Cyclic monophosphate cyclase in Firmicutes: from basic to practical approach

Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes such as cell wall homeostasis, biofilm formation, antibiotic and heat resistance, among others. In Lactococcus lactis, a Lactic Acid Bacterium used not only in the dairy industry 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 transporters, which belong to the Kup/HAK/KT family, are highly conserved in this species, being therefor 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 downregulated by c-di-AMP. On the other hand, different strains dereived 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 obtention of gdpP 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. Also, 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. 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 activity that promotes both humoral and cellular immune responses. Altoghether, 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. Finally, a first approach was done on c-di-AMP metabolism in another Lactic Acid Bacterium, Enterococcus faecalis. The use of this model bacterium permited not only to identify the basic characteristics of c-di-AMP metabolism but also to study the impact of GdpP on the virulence of E. faecalis by use of the infection model Galleria melonela