Structural and functional analysis of the CspB protease required for Clostridium spore germination
| Autor: | Chloe M. Adams, Brian E. Eckenroth, Sylvie Doublié, Emily E. Putnam, Aimee Shen |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
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Molecular Proteases Clostridium perfringens QH301-705.5 medicine.medical_treatment Immunology Peptidoglycan Biochemistry Microbiology Subtilase 03 medical and health sciences chemistry.chemical_compound Clostridium Bacterial Proteins Catalytic Domain Virology Genetics medicine Spore germination Humans Biology (General) Biology Molecular Biology 030304 developmental biology Spores Bacterial Serine protease 0303 health sciences Protease biology Clostridioides difficile 030306 microbiology fungi Subtilisin RC581-607 biology.organism_classification Protein Structure Tertiary chemistry Clostridium Infections biology.protein Parasitology Serine Proteases Immunologic diseases. Allergy Research Article |
| Zdroj: | PLoS Pathogens, Vol 9, Iss 2, p e1003165 (2013) PLoS Pathogens |
| ISSN: | 1553-7374 1553-7366 |
| Popis: | Spores are the major transmissive form of the nosocomial pathogen Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission. Author Summary Clostridium difficile is the leading cause of health-care associated diarrhea worldwide. C. difficile infections begin when its spores transform into vegetative cells during a process called germination. In Clostridium sp., germination requires that the spore cortex, a thick, protective layer, be removed by the cortex hydrolase SleC. While previous studies have shown that SleC activity depends on a subtilisin-like protease, CspB, the mechanisms regulating CspB function have not been characterized. In this study, we solved the first crystal structure of the Csp family of proteases and identified its key functional regions. We determined that CspB carries a unique jellyroll domain required for stabilizing the protein both in vitro and in C. difficile and a prodomain required for proper folding of the protease. Unlike all other prokaryotic subtilisin-like proteases, the prodomain remains bound to CspB and inhibits its protease activity until the germination signal is sensed. Our study provides new insight into how germination is regulated in C. difficile and may inform the development of inhibitors that can prevent germination and thus C. difficile transmission. |
| Databáze: | OpenAIRE |
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