10]. Together, these results suggest that the C. difficile ger isolates are defective in the earliest stages of spore germination and may be defective in recognizing TA as a germinant. Example 2. Identifying CspC as the germination-specific protease in C. difficile Determining the locations of SNPs that give rise to ger phenotype

The locations of the SNP(s) that gave rise to the germination-null phenotypes were determined using Illumina sequencing technology. The DNA sequence of the 10 ger isolates were compared to determine if all had mutations in the same locus or loci. All isolates had in common mutations in 7 loci, with 6 loci having conserved mutations among all isolates (Table 3). Interestingly, C. difficile gerl - ger 10 had several different mutations in the cspBAC locus (Table 4). In Clostridium perfringens, 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. C.

perfringens 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. In wild-type C. difficile, cspB and cspA coding sequences have been fused. Further, the 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.

Table 3. Locations of the SNPs common to all 10 C. difficile ger isolates. Gene numbering is based upon C. difficile R20291 gene numbering.

Nt: nucleotide

aa: amino acid

fs: frame shift

Table 4. Locations of C. difficile cspBAC mutations in the ger mutants

Strain Mutation(s)

gerl CspC G171R

ger2 CspC G171R

ger3 CspC G483R

ger4 CspC V272G / S443N

ger5 CspBA Q632stp

ger6 CspC S488N

ger 7 CspBA W359stp

ger8 CspC G171R

ger9 CspC G483R gerlO CspC G276

stp: stop codon

Defining the role of C. difficile cspC during spore germination

To investigate the role of CspC in C. difficile spore germination, we generated a site-directed mutation using TargeTron technology [4,25,26,27,28]. The resulting strain, C. difficile JSC 10 (cspC::ermB), is unable to initiate germination in response to TA (FIG. 3B) unless provided with the cspBAC locus expressed in trans from a plasmid (FIG. 3C); wild-type C. difficile UKl initiates germination in response to TA (FIG. 3A). Interestingly, when C. difficile JSC10 was

complemented with the cspBAC locus, spores generated from this strain appear to germinate more rapidly than do C. difficile UKl spores. When analyzed for Ca⁺⁺- DPA release, wild-type C. difficile UKl and C. difficile JSC 10 (pJS123) released Ca⁺⁺-DPA while C. difficile JSC10 was unable to release Ca⁺⁺-DPA (FIG. 3D). Mutations in cspC can alter germinant specificity

It was previously reported that a mutation in sleC prevents C. difficile spore germination [27]. Thus, mutations that affect germination do not necessarily indicate that the gene in which the mutation lies normally codes for a germinant receptor. To test the hypothesis that C. difficile CspC is a bona fide germinant receptor, we again mutagenized C. difficile UKl and allowed the mutagenized bacteria to form spores. The purified spores were plated on BHIS medium supplemented with 0.5 mM chenodeoxy cholic acid. We looked for colony formation after 48 hours of incubation at 37°C. Chenodeoxycholic acid is a competitive inhibitor of cholic acid-mediated germination for C. difficile UKl [18,19] and other C. difficile strains [29]. Thus, in order to form colonies, these spores must have acquired an altered germinant specificity. Colonies were isolated and the phenotype confirmed as described above. We sequenced cspC from these newly generated strains and identified a single mutation, G457R. When the cspCe457R allele was used to complement C. difficile JSC 10, we observed that this strain germinated in response to either TA or chenodeoxycholic acid (FIG. 4). These results indicate that C. difficile CspC is a receptor for bile acid germinants.

Spore germination and Ca⁺⁺-DPA release

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 (NH₄)₂S0₄, 6.6mM KH₂P0₄, 15mM NaCl, 59.5mM NaHC0₃ and 35.2mM Na₂HP0₄) supplemented with 10% TA and 1 mM glycine for 1 hour. Equal aliquots were incubated at 100°C as a measure of 100% Ca ^- DP A release (positive control) or incubated at 37°C in germination salts without TA addition (negative control). Spores were sedimented and the supernatant was analyzed at A₂₇₀ to measure the released Ca -DPA [46].

Example 3. Virulence studies in vivo.

Virulence studies

Female Syrian golden hamsters, 80g - 120g, were housed individually in cages and had ad libitum access to food and water for the duration of the

experiment. To induce susceptibility to C. difficile infection, hamsters were gavaged with 30 mg / kg clindamycin [31,47]. After 5 days, hamsters were gavaged with 1,000 spores of C. difficile UK1 or C. difficile JSC10 or C. difficile JSC10 pJS123, 10 animals per strain, and monitored for signs of disease (lethargy, poor fur coat and wet tail). Hamsters showing signs of disease were euthanized by C0₂ asphyxia followed by thoracotomy as a secondary means of death in accordance with Panel on Euthanasia of the American Veterinary Medical Association. Fecal samples were collected daily and cecum samples were collected on those hamsters requiring euthanasia. All animal studies were performed with prior approval from the Texas A&M University Institutional Animal Care and Use Committee.

Bile acid-mediated germination is important for C. difficile infection in hamsters

The in vivo signals that trigger C. difficile spore germination are unknown, though bile acids are obvious candidates [30]. To test whether bile acid-mediated germination is required for C. difficile infection, Syrian hamsters were treated with clindamycin to induce sensitivity to C. difficile colonization and infection; the Syrian hamster has been used for approximately 30 years to assess C. difficile virulence and recapitulates the most severe form of human C. difficile infection, pseudomembranous colitis [31]. Hamsters were gavaged with 1 ,000 C. difficile UK1 spores or C. difficile JSC 10 spores or C. difficile JSC 10 (pJS123) spores and monitored for signs of CDI. Animals infected with either C. difficile UK1 or C. difficile JSC 10 (pJS123) rapidly succumbed to disease. However, C. difficile JSC 10 was unable to cause fulminant CDI and exhibited reduced virulence (Chi-squared: p- value <0.02) (FIG. 5). These results show that bile acid-mediated germination is important for C. difficile disease and suggest that inhibiting C. difficile spore germination may have therapeutic potential. Statistical analyses

Experiments were performed in triplicate and, where indicated, error bars represent 1 standard deviation from the mean. A representative sample for the initiation of germination experiments at A₆₀o is shown, error bars obscure the data. The data varied by <5%. Statistical significance of Ca⁺⁺-DPA release was performed using the Student's T-test. Differences in hamster survival between those infected with C. difficile JSC 10 and either C. difficile UK1 or C. difficile JSC 10 pJS123 were analyzed using the Log-rank test (GraphPad Prism).

Differential Radial Capillary Action of Ligand Assay (DRaCALA).

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 μΐ,) onto dry untreated nitrocellulose (GE Healthcare) in triplicate and allowed to dry completely before quantification. 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.

In example embodiments, the protein is a CspC or a bile acid-binding fragment of CspC. In other embodiments, the DRaCALA system involves a C. difficile spore. In example embodiments, the test compound is detectably labeled, for example, with a radiolabel.

It should be understood that for all numerical bounds describing some parameter in this application, such as "about," "at least," "less than," and "more than," the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description at least 1, 2, 3, 4, or 5 also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.

For all patents, applications, and other references cited herein, such as nonpatent literature and reference sequence information, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited. Where any conflict exits between a document incorporated by reference and the present application, this application will control. Headings used in this application are for convenience only and do not affect the interpretation of this application.

Preferred features of each of the aspects provided by the invention are applicable to all of the other aspects of the invention mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features (e.g. elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the invention including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed invention piecemeal without departing from the invention. For example, for material that are disclosed, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein, as are methods of making and using such compounds. Thus, if a class of elements

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. Thus, is this example, 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,

B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, elements of a composition of matter, and steps of method of making or using the compositions.

The forgoing aspects of the invention, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art. Thus, to the extent an element is described in one or more references known to the person having ordinary skill in the art, they may be excluded from the claimed invention by, inter alia, a negative proviso or disclaimer of the feature or combination of features.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. References

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Claims

CLAIMS What is claimed is:

1. A method of identifying a test compound that binds a germination-related protease-like protein (CspC), comprising 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, wherein the presence of the complex identifies the test compound as a compound that binds CspC.

2. The method of Claim 1 , wherein the bile acid-binding fragment of CspC comprises a sequence at least 60% identical to SEQ ID NO: 1.

3. The method of any one of the preceding claims, wherein the bile acid- binding fragment of CspC is on the surface of a bacterial spore or is embedded in the spore cortex.

4. The method of any one of the preceding claims, wherein the bile acid- binding fragment of CspC is in a cell-free system.

5. The method of any one of the preceding claims, wherein the test compound is detectably labeled.

6. The method of any one of the preceding claims, wherein the bile acid- binding fragment of CspC is detectably labeled.

7. The method of Claim 5 or 6, wherein the detectable label is a fluorescent label, dye label, isotopic label, radio label, or a combination thereof.

8. The method of Claim 7, wherein the detectable label is a fluorescent label.

9. The method of any one of the preceding claims, wherein 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 method of any one of the preceding claims, wherein the test compound is a small molecule or biological macromolecule.

The method of Claim 10, wherein the test compound is a small molecule and the small molecule is a bile acid.

The method of any one of the preceding claims, wherein the mixture further comprises a bile acid.

The method of Claim 12, wherein the bile acid is taurocholic acid, or a salt or an ester thereof.

The method of Claim 12, wherein the bile acid is chenodeoxycholic acid, or a salt or an ester thereof.

The method of any one of Claims 12-14, wherein the bile acid is detectably labeled.

The method of Claim 15, wherein the detectable label is a fluorescent label, dye label, isotopic label, radio label, or a combination thereof.

The method of any one of Claims 12-16, wherein 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 method of any one of Claims 12-17, further comprising detecting displacement of the bile acid from a complex of the bile acid with the bile acid-binding fragment of CspC.

19. The method of any one of the preceding claims, wherein the mixture further comprises glycine.

20. The method of Claim 19, wherein the mixture further comprises any amino acid except glycine.

21. The method of Claim 20, wherein the mixture further comprises histidine.

22. The method of any one of Claims 21-21, wherein the bile acid is present at a concentration of about 0.05 to about 5% W/V.

23. The method of any one of the preceding claims, wherein the test agent is a modulator of germination of a spore of a Clostridium species.

24. The method of Claim 23, wherein the modulator is an agonist of germination of a spore of a Clostridium species.

25. The method of Claim 23, wherein the modulator is an antagonist of

germination of a spore of a Clostridium species.

26. The method of any one of Claims 23-25, wherein the Clostridium species is a Clostridium difficile.

27. The method of Claim 26, wherein the Clostridium difficile is an epidemic strain.

28. The method of Claim 27, wherein the Clostridium difficile is the

BI/NAP1/027 strain.

29. An isolated antibody, or antigen-binding fragment thereof, that specifically binds a CspC or a bile acid-binding fragment of CspC.

30. The antibody of Claim 29, wherein the CspC comprises a sequence at least 60% identical to SEQ ID NO: 1, or a fragment thereof.

31. An isolated bile acid-binding fragment of CspC.

32. A method of inhibiting germination of a spore of a Clostridium species comprising contacting the spore with the antibody of Claim 29 or 30, wherein said antibody is an antagonist antibody; or the bile acid-binding fragment of CspC of Claim 31.

33. A method of treating and/or preventing a Clostridium infection in a

mammalian subject in need thereof comprising administering to the subject a therapeutically effective amount of the antibody of Claim 29 or 30; or the bile acid-binding fragment of CspC of Claim 31.

34. The antibody of Claim 29 or 30; or the bile acid-binding fragment of CspC of Claim 31 for use in a method of treating and/or preventing a Clostridium infection in a mammalian subject in need thereof.

35. A method of treating and/or preventing a Clostridium infection in a

mammalian subject in need thereof comprising administering to the subject a therapeutically effective mount of a modulator of germination of a spore of a Clostridium species, wherein the modulator is identified by the method of any one of Claims 23 to 28.

36. The method of Claim 35, wherein the modulator is an antagonist of

germination of a spore of a Clostridium species.

37. The method of Claim 35, wherein the modulator is an agonist of germination of a spore of a Clostridium species.

38. The method of any one of Claims 35-37, wherein the modulator is

administered to the subject concurrently or sequentially with an antibiotic.

39. The method of Claim 38, wherein the antibiotic is metronidazole,

vacomycin, or fidaxomicin.

40. The method of any one of Claims 35-39, wherein the subject is a human.

41. The modulator of germination of a spore of a Clostridium species identified by the method of any one of Claims 23 to 28 for use in a method of treating and/or preventing a Clostridium infection in a mammalian subject in need thereof.

42. The method of Claim 1 , wherein the test compound is a bile acid .

43. The method of any one of Claims 1-28, wherein the bile-acid binding

fragment of CspC is associated with a Clostridium spore, and further comprising detecting the release of Ca⁺⁺-dipicolinic acid (DP A) from the spore.

44. The method of any one of Claims 1-28, wherein the bile-acid binding

fragment of CspC is associated with a Clostridium spore, and further comprising monitoring for spore germination.

45. An isolated Clostridium, plasmid, or plasmid-containing Clostridium, comprising one or more of the mutations listed in Table 2, Table 3, or Table 4.