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

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

Complex c-di-GMP Signaling Networks Mediate Transition between Virulence Properties and Biofilm Formation in Salmonella enterica Serovar Typhimurium

Upon Salmonella enterica serovar Typhimurium infection of the gut, an early line of defense is the gastrointestinal epithelium which senses the pathogen and intrusion along the epithelial barrier is one of the first events towards disease. Recently, we showed that high intracellular amounts of the secondary messenger c-di-GMP in S. typhimurium inhibited invasion and abolished induction of a pro-inflammatory immune response in the colonic epithelial cell line HT-29 suggesting regulation of transition between biofilm formation and virulence by c-di-GMP in the intestine. Here we show that highly complex c-di-GMP signaling networks consisting of distinct groups of c-di-GMP synthesizing and degrading proteins modulate the virulence phenotypes invasion, IL-8 production and in vivo colonization in the streptomycin-treated mouse model implying a spatial and timely modulation of virulence properties in S. typhimurium by c-di-GMP signaling. Inhibition of the invasion and IL-8 induction phenotype by c-di-GMP (partially) requires the major biofilm activator CsgD and/or BcsA, the synthase for the extracellular matrix component cellulose. Inhibition of the invasion phenotype is associated with inhibition of secretion of the type three secretion system effector protein SipA, which requires c-di-GMP metabolizing proteins, but not their catalytic activity. Our findings show that c-di-GMP signaling is at least equally important in the regulation of Salmonella-host interaction as in the regulation of biofilm formation at ambient temperature

Recent advances in the molecular mechanisms causing primary generalized glucocorticoid resistance

Primary Generalized Glucocorticoid Resistance is a rare condition characterized by generalized, partial, target tissue insensitivity to glucocorticoids owing to inactivating mutations, insertions or deletions in the human glucocorticoid receptor (hGR) gene (NR3C1). Recent advances in molecular and structural biology have enabled us to elucidate the molecular mechanisms of action of the mutant receptors and to understand how certain conformational alterations of the defective hGRs result in generalized glucocorticoid resistance. Furthermore, our ever-increasing understanding of the molecular mechanisms of glucocorticoid action indicates that the glucocorticoid signaling pathway is a stochastic system that plays a fundamental role in maintaining both basal and stress-related homeostasis. In this review, we summarize the clinical manifestations and molecular pathogenesis of Primary Generalized Glucocorticoid Resistance, we present our recent findings from the functional characterization of three novel heterozygous point mutations in the NR3C1 gene, and we discuss the diagnostic approach and therapeutic management of the condition. When the condition is suspected, we recommend sequencing analysis of the NR3C1 gene as well as of other genes encoding proteins involved in the glucocorticoid signal transduction. The tremendous progress of next-generation sequencing will undoubtedly uncover novel hGR partners or cofactors

Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes

International audienceExtracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases

Transcriptomics in tissue glucocorticoid sensitivity

Background Synthetic glucocorticoids are widely used in the treatment of several inflammatory, autoimmune and lymphoproliferative disorders. However, considerable variation in response to therapeutic doses of glucocorticoids has been documented among individuals. The aim of our study was to identify novel glucocorticoid sensitivity-determining genes using genome-wide expression profiling in healthy subjects. Methods One hundred one healthy subjects [mean age +/- standard error of the mean (SEM); 26.52 +/- 0.50 years] were given 0.25 mg dexamethasone at midnight, and serum cortisol concentrations were determined at 08:00 hours the following morning. Subjects were stratified into the 10% most glucocorticoid-sensitive and 10% most glucocorticoid-resistant according to the serum cortisol concentrations. Genomic DNA, RNA and plasma samples were obtained in the 22 subjects one month later. Results Transcriptomic analysis showed variability between glucocorticoid-resistant and glucocorticoid-sensitive subjects. One hundred thirty-three genes were upregulated and 49 downregulated in the glucocorticoid-resistant compared to the glucocorticoid-sensitive group. Further analysis revealed differences between 3 glucocorticoid-resistant and 3 glucocorticoid-sensitive subjects. The majority of the 1058 upregulated genes and 1139 downregulated genes were found to participate in telomere maintenance, systemic lupus erythematosus and Alzheimer’s disease. Interestingly, Synuclein A, a key molecule in Parkinson’s disease, was upregulated in the subgroup of glucocorticoid-sensitive subjects. Conclusions We have identified differences in tissue sensitivity to glucocorticoids among healthy subjects at the transcriptomic level. These differences are associated with differential expression of genes related to autoimmune and neurological disorders

Transient generalized glucocorticoid hypersensitivity

Background Transient generalized glucocorticoid hypersensitivity is a rare disorder characterized by increased tissue sensitivity to glucocorticoids and compensatory hypo-activation of the hypothalamic-pituitary-adrenal axis. The condition itself and the underlying molecular mechanisms have not been elucidated. Objective To present the clinical manifestations, endocrinologic evaluation and transcriptomic profile in a patient with transient generalized glucocorticoid hypersensitivity. Design and Results A 9-year-old girl presented with an 8-month history of clinical manifestations suggestive of Cushing syndrome. Endocrinologic evaluation revealed undetectable 08: 00 h ACTH (<1 pg/mL) and cortisol (0.025 mu g/dL) concentrations, which remained decreased throughout the 24-h period and did not respond to stimulation with ovine CRH. The disease gradually resolved spontaneously over the ensuing 3 months. Sequencing of the human glucocorticoid receptor gene revealed no mutations or polymorphisms. Western blot analysis in peripheral blood mononuclear cells revealed equal protein expression of hGR alpha of the patient in the disease and postresolution phases compared with a control subject. Transcriptomic analysis in peripheral blood mononuclear cells in the disease and postresolution phases identified 903 differentially expressed genes. Of these, 106 genes were up-regulated and 797 were down-regulated in the disease compared with the resolution phase. Bioinformatics analysis on the differentially expressed gene networks revealed Nuclear Factor-kappa B as the predominant transcription factor influencing the expression of the majority of differentially expressed genes. Conclusions Our findings indicate that a transient postreceptor defect, or a virus-or bacterium-encoded molecule, may have enhanced glucocorticoid signal transduction, leading to transient generalized glucocorticoid hypersensitivity and hypo-activation of the HPA axis