Role of c-di-GMP signalling in bacterial-host interactions

Sammanfattning: Bacteria have various ways to sense environmental signals and to adapt their behavior and physiology through different signaling systems. Secondary messenger signaling, amplified by enzymatic activity, rapidly transmits a signal in the cell, resulting in allosteric functional control. Cyclic diguanosine monophosphate (c-di-GMP) is a novel global secondary messenger that is found exclusively in bacteria and is involved in fundamental bacterial behavior such as motility, sessility and virulence. Regulation of virulence by c-di-GMP signaling is crucial for many pathogens. The aim of this thesis was to study the potential role of c-di-GMP in bacterial-host interactions using Salmonella enterica serovar Typhimurium as a model system. We wanted to study the effect of c-di-GMP on virulence phenotypes and to identify the components and mechanisms through which c-di-GMP mediates its effects. Using the colon carcinoma cell line HT-29 we found that high levels of intracellular c-di-GMP inhibited invasion of S. typhimurium into epithelial cells, and induction of production of the proinflammatory cytokine interleukine-8 (IL-8) from epithelial cells. This suggests that c-di-GMP negatively regulates acute virulence phenotypes of S. typhimurium. Inhibition of virulence phenotypes is partially mediated through biofilm components; the exopolysaccharides cellulose and capsule, as well as the biofilm regulator CsgD. C-di-GMP also interferes with the secretion of SopE2, a S. typhimurium effector protein, as well as of flagellin, both of which are secreted by Type Three Secretion Systems. GGDEF and EAL domain proteins are diguanylate cyclases and phosphodiesterases that synthesize and degrade c-di-GMP, respectively. These proteins amplify the primary signal through a local or global change in the c-di-GMP concentration, and their specific activity determines the phenotypic output. We did a comprehensive study of S. typhimurium mutants of GGDEF/EAL domain proteins that revealed distinct groups of proteins involved in invasion, IL-8 production and colonization in streptomycin-treated mice. The distinct groups of proteins suggest non-redundancy and specific, localized activity of the secondary messenger towards regulatory targets. C-di-GMP is involved in the regulation of biofilm formation. However, the role of biofilm formation in bacterial-host interactions of commensal Escherichia coli has not been studied in detail. So, we investigated the effect of the extracellular matrix components cellulose and curli fimbriae to bacterial adherence, internalization and induction of the pro-inflammatory cytokine IL-8 in HT-29 cells. Cellulose and curli had differential effects; while curli fimbriae promoted adherence, internalization and IL-8 production, cellulose expression in the curli expressing background inhibited these phenotypes. Curli-bound flagellin was highly immunostimulatory. In addition, our studies revealed two highly immunostimulatory flagellin sequences from commensal E. coli isolates. These flagellin sequences belong to the EC2 group of E. coli flagellins, which are closely related to S. typhimurium FliC flagellin, presumably already present in a common ancestor of E. coli and S. typhimurium.