(489al) A Secreted Factor From Human Intestinal Epithelial Cells Increases Enterohemorrhagic Escherichia Coli Virulence

Introduction of pathogens such as enterohemorrhagic Escherichia coli (EHEC) disturbs the homeostasis in human gastro-intestinal (GI) tract and rapidly leads to colonization and infection. Work in our laboratory has focused on investigating intra- and inter-kingdom signaling in host cells, commensal bacteria, and the pathogen EHEC. The objective of this study was to identify the mechanisms through which EHEC recognizes host cell environment. We hypothesized that EHEC cells recognizes specific molecule(s) in the GI tract microenvironment to activate its virulence machinery in vivo. We have previously shown that human hormones such as norepinephrine can be opportunistically utilized by EHEC for infection. Here we investigated the role of intestinal epithelial cell-secreted molecules in promoting EHEC virulence and infection. Gene expression studies with EHEC exposed to spent intestinal epithelial cell medium significantly increased the expression of 34 of 41 virulence genes belonging to the LEE pathogenicity island, and also of 22 genes involved in cell division and adhesion. Several genes involved in the uptake of the quorum sensing signal autoinducer-2 (AI-2) that increases EHEC virulence, and genes involved in iron uptake and metabolism were also up-regulated. These observations were corroborated by in vitro attachment assays where spent medium caused a 5-fold increase in EHEC attachment to HCT-8 cells. Through further experiments, we have shown that the secreted molecule(s) that contribute to increased EHEC virulence are protein-based and are larger than 3 kDa (i.e., not small peptides). Current work focuses on size-exclusion chromatography and mass spectrometry to identify the molecule(s) recognized by EHEC to increase its virulence. Our results provide the basis of an inter-kingdom signaling-based framework where recognition of host cell molecules by pathogens initiates infection in the GI tract.