The major outer sheath protein (Msp) is the most prominent surface antigen of the periodontal pathogen Treponema denticola. It mediates adhesion to extracellular matrix and dysregulation of cytoskeletal homeostasis of host cells. Disassembly of actin filaments and the coincident subcortical de novo synthesis of actin filaments in fibroblasts upon exposure to Msp were investigated with a barbed-end fluorescent labeling method. The functional impact of actin cytoskeleton disorganization was determined with a scratch wound migration assay in fibroblast monolayers and a videomicroscopy migration assay in neutrophils. Msp pretreatment had a significant inhibitory effect on the migration of the fibroblasts across a collagen substratum and inhibited the neutrophil chemotactic migration towards a chemoattractant. In a study originally aimed to find the biologically active domains of Msp that may perturb actin, short peptides were selected from the deduced and predicted surface exposed regions of Msp and investigated for their role in actin dynamics and cell motility. A novel BSA-conjugated peptide (P34BSA) was found serendipitously to induce stress fiber formation and stability in fibroblasts. This activity was found to be mediated by Rho activation and cofilin phosphorylation, which are important tandem signaling pathways in the regulation of a variety of actin-dependent cellular functions. P34BSA was internalized by the cells. Yet, a mechanistic study using low-temperature treatments and depletion of cholesterol with methyl-β-cyclodextrin (MβCD) revealed that P34BSA most likely induces actin stress fiber formation extracellularly through a Rho-dependent signaling pathway. P34BSA induced Rho activation via binding of guanosine nucleotide exchange factor p114RhoGEF to RhoA, one of many exchange factors that have been shown to play a role in activating Rho signaling. Pretreatment with P34BSA partially protected the fibroblasts against the actin-disrupting effects of cytochalasin D and latrunculin B, and the cells maintained most of their actin filaments. P34BSA treatment caused retardation of fibroblast migration on a collagen substratum. It also inhibited the chemotactic movement of neutrophils towards the chemoattractant fMLP.
P34 may represent a novel amino acid sequence of a bacterial virulence protein that, when conjugated to BSA, can be used as a chemical reagent to investigate RhoA signaling pathways in host cells.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/17724 |
| Date | 23 September 2009 |
| Creators | Amin, Mohsen |
| Contributors | Ellen, Richard P. |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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