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The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile.

ABSTRACT: Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores. Previous studies have identified cholic amide m-sulfonic acid, CamSA, as an inhibitor of spore germination. However, CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series of cholic acid amides were synthesized and tested against R20291. The best compound in the series was the simple phenyl amide analogue which possessed an IC50 value of 1.8 ?M, more than 225 times as potent as the natural germination inhibitor, chenodeoxycholate. This is the most potent inhibitor of C. difficile spore germination described to date. QSAR and molecular modeling analysis demonstrated that increases in hydrophobicity and decreases in partial charge or polar surface area were correlated with increases in potency.

SUBMITTER: Sharma SK

PROVIDER: S-EPMC6192251 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile.

Sharma Shiv K SK Yip Christopher C Esposito Emilio Xavier EX Sharma Prateek V PV Simon Matthew P MP Abel-Santos Ernesto E Firestine Steven M SM

Journal of medicinal chemistry 20180730 15

Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores. Previous studies have identified cholic amide m-sulfonic acid, CamSA, as an inhibitor of spore germination. However, CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series of cholic acid amides were synth ...[more]

PMID: 30004695

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Project description:UnlabelledBacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway.ImportanceClostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway.

Project description:Clostridium difficile causes considerable health care-associated gastrointestinal disease that is transmitted by its metabolically dormant spore form. Upon entering the gut, C. difficile spores germinate and outgrow to produce vegetative cells that release disease-causing toxins. C. difficile spore germination depends on the Csp family of (pseudo)proteases and the cortex hydrolase SleC. The CspC pseudoprotease functions as a bile salt germinant receptor that activates the protease CspB, which in turn proteolytically activates the SleC zymogen. Active SleC degrades the protective cortex layer, allowing spores to outgrow and resume metabolism. We previously showed that the CspA pseudoprotease domain, which is initially produced as a fusion to CspB, controls the incorporation of the CspC germinant receptor in mature spores. However, study of the individual Csp proteins has been complicated by the polar effects of TargeTron-based gene disruption on the cspBA-cspC operon. To overcome these limitations, we have used pyrE-based allelic exchange to create individual deletions of the regions encoding CspB, CspA, CspBA, and CspC in strain 630Δerm Our results indicate that stable CspA levels in sporulating cells depend on CspB and confirm that CspA maximizes CspC incorporation into spores. Interestingly, we observed that csp and sleC mutants spontaneously germinate more frequently in 630Δerm than equivalent mutants in the JIR8094 and UK1 strain backgrounds. Analyses of this phenomenon suggest that only a subpopulation of C. difficile 630Δerm spores can spontaneously germinate, in contrast with Bacillus subtilis spores. We also show that C. difficile clinical isolates that encode truncated CspBA variants have sequencing errors that actually produce full-length CspBA.IMPORTANCEClostridium difficile is a leading cause of health care-associated infections. Initiation of C. difficile infection depends on spore germination, a process controlled by Csp family (pseudo)proteases. The CspC pseudoprotease is a germinant receptor that senses bile salts and activates the CspB protease, which activates a hydrolase required for germination. Previous work implicated the CspA pseudoprotease in controlling CspC incorporation into spores but relied on plasmid-based overexpression. Here we have used allelic exchange to study the functions of CspB and CspA. We determined that CspA production and/or stability depends on CspB and confirmed that CspA maximizes CspC incorporation into spores. Our data also suggest that a subpopulation of C. difficile spores spontaneously germinates in the absence of bile salt germinants and/or Csp proteins.

Dataset Information

The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile.

Publications

The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile.

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