Browsing by Subject "AI-2"
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Item Influence of autoinducer 2 (ai-2) and ai-2-like inhibitors generated from ground beef on escherichia coli o157:h7 protein expression(2009-05-15) Soni, Kamleshkumar A.Autoinducer 2 (AI-2) molecules produced by bacterial cells are thought to be involved in controlling a variety of bacterial cellular processes by coordinated gene and protein expression. Previous work in our laboratory has shown that ground beef contains compounds that can interfere with AI-2-mediated bioluminescence expression in Vibrio. harveyi. The underlying hypothesis of this work was that AI-2 molecules affect the protein expression in Escherichia coli O157:H7 and AI-2 inhibitory molecules negate the influence of AI-2 molecules. The main objectives of this study were to identify, characterize, and isolate the factors responsible for inhibition of AI-2 molecules from ground beef extracts, elucidate the role of LuxS/AI-2 cell signaling system in E. coli O157:H7 protein expression, and determine if inhibitory factors present in ground beef extract can negate the influence of AI-2 molecules on the protein expression. Using a solvent extraction procedure and gas chromatography analysis, AI-2 inhibitory factors present in ground beef extracts were identified as both medium and long chain fatty acids. When identified fatty acids were tested at different concentrations for AI-2 inhibition, AI-2 inhibition ranging from 25% to 90% was observed. Both ground beef extracts and mixture of selected fatty acids also resulted in 2- to 4-fold reduced AI-2 influenced biofilm formation by E. coli K12 cells. Identification of LuxS/AI-2-mediated protein expression in E. coli O157:H7 was conducted using two dimensional gel electrophoresis. Protein expression analysis showed that the LuxS/AI-2 system modulates the expression of proteins involved in different cellular processes such as carbohydrate and amino acid metabolism, stress response, and formation of flagella and motility. When AI-2 inhibitory factors were added along with AI-2 molecules, the expression patterns of three AI-2-influenced proteins (GlmS, SpeE, and NikA) were changed suggesting that AI-2 inhibitors can negate the influence of AI-2 molecules on protein expression of selected proteins. Collectively, these results highlight that proteins associated with different cellular processes in E. coli O157:H7 can be modulated depending on whether cells are in contact with AI-2 molecules in the presence or absence of AI-2 inhibitory factors.Item 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 Role of AI-2 in oral biofilm formation using microfluidic devices(2009-05-15) Kim, Sun HoBiofilms are highly organized bacterial structures that are attached to a surface. They are ubiquitous in nature and may be detrimental, causing numerous types of illnesses in living organisms. Biofilms in the human oral cavity are the main cause of dental caries and periodontal diseases and can act as a source for pathogenic organisms to spread within the body and cause various types of systemic diseases. Streptococcus mutans is the primary etiological agent of dental caries, the single most chronic childhood disease. In many cases, quorum sensing (QS) is required for initial formation and subsequent development of biofilms and the signaling molecule autoinducer 2 (AI- 2) has been well studied as an inter-species QS signaling molecule. However, recent reports also suggest that AI-2-mediated signaling is important for intra-species biofilm formation in both Gram-negative and positive bacteria. Therefore, there is significant interest in understanding the role of different QS signals such as AI-2 in oral biofilm formation. Microfluidic devices provide biomimetic environments and offer a simple method for executing multiple stimuli experiments simultaneously, thus, can be an extremely powerful tool in the study of QS in biofilms. In this study, we report conditions that support the development of S. mutans biofilms in microchannel microfluidic devices, and the effects of extracellular addition of chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione (DPD; precursor of AI-2) on mono-species S. mutans luxS (AI-2 deficient strain) biofilm formation using a gradient generating microfluidic device. S. mutans wild type (WT) and luxS biofilms were developed in nutrient rich medium (25% brain heart infusion medium, BHI + 1% sucrose) for up to 48 h. Maximum biofilm formation with both strains was observed after 24 h, with distinct structure and organization. No changes in S. mutans luxS biofilm growth or structure were observed upon exposure to different concentrations of AI-2 in a gradient generating device (0 to 5 M). These results were also validated by using a standard 96-well plate assay and by verifying the uptake of AI-2 by S. mutans luxS. Our data suggest that extracellular addition of AI-2 does not complement the luxS deletion in S. mutans with respect to biofilm formation.