Browsing by Subject "indole"
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Item The effects of low-shear modeled microgravity on Streptococcus pneumoniae and adherent-invasive Escherichia coli(2007-07-20) Christopher Ashley Allen; Dr. Alfredo Torres; Dr. Ray Stowe III; Dr. Duane Pierson; Dr. David Niesel; Dr. Ashok ChopraThe effects of low-shear modeled microgravity (LSMMG) were investigated on Streptococcus pneumoniae global gene expression and on adherent-invasive Escherichia coli (AIEC) physiology and colonization properties. Habitation in space exposes both humans and microbes to microgravity conditions which are characterized by reductions in fluid shear forces. Areas of low-shear stress are also encountered in physiologically relevant regions of the body including the respiratory, gastrointestinal, and urogenital tracts. The LSMMG environment impacts both bacterial physiology and virulence properties and can be modeled using rotating-wall bioreactors known as high-aspect ratio vessels (HARVs). \r\nPrevious studies have evaluated the global transcriptional profiles of Gram-negative bacteria; however, no Gram-positive species have been examined. Microarray analysis of S. pneumoniae strain TIGR4 (serotype 4), after growth under LSMMG, revealed a dramatic down-shift in gene expression based on cluster analysis. Within this group of responsive genes, statistical analyses revealed that the expression of 81 genes was significantly altered. These genes were found to be associated with 7 different functional categories, including many which were uncharacterized. Several gene groups shared common functional operons and regulons such as those involved in competence induction, antimicrobial peptide production, and carbohydrate uptake. \r\nWhile previous studies examining the effects of LSMMG on bacteria have focused on well-characterized strains of both commensal and pathogenic species, there is limited information regarding the effects of LSMMG on clinical isolates associated with Crohn’s Disease, an inflammatory bowel pathology. Analysis of wild-type AIEC strain O83:H1 and an isogenic rpoS mutant (CAA001), after growth under LSMMG, revealed alterations in environmental stress resistance and increased adherence. Altered resistances to thermal and osmotic stresses were observed by LSMMG-grown AIEC O83:H1, while resistance to oxidative and acid stresses appeared to be rpoS-dependent. Further, CAA001 displayed a hyper-adherent phenotype while grown under LSMMG. TnphoA mutagenesis was used to abolish the hyper-adherent phenotype of CAA001 under LSMMG, and the insertion was mapped within the tnaB gene, encoding tryptophan permease. Complementation of the tnaB gene in the rpoS tnaB double-mutant restored adherence capabilities. These findings extend our understanding of how mechanical forces (e.g. LSMMG) can affect the functions of Gram-positive and Gram-negative species.\r\nItem Inter-Kingdom Signaling Interactions in Enterohemorrhagic Escherichia coli Infections(2011-10-21) Bansal, TarunThe overall goal of this research was to understand the role of inter-kingdom signaling in enterohemorrhagic Escherichia coli (EHEC) infections of the human gastro-intestinal (GI) tract from the perspective of both the invading pathogen and the human intestinal epithelial cells, which they colonize. Differential gene expression of EHEC was studied upon exposure to the human neuroendocrine hormones epinephrine and norepinephrine. We determined that these hormones increase EHEC chemotaxis, motility, biofilm formation, colonization of host cells, and virulence gene expression. We also studied the EHEC response to the GI tract commensal bacterial signaling molecules indole and autoinducer-2 (AI-2). We observed that indole decreases all the EHEC phenotypes that are increased by the human hormones and represses EHEC virulence. However, the effect of AI-2 was similar to that observed with hormones and opposite to that observed with indole, i.e. AI-2 increases EHEC virulence phenotypes. We studied changes in host cell transcriptome in the presence of the commensal bacterial signal indole. Indole increases expression of genes involved in tight junction and gap junction formation, and production of mucins and actin cytoskeleton genes. Indole also down-regulates genes encoding for pro-inflammatory cytokines, chemokines, and Toll-like receptors. The gene expression results were confirmed with phenotypic assays where we observed an increase in trans-epithelial resistance, increase in the anti-inflammatory cytokine IL-10, decrease in the pro-inflammatory cytokine IL-8, decrease in the activity of the pro-inflammatory transcription factor NF-?B, and decrease in colonization by EHEC of the indole-pre-treated HCT-8 cells. We established that factors secreted by epithelial cells are important determinants of EHEC virulence. Gene expression studies showed that 34 out of 41 LEE virulence genes were induced when EHEC was cultured in conditioned medium. In addition, the data showed increased expression of the shiga toxin-2 prophage 933W. These changes in gene expression were corroborated by a 5-fold increase in HCT-8 cell colonization and increased intracellular Stx2 phage titers. We determined that the HCT-8-secreted factor(s) was protein-based and that it was greater than 3 kDa in size. In conclusion, we have characterized the pathogen response to various eukaryotic and prokaryotic GI tract signals. We have established, for the first time, that the commensal bacterial signal indole is an inter-kingdom signal for the host epithelial cells. Overall, our studies provide a greater understanding of host-pathogen interactions.Item Novel Methodology Towards the Indole Core and Iron-Catalyzed Electrophilic Hydroamination of Alkenes(2014-04-18) Huehls, Christopher BryanIndole-containing structures and their generation still draw much attention because of their ubiquity in natural products, medicinal compounds, and organic materials. Given this prevalence, synthetic methods toward these structural motifs are numerous, each with a distinct set of advantages and disadvantages. One significant challenge is the generation of 3,3-disubstituted indolines that are frequently encountered in indole cores. The discovery, optimization, and scope of a C3-quaternary indolenine synthesis, as well as, efforts to expand the methodology for the generation of higher order indole-containing polycycles, will be discussed. This novel reactivity also lead to a generalized synthesis of ?,?-unsaturated N-aryl ketonitrones which has few literature examples. A modest start to realizing the success of 3-substituted non-N-protected indoles as a Michael donor will also be explored. Green chemistry continues to play an important role in creating a sustainable world. At the core of green chemistry is the reduction or elimination of the use or generation of hazardous substances. Catalysis by definition is green by reduction; however, many of the catalytic systems utilize toxic metals that can hamper or cause further concerns with allowable limits on industrial scales. Iron catalyzed reactions seek to replace these toxic metals with a benign one that is also relatively cheap. Nitrogen containing compounds are an important feed stock for the pharmaceutical and other industries. The iron catalyzed intermolecular hydroamination of alkenes with electrophilic amines will be discussed.Item Systems Biology of Microbiota Metabolites and Adipocyte Transcription Factor Network(2013-07-17) Choi, KyungohThe overall goal of this research is to understand roles of gut microbiota metabolites and adipocyte transcription factor (TF) network in health and disease by developing systematic analysis methods. As microbiota can perform diverse biotransformation reactions, the spectrum of metabolites present in the gastrointestinal (GI) tract is extremely complex but only a handful of bioactive microbiota metabolites have been identified. We developed a metabolomics workflow that integrates in silico discovery with targeted mass spectrometry. A computational pathway analysis where microbiota metabolisms are modeled as a single metabolic network is utilized to predict a focused set of targets for multiple reaction monitoring (MRM) analysis. We validated our methodology by predicting, quantifying in murine cecum and feces and characterizing tryptophan (TRP)-derived metabolites as ligands for the aryl hydrocarbon receptor. The adipocyte process of lipid droplet accumulation and differentiation is regulated by multiple TFs that function together in a network. Although individual TF activation is previously reported, construction of an integrated network has been limited due to different measurement conditions. We developed an integrated network model of key TFs - PPAR?, C/EBP?, CREB, NFAT, FoxO1, and SREBP-1c - underlying adipocyte differentiation. A hypothetic model was determined based on literature, and stochastic simulation algorithm (SSA) was applied to simulate TF dynamics. TF activation profiles at different stages of differentiation were measured using 3T3-L1 reporter cell lines where binding of a TF to its DNA binding element drives expression of the Gaussia luciferase gene. Reaction trajectories calculated by SSA showed good agreement with experimental measurement. The TF model was further validated by perturbing dynamics of CREB using forskolin, and comparing the predicted response with experimental data. We studied the molecular recognition mechanism underlying anti-inflammatory function of a bacterial metabolite, indole in DC2.4 cells. The indole treatment attenuated the fraction of cells that were producing the pro-inflammatory cytokine, TNF? and knockdown of nuclear receptor related 1 (Nurr1; NR4A2) resulted in less indole-derived suppression of TNF? production. The first discovery of NR4A2 as a molecular mediator of the endogenous metabolite, indole is expected to provide a new strategy for treatment of inflammatory disorders.