Virginia Tech
Investigating the enzyme activity of a <i>Clostridioides difficile</i> amidase complex
Abstract
dc:description.abstract<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.
Degree
thesis:*- Name thesis:degree_name
- Master of Science
- Level thesis:degree_level
- masters
- Discipline thesis:degree_discipline
- Biological Sciences
- Department dc:contributor.department
- Biological Sciences
- Grantor dc:publisher
- Virginia Tech
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Kohler, Brian Jacob
- Chair dc:contributor.committeechair
-
- Popham, David L.
- Committee members dc:contributor.committeemember
-
- Jutras, Brandon L.
- Melville, Stephen B.
Subjects
dc:subject × 4Rights
dc:rights- Statement dc:rights
-
- In Copyright
- Licence dc:rights.uri
- Language dc:language.iso
- en
Identifiers
dc:identifier.*- Dc Identifier Other
- vt_gsexam:38230
- OAI identifier oai:identifier
- oai:vtechworks.lib.vt.edu:10919/115840