<i>Clostridioides difficile</i> is a highly antibiotic resistant and infectious endospore-forming bacterium, responsible for an estimated 450,000 cases per year. The formation of an endospore is necessary for the survival of the bacterium while in-transit between hosts and while passing through the toxic environment of the host's stomach. Essential to the endospore's resistance is a thick layer of highly modified peptidoglycan called the cortex. While the endospore cortex is forming, the enzymes CwlD and PdaA convert N-acetylmuramic acid (NAM) into muramic--lactam (MAL). MAL serves as a recognition element for germination-specific lytic enzymes that degrade cortex peptidoglycan layers during germination. Without the MAL residues the endospore cannot complete germination. Unique to the Peptostreptococcaceae family, which includes C. difficile, is the lipoprotein GerS, which is required for the function of CwlD. The interaction between these two proteins is poorly understood. In this work, attempts to complement a Bacillus subtilis cwlD mutant using C. difficile gerS and/or cwlD were unsuccessful. No MAL residues were produced, and spores produced were incapable of completing germination. In vitro assays of CwlD activity on purified peptidoglycan revealed binding and activity of C. difficile CwlD, which were significantly increased when in complex with GerS. The ability of C. difficile CwlD+GerS to function in vitro but not to complement in B. subtilis suggests that in vivo activity is blocked by some factor in this heterologous system. Such a factor might be the in vivo ionic environment or a failure to properly localize within the forespore in B. subtilis cells. Furthering the understanding of C. difficile's germination machinery will potentially provide new targets for therapies. / Master of Science / Antibiotics have saved countless lives since their initial discovery and subsequent use to kill harmful bacteria. However, they have also led to the rise of antibiotic associated diarrhea (AAD), which can be fatal. AAD is caused by antibiotic resistant bacteria that can infect the gut after a large number of bacteria, which exist normally in a healthy gut, are killed by antibiotics. The number one cause of AAD is Clostridioides difficile, which accounts for approximately 450,000 cases a year in the United States, and millions of cases worldwide. C. difficile is highly antibiotic resistant and can exist in the environment for decades as an endospore, protected from many types of disinfectants. This bacterium is commonly spread in hospital settings where it can survive many of the cleaning regiments to infect vulnerable patients. Our work focused on how one of the structures of the endospore is modified in C. difficile to better understand a part of the machinery necessary for causing infection. Studying how the bacteria produces an endospore can shed light on targets for new treatments.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/115840 |
| Date | 24 July 2023 |
| Creators | Kohler, Brian Jacob |
| Contributors | Biological Sciences, Popham, David L., Jutras, Brandon L., Melville, Stephen B. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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