The 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-κB, 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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2010-08-8177 |
| Date | 2010 August 1900 |
| Creators | Bansal, Tarun |
| Contributors | Jayaraman, Arul, Wood, Thomas K. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | thesis, text |
| Format | application/pdf |
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