Analysis of the human endothelial cell response to Borrelia burgdorferi wild-type and deleted in the gene encoding the integrin ligand P66 [experiment B]. Homo sapiens

The overall goal of these experiments was to determine how human cells respond to ontegrin binding by Borrelia burgdorferi. Lyme disease is the most common vector-borne infection in the Northern hemisphere, yet little is understood about the pathogenic mechanisms of the causative agent, Borrelia burgdorferi. Cells in culture were infected with the bacteria that do and do not express the beta3 integrin ligand P66, which are otherwise isogenic. As additional controls, uninfected cells were also included in the analyses. Overall design: The cell line used for primary data analyses provided here was Ea.hy926, a macrovascular line (Edgell, C. J.,et al. 1990. Endothelium specific Weibel-Palade bodies in a continuous human cell line, EA.hy926. In vitro Cell. & Dev. Biol. 26:1167-1172, and Edgell, C. J., et al. 1983. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc. Natl. Acad. Sci. 80:3734-3737). Epithelial cells HEK-293 (which does not express b3 integrins) and HEK-293 transfected to express integrin αvβ3 were also analyzed. Infection times were 1 hour or 3 hours for cells lifted with EDTA and washed prior to bacterial infection. RNA was harvested and reverse transcribed. Labeled cDNAs were used to probe 2 color arrays. In each of at least three biological replicate experiments, for each time point, three comparisons were made. First, cells infected with B. burgdorferi with a marker inserted adjacent to the p66 gene (p66+) were compared to the cells infected with B. burgdorferi with the p66 gene interrupted by the marker (Coburn, J. and Cugini, C. (2003) Targeted mutation of P66 of Borrelia burgdorferi disrupts attachment to integrin αvβ3. Proc. Natl. Acad. Sci. 100:7301-7306). Second, the p66+-infected cells were compared to the uninfected cells. Third, the p66- mutant-infected cells were compared to the uninfected cells. Arrays used were generated at the Tufts University core faculity using Operon's human v2.1.2 plus v2.1.4 cDNA library (Operon Technologies, Inc., Alameda, CA). Only data generated from spots that in all three experiments were of sufficient quality to allow interpretation are included. IEa.hy926 endothelial cell lines, or HEK-293 epithelial cells, or transfected HEK-293 derivative expressing integrin avb3