Title

The Regulation and Characterization of the Clostridioides difficile Infection Through the Stringent Response Mechanism

Description/Abstract

Clostridioides difficile Infection (CDI) is one of the widest spread hospital-acquired infections in first world. It persists as dormant spores in aerobic environments, which colonizes the gut upon host ingestion resulting in the secretion of cytotoxins. Symptoms range from watery diarrhea to life threatening pseudomembranous colitis. Notably, C. difficile is incredibly resistant to a wide array of antibiotic treatments. We focused our attention to the stringent response pathway, which is a stress-induced signaling pathway. Bacterial cells accumulate two alarmones, guanosine tetraphosphdate (ppGpp) and guanosine pentaphosphate (pppGpp) in the cytoplasm via enzymes belonging to the Rel/Spo homolog (RSH) family. These small molecules facilitate bacterial survival during stresses while also regulating virulence factor production. For the first time, we’ve confirmed C. difficile genome to encode rsh and relQ, suggesting that this organism can also mount the stringent response cascade. RSH, a long bifunctional synthetase and hydrolase domain harboring protein and RelQ, a synthetase domain-only carrying protein regulate the stringent response in different Gram-positive species. Through radiolabeled thin layer chromatography, we have also shown C. difficile RSH (CdRSH) to be a unique ppGpp synthetase in vitro. We are currently utilizing site overlap extension and site directed mutagenesis techniques to render C. difficile RSH catalytically inactive. Recent work has also included the designing and cloning of truncation constructs from the gene sequence of full-length C. difficile rsh. By cloning the N-terminal catalytic region of CdRSH, we aim to characterize the mechanism(s) by which RSH’s C-terminal regulatory region inversely regulates the enzyme’s opposing catalytic activities.

Presenting Author Name/s

Anthony Resek

Faculty Advisor

Erin Purcell

Presentation Type

Poster

Disciplines

Biochemistry | Biochemistry, Biophysics, and Structural Biology

Session Title

Poster Session

Location

Learning Commons, Atrium

Start Date

2-8-2020 8:00 AM

End Date

2-8-2020 12:30 PM

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Feb 8th, 8:00 AM Feb 8th, 12:30 PM

The Regulation and Characterization of the Clostridioides difficile Infection Through the Stringent Response Mechanism

Learning Commons, Atrium

Clostridioides difficile Infection (CDI) is one of the widest spread hospital-acquired infections in first world. It persists as dormant spores in aerobic environments, which colonizes the gut upon host ingestion resulting in the secretion of cytotoxins. Symptoms range from watery diarrhea to life threatening pseudomembranous colitis. Notably, C. difficile is incredibly resistant to a wide array of antibiotic treatments. We focused our attention to the stringent response pathway, which is a stress-induced signaling pathway. Bacterial cells accumulate two alarmones, guanosine tetraphosphdate (ppGpp) and guanosine pentaphosphate (pppGpp) in the cytoplasm via enzymes belonging to the Rel/Spo homolog (RSH) family. These small molecules facilitate bacterial survival during stresses while also regulating virulence factor production. For the first time, we’ve confirmed C. difficile genome to encode rsh and relQ, suggesting that this organism can also mount the stringent response cascade. RSH, a long bifunctional synthetase and hydrolase domain harboring protein and RelQ, a synthetase domain-only carrying protein regulate the stringent response in different Gram-positive species. Through radiolabeled thin layer chromatography, we have also shown C. difficile RSH (CdRSH) to be a unique ppGpp synthetase in vitro. We are currently utilizing site overlap extension and site directed mutagenesis techniques to render C. difficile RSH catalytically inactive. Recent work has also included the designing and cloning of truncation constructs from the gene sequence of full-length C. difficile rsh. By cloning the N-terminal catalytic region of CdRSH, we aim to characterize the mechanism(s) by which RSH’s C-terminal regulatory region inversely regulates the enzyme’s opposing catalytic activities.