Role of GroEL in pathological angiogenesis during bartonellosis

University Of Montana, Missoula MT

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Abstract

? DESCRIPTION (provided by applicant): Three Bartonella species cause pathological angiogenesis and tumorigenesis during infection of humans; disease manifestations that are extremely unusual for bacterial pathogens. Our long-term goals are to elucidate the cellular and molecular basis for aberrant neovascularization and tumor formation during bartonelloses. Our preliminary studies have shown that the GroEL (chaperonin 60) protein of Bartonella bacilliformis (Bb) is a potent mitogen for human vascular endothelial cells. Bacterial GroEL's have long been recognized as potent immunogens and effectors that can modulate growth, activation states, apoptosis, cytokine production and migration of eukaryotic cells. We hypothesize that Bb GroEL, in addition to being mitogenic, induces expression of cytokines and cell-surface adhesion molecules, thereby influencing migratory behavior and stimulating morphogenesis in cells of the vasculature. These hypotheses will be addressed in three specific aims. In aim 1, we will dissect the mechanism of Bb GroEL's mitogenic activity by mapping its functional domains using Gateway technology and deletional mutagenesis. A second goal is to determine if GroES is mitogenic and/or if it enhances GroEL's activity, as determined by in vitro cell-growth assays. Results of aim 1 will increase our understanding of GroEL's mechanism and will elucidate the role of the GroES co- chaperonin. In aim 2, we will analyze pro-inflammatory cytokines, chemokines and angiogenic factors (IL-1?, IL-8, angiopoietin-2, GRO-?, etc.) produced by human umbilical vein endothelial cells (HUVECs) and monocytic (THP-1) cells in response to a Bb infection and Bb GroEL using ELISAs. The activation state of NF-?B during a Bb infection and Bb GroEL stimulation will be determined by a NF-?B reporter system in host cells and a colorimetric ELISA. Results of aim 2 will elucidate the inflammatory cytokines and angiogenic growth factors involved in the pathological angiogenesis that occurs during bartonellosis. In addition, the results will allow us to formulate a working model of how the process is regulated, and it will provide a means of comparison with other bacterium-induced vascular pathologies. In aim 3, we will determine whether Bb GroEL elicits proangiogenic cellular effects on HUVECs. First, the migratory behavior of stimulated HUVECs will be examined using a Transwell cell culture system and microscopy. Upregulation of cell-surface adhesion molecules (E-selectin, VCAM-1 and ICAM-1) will also be followed in Bb GroEL-stimulated HUVECs by FACS analysis. Finally, formation of capillary-like tube formation by stimulated HUVECs grown in matrigel will be assessed microscopically. Results of aim 3 will shed light on HUVEC morphogenesis, migration and altered cell-surface adhesion profiles in response to Bb GroEL; all of which could contribute to neovascularization. We believe this system provides an exceptional model for studying vascular pathologies that can manifest during chronic infections by Helicobacter pylori (gastrointestinal ulcers, cancer), Chlamydia (possibly cancers of the female genital tract, atherosclerosis) and other Bartonella (bacillary angiomatosis and peliosis during AIDS).