The Regulation and Characterization of the \u3ci\u3eClostridioides difficile\u3c/i\u3e Infection Through the Stringent Response Mechanism

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

Single-Cell Characterization of \u3ci\u3eClostridioidies difficile\u3c/i\u3e Motility Using Anaerobic Live Cell Microscopy

Clostridioidies difficile (C.difficile) is an anaerobic bacterium responsible for CDI (Clostridioidies difficile infection), a common problem in hospitals and for people using antibiotics, due to the bacteria’s resistance to common treatments and ready appetite for sugar byproducts in the intestine. C.difficile has proven to be resistant to multiple antibiotic families, including beta lactams and fluroquinolones. The bacteria have two forms: dormant spores that persist in the environment and spread the infection, and vegetative cells, which proliferate within the host colon and produce virulent toxins. Little is known about the behavior of vegetative cells within hosts, because they are strict anaerobes and killed by the environmental oxygen. It is known that the organism is a motile, flagellated bacterium which swims through liquids and can even traverse the surface of solids through the extension and retraction of pili in vitro. Here, we demonstrate a novel methodology for capturing active imagery of the microbe at the level of individual cells, and show that the epidemic C.difficile strain R20291 has its motility regulated by the presence of different sources of energy that it is cultured in. This provides a superior ability to analyze distribution patterns of bacteria outside the anaerobic chamber, in addition to showing that this distribution is highly regulated by the nutritional substrate available to the microbe. Different concentrations of both arabinose and glucose showed no difference in motility for C.difficile, whereas higher concentrations of the mucus component N-actylneuraminic acid (Neu5Ac) caused significant reductions in movement and direction changes in the bacteria. This suggests that C.difficile responds to substrate nutrient type and concentration through changes in behavior and may actively target gut mucus as a colonization site