Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach

Autores
Quintana, Ingrid M.
Año de publicación
2018
Idioma
inglés
Tipo de recurso
tesis doctoral
Estado
versión publicada
Colaborador/a o director/a de tesis
Magni, Christian
Stülke, Jörg
Descripción
Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes such as cell wall homeostasis, biofilm formation, antibiotics and heat resistance, among others. In Lactococcus lactis and Enterococcus faecalis, Lactic Acid Bacteria used not only as research models but also as a cell factory in biotechnological processes, the only reported interaction partner of c-di-AMP is the pyruvate carboxylase enzyme, PyrCarb. Nevertheless, in the last year investigations directed its main role towards potassium metabolism. In this thesis, KupA and KupB, two potassium transporters encoded in L. lactis IL1403 genome, are described for the first time. According to an in silico analysis, these proteins, which belong to the Kup/HAK/KT family, are highly conserved in this species, being therefore a strain independent potassium uptake system. In addition, evidence shows that both proteins are able to uptake this cation with high affinity, and we demonstrate that KupA as well as KupB bind to and are down-regulated by c-di-AMP. On the other hand, different strains derived from L. lactis IL1403 were developed aiming to modify intracellular pools of c-di-AMP in a stable system. One strategy for the reduction of c-di-AMP levels was the obtaining of ΔgdpP mutants via homologous recombination. Maintenance of this second messenger levels close to wild type ones, suggested the presence of another c-di-AMP degrading enzyme. A first description of a putative enzyme with this activity, encoded by yheB gene was done by BNPP assay. In addition, by use of a pH inducible vector, construction of strain L. lactis LL03 with concentrations of this second messenger above 15 times basal levels was possible. This system was therefore selected for further investigations on the development of a vaccine prototype against Chagas disease. On the other hand, L. lactis is a promising candidate for the development of mucosal vaccines with more than 20 years of experimental research. Moreover, c-di-AMP has been reported as a strong mucosal adjuvant promoting both humoral and cellular immune responses. Altogether, in this thesis the development of a recombinant L. lactis strain is reported, able to produce both an antigen as well as an adjuvant in order to develop a novel vaccine prototype against the Trypanosoma cruzi parasite, the causal agent of Chagas disease. This is a tropical disease originated in a specific area of South America but currently spreading in four continents. Finally, an initial approach was done on c-di-AMP metabolism in E. faecalis. The presence of a Kup transporter was also corroborated in this species, and some basic characteristics of the c-di-AMP degradative pathway were explored via a ΔgdpP mutant construction. Finally, the impact of GdpP on the virulence of E. faecalis was analyzed by use of the infection model Galleria mellonella.
Fil: Fil: Quintana, Ingrid M. Centro de Estudios Interdisciplinarios. Universidad Nacional de Rosario; Argentina
Materia
c-di-AMP
Lactococcus lactis
Metabolic processes
Nivel de accesibilidad
acceso abierto
Condiciones de uso
Atribución-NoComercial 2.5 Argentina (CC BY-NC 2.5 AR)
Repositorio
RepHipUNR (UNR)
Institución
Universidad Nacional de Rosario
OAI Identificador
oai:rephip.unr.edu.ar:2133/15229

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oai_identifier_str oai:rephip.unr.edu.ar:2133/15229
network_acronym_str RepHipUNR
repository_id_str 1550
network_name_str RepHipUNR (UNR)
spelling Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approachQuintana, Ingrid M.c-di-AMPLactococcus lactisMetabolic processesCyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes such as cell wall homeostasis, biofilm formation, antibiotics and heat resistance, among others. In Lactococcus lactis and Enterococcus faecalis, Lactic Acid Bacteria used not only as research models but also as a cell factory in biotechnological processes, the only reported interaction partner of c-di-AMP is the pyruvate carboxylase enzyme, PyrCarb. Nevertheless, in the last year investigations directed its main role towards potassium metabolism. In this thesis, KupA and KupB, two potassium transporters encoded in L. lactis IL1403 genome, are described for the first time. According to an in silico analysis, these proteins, which belong to the Kup/HAK/KT family, are highly conserved in this species, being therefore a strain independent potassium uptake system. In addition, evidence shows that both proteins are able to uptake this cation with high affinity, and we demonstrate that KupA as well as KupB bind to and are down-regulated by c-di-AMP. On the other hand, different strains derived from L. lactis IL1403 were developed aiming to modify intracellular pools of c-di-AMP in a stable system. One strategy for the reduction of c-di-AMP levels was the obtaining of ΔgdpP mutants via homologous recombination. Maintenance of this second messenger levels close to wild type ones, suggested the presence of another c-di-AMP degrading enzyme. A first description of a putative enzyme with this activity, encoded by yheB gene was done by BNPP assay. In addition, by use of a pH inducible vector, construction of strain L. lactis LL03 with concentrations of this second messenger above 15 times basal levels was possible. This system was therefore selected for further investigations on the development of a vaccine prototype against Chagas disease. On the other hand, L. lactis is a promising candidate for the development of mucosal vaccines with more than 20 years of experimental research. Moreover, c-di-AMP has been reported as a strong mucosal adjuvant promoting both humoral and cellular immune responses. Altogether, in this thesis the development of a recombinant L. lactis strain is reported, able to produce both an antigen as well as an adjuvant in order to develop a novel vaccine prototype against the Trypanosoma cruzi parasite, the causal agent of Chagas disease. This is a tropical disease originated in a specific area of South America but currently spreading in four continents. Finally, an initial approach was done on c-di-AMP metabolism in E. faecalis. The presence of a Kup transporter was also corroborated in this species, and some basic characteristics of the c-di-AMP degradative pathway were explored via a ΔgdpP mutant construction. Finally, the impact of GdpP on the virulence of E. faecalis was analyzed by use of the infection model Galleria mellonella.Fil: Fil: Quintana, Ingrid M. Centro de Estudios Interdisciplinarios. Universidad Nacional de Rosario; ArgentinaMagni, ChristianStülke, Jörg2018-06-11info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_db06info:ar-repo/semantics/tesisDoctoralapplication/pdfhttp://hdl.handle.net/2133/15229enginfo:eu-repo/semantics/openAccessAtribución-NoComercial 2.5 Argentina (CC BY-NC 2.5 AR)http://creativecommons.org/licenses/by-nc-nd/2.5/ar/Licencia RepHipreponame:RepHipUNR (UNR)instname:Universidad Nacional de Rosario2025-09-04T09:43:45Zoai:rephip.unr.edu.ar:2133/15229instacron:UNRInstitucionalhttps://rephip.unr.edu.ar/Universidad públicaNo correspondehttps://rephip.unr.edu.ar/oai/requestrephip@unr.edu.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:15502025-09-04 09:43:46.095RepHipUNR (UNR) - Universidad Nacional de Rosariofalse
dc.title.none.fl_str_mv Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach
title Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach
spellingShingle Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach
Quintana, Ingrid M.
c-di-AMP
Lactococcus lactis
Metabolic processes
title_short Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach
title_full Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach
title_fullStr Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach
title_full_unstemmed Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach
title_sort Cyclic di-nucleotide monophosphate cyclase in Firmicutes: from basic to practical approach
dc.creator.none.fl_str_mv Quintana, Ingrid M.
author Quintana, Ingrid M.
author_facet Quintana, Ingrid M.
author_role author
dc.contributor.none.fl_str_mv Magni, Christian
Stülke, Jörg
dc.subject.none.fl_str_mv c-di-AMP
Lactococcus lactis
Metabolic processes
topic c-di-AMP
Lactococcus lactis
Metabolic processes
dc.description.none.fl_txt_mv Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes such as cell wall homeostasis, biofilm formation, antibiotics and heat resistance, among others. In Lactococcus lactis and Enterococcus faecalis, Lactic Acid Bacteria used not only as research models but also as a cell factory in biotechnological processes, the only reported interaction partner of c-di-AMP is the pyruvate carboxylase enzyme, PyrCarb. Nevertheless, in the last year investigations directed its main role towards potassium metabolism. In this thesis, KupA and KupB, two potassium transporters encoded in L. lactis IL1403 genome, are described for the first time. According to an in silico analysis, these proteins, which belong to the Kup/HAK/KT family, are highly conserved in this species, being therefore a strain independent potassium uptake system. In addition, evidence shows that both proteins are able to uptake this cation with high affinity, and we demonstrate that KupA as well as KupB bind to and are down-regulated by c-di-AMP. On the other hand, different strains derived from L. lactis IL1403 were developed aiming to modify intracellular pools of c-di-AMP in a stable system. One strategy for the reduction of c-di-AMP levels was the obtaining of ΔgdpP mutants via homologous recombination. Maintenance of this second messenger levels close to wild type ones, suggested the presence of another c-di-AMP degrading enzyme. A first description of a putative enzyme with this activity, encoded by yheB gene was done by BNPP assay. In addition, by use of a pH inducible vector, construction of strain L. lactis LL03 with concentrations of this second messenger above 15 times basal levels was possible. This system was therefore selected for further investigations on the development of a vaccine prototype against Chagas disease. On the other hand, L. lactis is a promising candidate for the development of mucosal vaccines with more than 20 years of experimental research. Moreover, c-di-AMP has been reported as a strong mucosal adjuvant promoting both humoral and cellular immune responses. Altogether, in this thesis the development of a recombinant L. lactis strain is reported, able to produce both an antigen as well as an adjuvant in order to develop a novel vaccine prototype against the Trypanosoma cruzi parasite, the causal agent of Chagas disease. This is a tropical disease originated in a specific area of South America but currently spreading in four continents. Finally, an initial approach was done on c-di-AMP metabolism in E. faecalis. The presence of a Kup transporter was also corroborated in this species, and some basic characteristics of the c-di-AMP degradative pathway were explored via a ΔgdpP mutant construction. Finally, the impact of GdpP on the virulence of E. faecalis was analyzed by use of the infection model Galleria mellonella.
Fil: Fil: Quintana, Ingrid M. Centro de Estudios Interdisciplinarios. Universidad Nacional de Rosario; Argentina
description Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes such as cell wall homeostasis, biofilm formation, antibiotics and heat resistance, among others. In Lactococcus lactis and Enterococcus faecalis, Lactic Acid Bacteria used not only as research models but also as a cell factory in biotechnological processes, the only reported interaction partner of c-di-AMP is the pyruvate carboxylase enzyme, PyrCarb. Nevertheless, in the last year investigations directed its main role towards potassium metabolism. In this thesis, KupA and KupB, two potassium transporters encoded in L. lactis IL1403 genome, are described for the first time. According to an in silico analysis, these proteins, which belong to the Kup/HAK/KT family, are highly conserved in this species, being therefore a strain independent potassium uptake system. In addition, evidence shows that both proteins are able to uptake this cation with high affinity, and we demonstrate that KupA as well as KupB bind to and are down-regulated by c-di-AMP. On the other hand, different strains derived from L. lactis IL1403 were developed aiming to modify intracellular pools of c-di-AMP in a stable system. One strategy for the reduction of c-di-AMP levels was the obtaining of ΔgdpP mutants via homologous recombination. Maintenance of this second messenger levels close to wild type ones, suggested the presence of another c-di-AMP degrading enzyme. A first description of a putative enzyme with this activity, encoded by yheB gene was done by BNPP assay. In addition, by use of a pH inducible vector, construction of strain L. lactis LL03 with concentrations of this second messenger above 15 times basal levels was possible. This system was therefore selected for further investigations on the development of a vaccine prototype against Chagas disease. On the other hand, L. lactis is a promising candidate for the development of mucosal vaccines with more than 20 years of experimental research. Moreover, c-di-AMP has been reported as a strong mucosal adjuvant promoting both humoral and cellular immune responses. Altogether, in this thesis the development of a recombinant L. lactis strain is reported, able to produce both an antigen as well as an adjuvant in order to develop a novel vaccine prototype against the Trypanosoma cruzi parasite, the causal agent of Chagas disease. This is a tropical disease originated in a specific area of South America but currently spreading in four continents. Finally, an initial approach was done on c-di-AMP metabolism in E. faecalis. The presence of a Kup transporter was also corroborated in this species, and some basic characteristics of the c-di-AMP degradative pathway were explored via a ΔgdpP mutant construction. Finally, the impact of GdpP on the virulence of E. faecalis was analyzed by use of the infection model Galleria mellonella.
publishDate 2018
dc.date.none.fl_str_mv 2018-06-11
dc.type.none.fl_str_mv info:eu-repo/semantics/doctoralThesis
info:eu-repo/semantics/publishedVersion
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info:ar-repo/semantics/tesisDoctoral
format doctoralThesis
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/2133/15229
url http://hdl.handle.net/2133/15229
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
Atribución-NoComercial 2.5 Argentina (CC BY-NC 2.5 AR)
http://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Licencia RepHip
eu_rights_str_mv openAccess
rights_invalid_str_mv Atribución-NoComercial 2.5 Argentina (CC BY-NC 2.5 AR)
http://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Licencia RepHip
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