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Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection.

Francis MB, Allen CA, Shrestha R, Sorg JA - PLoS Pathog. (2013)

Bottom Line: 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 may represent an attractive target for novel therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
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 may represent an attractive target for novel therapeutics.

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Related in: MedlinePlus

Mutations in C.difficile cspC can alter germination specificity.Purified C. difficile JSC10 (cspC::ermB) pJS144 (pcspBACG457R) spores were suspended in BHIS medium (•) or BHIS medium supplemented with 1 mM chenodeoxycholic acid (▴) or 5 mM chenodeoxycholic acid (▾) or 10 mM TA (▪) and the initiation of germination was followed at A600.
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ppat-1003356-g004: Mutations in C.difficile cspC can alter germination specificity.Purified C. difficile JSC10 (cspC::ermB) pJS144 (pcspBACG457R) spores were suspended in BHIS medium (•) or BHIS medium supplemented with 1 mM chenodeoxycholic acid (▴) or 5 mM chenodeoxycholic acid (▾) or 10 mM TA (▪) and the initiation of germination was followed at A600.

Mentions: 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 UK1 and allowed the mutagenized bacteria to form spores. The purified spores were plated on BHIS medium supplemented with 0.5 mM chenodeoxycholic 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 UK1 [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 cspCG457R allele was used to complement C. difficile JSC10, we observed that this strain germinated in response to either TA or chenodeoxycholic acid (Figure 4). These results suggest that C. difficile CspC is a receptor for bile acid germinants.


Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection.

Francis MB, Allen CA, Shrestha R, Sorg JA - PLoS Pathog. (2013)

Mutations in C.difficile cspC can alter germination specificity.Purified C. difficile JSC10 (cspC::ermB) pJS144 (pcspBACG457R) spores were suspended in BHIS medium (•) or BHIS medium supplemented with 1 mM chenodeoxycholic acid (▴) or 5 mM chenodeoxycholic acid (▾) or 10 mM TA (▪) and the initiation of germination was followed at A600.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649964&req=5

ppat-1003356-g004: Mutations in C.difficile cspC can alter germination specificity.Purified C. difficile JSC10 (cspC::ermB) pJS144 (pcspBACG457R) spores were suspended in BHIS medium (•) or BHIS medium supplemented with 1 mM chenodeoxycholic acid (▴) or 5 mM chenodeoxycholic acid (▾) or 10 mM TA (▪) and the initiation of germination was followed at A600.
Mentions: 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 UK1 and allowed the mutagenized bacteria to form spores. The purified spores were plated on BHIS medium supplemented with 0.5 mM chenodeoxycholic 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 UK1 [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 cspCG457R allele was used to complement C. difficile JSC10, we observed that this strain germinated in response to either TA or chenodeoxycholic acid (Figure 4). These results suggest that C. difficile CspC is a receptor for bile acid germinants.

Bottom Line: 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 may represent an attractive target for novel therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
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 may represent an attractive target for novel therapeutics.

Show MeSH
Related in: MedlinePlus