{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/135503"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/135503","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Investigating the Roles of CwlD and GerS in <i>Clostridioides difficile</i> Spore Cortex Modification","abstract":"Clostridioides difficile is a notable nosocomial pathogen distinguished by its capacity to produce spores, essential for its survival and dissemination. The spore cortex, consisting of a modified peptidoglycan layer, is essential for dormancy and germination. This work examines the enzyme CwlD, which facilitates the transformation of N-acetylmuramic acid (NAM) into muramic-δlactam (MAL)—a change crucial for adequate cortex production and germination. The lipoprotein GerS is essential for CwlD function in C. difficile; however, no GerS homolog is present in Bacillus subtilis, a well-established model for spore biology. To assess the species requirements for CwlD and GerS, we expressed FLAG-tagged versions of C. difficile CwlD, both in the presence and absence of GerS, in a B. subtilis ΔcwlD background. Despite the effective assembly and integration of all genetic constructs, no expression of FLAG-tagged protein was seen. The data indicates that factors such as translational inefficiency or protein degradation may impede the expression of these heterologous proteins in B. subtilis. This contrasts with prior research demonstrating observable production of variants even though they remained non-functional in B. subtilis. These findings highlight the significance of the native cellular environment for the functional expression of CwlD–GerS and enhance the comprehension of species-specific regulatory mechanisms in spore life. These results may guide future therapy strategies aimed at targeting C. difficile germination by inhibiting CwlD-GerS-mediated MAL production. This would impede spore germination, presenting a potential approach to avoid infection initiation and diminish recurrence by maintaining remaining spores in a dormant state.","abstract_html":"Clostridioides difficile is a notable nosocomial pathogen distinguished by its capacity to produce spores, essential for its survival and dissemination. The spore cortex, consisting of a modified peptidoglycan layer, is essential for dormancy and germination. This work examines the enzyme CwlD, which facilitates the transformation of N-acetylmuramic acid (NAM) into muramic-δlactam (MAL)—a change crucial for adequate cortex production and germination. The lipoprotein GerS is essential for CwlD function in C. difficile; however, no GerS homolog is present in Bacillus subtilis, a well-established model for spore biology. To assess the species requirements for CwlD and GerS, we expressed FLAG-tagged versions of C. difficile CwlD, both in the presence and absence of GerS, in a B. subtilis ΔcwlD background. Despite the effective assembly and integration of all genetic constructs, no expression of FLAG-tagged protein was seen. The data indicates that factors such as translational inefficiency or protein degradation may impede the expression of these heterologous proteins in B. subtilis. This contrasts with prior research demonstrating observable production of variants even though they remained non-functional in B. subtilis. These findings highlight the significance of the native cellular environment for the functional expression of CwlD–GerS and enhance the comprehension of species-specific regulatory mechanisms in spore life. These results may guide future therapy strategies aimed at targeting C. difficile germination by inhibiting CwlD-GerS-mediated MAL production. This would impede spore germination, presenting a potential approach to avoid infection initiation and diminish recurrence by maintaining remaining spores in a dormant state.","abstract_has_math":false,"creators":["Nawar, Shaeri"],"institution":"Virginia Tech","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Biological Science","degree_department":"Biological Sciences","school":null,"contributors":[],"advisors":[],"committee_chairs":["Popham, David L."],"committee_members":["Stevens, Ann M.","Caswell, Clayton C.","Melville, Stephen B."],"year":2025,"date_issued":"2025-05-12","date_published":"2025-05-12","updated_at":"2026-07-22T22:19:16Z","subjects":["Clostridioides difficile","cortex","peptidoglycan","CwlD","GerS","sporulation","germination","Bacillus subtilis","spores"],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/10919/135503","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Popham, David L."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Stevens, Ann M.","Caswell, Clayton C.","Melville, Stephen B."]},{"key":"dc:contributor.department","label":"Department","values":["Biological Sciences"]},{"key":"dc:creator","label":"Author","values":["Nawar, Shaeri"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-06-13T12:36:34Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-06-13T12:36:34Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-05-12"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.dcmitype","label":"Dc Type Dcmitype","values":["Text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biological Science"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Clostridioides difficile","cortex","peptidoglycan","CwlD","GerS","sporulation","germination","Bacillus subtilis","spores"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10919/135503"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Clostridioides difficile is a notable nosocomial pathogen distinguished by its capacity to produce spores, essential for its survival and dissemination. The spore cortex, consisting of a modified peptidoglycan layer, is essential for dormancy and germination. This work examines the enzyme CwlD, which facilitates the transformation of N-acetylmuramic acid (NAM) into muramic-δlactam (MAL)—a change crucial for adequate cortex production and germination. The lipoprotein GerS is essential for CwlD function in C. difficile; however, no GerS homolog is present in Bacillus subtilis, a well-established model for spore biology. To assess the species requirements for CwlD and GerS, we expressed FLAG-tagged versions of C. difficile CwlD, both in the presence and absence of GerS, in a B. subtilis ΔcwlD background. Despite the effective assembly and integration of all genetic constructs, no expression of FLAG-tagged protein was seen. The data indicates that factors such as translational inefficiency or protein degradation may impede the expression of these heterologous proteins in B. subtilis. This contrasts with prior research demonstrating observable production of variants even though they remained non-functional in B. subtilis. These findings highlight the significance of the native cellular environment for the functional expression of CwlD–GerS and enhance the comprehension of species-specific regulatory mechanisms in spore life. These results may guide future therapy strategies aimed at targeting C. difficile germination by inhibiting CwlD-GerS-mediated MAL production. This would impede spore germination, presenting a potential approach to avoid infection initiation and diminish recurrence by maintaining remaining spores in a dormant state."]},{"key":"dc:description.abstractgeneral","label":"General Abstract","values":["Clostridioides difficile is a bacterium that is dangerous and is known to cause severe intestinal infections, particularly in individuals who are taking antibiotics or residing in hospitals. This bacterium's capacity to generate resistant latent spores that can endure severe conditions and induce recurrent infections is what renders it particularly challenging to eradicate. Two proteins, CwlD and GerS, collaborate to produce a unique molecule in the spore cell wall structure known as muramic-δ-lactam (MAL) in C. difficile. MAL is essential for the correct development of spores, which subsequently \"wake up\" (germinate) and produce infection. We attempted to produce these proteins in Bacillus subtilis, a closely related and extensively studied bacterium, to better understand their functionality. Although the genes were successfully inserted, we discovered that the proteins were either not produced or were not functioning normally. This implies that C. difficile may necessitate specific cellular factors that are not present in B. subtilis to sustain these proteins. By comprehending the mechanisms of these proteins, we can identify novel methods for preventing the germination of C. difficile spores. In the future, the prevention of this process could result in more effective treatments that prevent infection or its recurrence."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["ETD"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Investigating the Roles of CwlD and GerS in <i>Clostridioides difficile</i> Spore Cortex Modification"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Popham, David L."],"dc:contributor.committeemember":["Stevens, Ann M.","Caswell, Clayton C.","Melville, Stephen B."],"dc:contributor.department":["Biological Sciences"],"dc:creator":["Nawar, Shaeri"],"dc:date.accessioned":["2025-06-13T12:36:34Z"],"dc:date.available":["2025-06-13T12:36:34Z"],"dc:date.issued":["2025-05-12"],"dc:description.abstract":["Clostridioides difficile is a notable nosocomial pathogen distinguished by its capacity to produce spores, essential for its survival and dissemination. The spore cortex, consisting of a modified peptidoglycan layer, is essential for dormancy and germination. This work examines the enzyme CwlD, which facilitates the transformation of N-acetylmuramic acid (NAM) into muramic-δlactam (MAL)—a change crucial for adequate cortex production and germination. The lipoprotein GerS is essential for CwlD function in C. difficile; however, no GerS homolog is present in Bacillus subtilis, a well-established model for spore biology. To assess the species requirements for CwlD and GerS, we expressed FLAG-tagged versions of C. difficile CwlD, both in the presence and absence of GerS, in a B. subtilis ΔcwlD background. Despite the effective assembly and integration of all genetic constructs, no expression of FLAG-tagged protein was seen. The data indicates that factors such as translational inefficiency or protein degradation may impede the expression of these heterologous proteins in B. subtilis. This contrasts with prior research demonstrating observable production of variants even though they remained non-functional in B. subtilis. These findings highlight the significance of the native cellular environment for the functional expression of CwlD–GerS and enhance the comprehension of species-specific regulatory mechanisms in spore life. These results may guide future therapy strategies aimed at targeting C. difficile germination by inhibiting CwlD-GerS-mediated MAL production. This would impede spore germination, presenting a potential approach to avoid infection initiation and diminish recurrence by maintaining remaining spores in a dormant state."],"dc:description.abstractgeneral":["Clostridioides difficile is a bacterium that is dangerous and is known to cause severe intestinal infections, particularly in individuals who are taking antibiotics or residing in hospitals. This bacterium's capacity to generate resistant latent spores that can endure severe conditions and induce recurrent infections is what renders it particularly challenging to eradicate. Two proteins, CwlD and GerS, collaborate to produce a unique molecule in the spore cell wall structure known as muramic-δ-lactam (MAL) in C. difficile. MAL is essential for the correct development of spores, which subsequently \"wake up\" (germinate) and produce infection. We attempted to produce these proteins in Bacillus subtilis, a closely related and extensively studied bacterium, to better understand their functionality. Although the genes were successfully inserted, we discovered that the proteins were either not produced or were not functioning normally. This implies that C. difficile may necessitate specific cellular factors that are not present in B. subtilis to sustain these proteins. By comprehending the mechanisms of these proteins, we can identify novel methods for preventing the germination of C. difficile spores. In the future, the prevention of this process could result in more effective treatments that prevent infection or its recurrence."],"dc:description.degree":["Master of Science"],"dc:format.medium":["ETD"],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["https://hdl.handle.net/10919/135503"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["Clostridioides difficile","cortex","peptidoglycan","CwlD","GerS","sporulation","germination","Bacillus subtilis","spores"],"dc:title":["Investigating the Roles of CwlD and GerS in <i>Clostridioides difficile</i> Spore Cortex Modification"],"dc:type":["Thesis"],"dc:type.dcmitype":["Text"],"thesis:degree_discipline":["Biological Science"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:16Z"}