Treponema denticola Major Outer Sheath Protein Inhibits Collagen Binding to Fibroblasts

Phagocytic processes are driven by a finely controlled rearrangement of the actin cytoskeleton. The major outer sheath protein (Msp) of T. denticola (Td) induces actin reorganization in human gingival fibroblasts (HGF), typified by disruption of stress fibers and assembly of filaments subjacent to the plasma membrane. Objective: to determine the effect of Td Msp on the binding step of collagen phagocytosis by HGF. Methods: Msp was prepared from Td 35405; HGFs and Rat-2 fibroblasts were pretreated with Msp (different doses and times), and incubated with collagen-coated fluorescent latex beads. The binding step of phagocytosis was measured by flow cytometry by analyzing the percentage of cells that bound fluorescent beads. Results: Exposure of fibroblasts to Msp led to a significant decrease in collagen bead binding. Rat-2 cells were equivalent to HGF in terms of Msp-mediated effects on collagen binding. The inhibition induced by Msp on the binding step of collagen phagocytosis was dose- and duration- dependent and due to a heat-labile molecule. The Msp effect was reversible, but not completely, within 4 h after washout of the Msp. Fibroblasts remained viable (excluded propidium iodide) after Msp treatment. Msp-treated cells lost stress fibers and formed brightly staining subcortical actin filaments near the plasma membrane. Bead wash-out experiments established that Msp’s reduction in the number of bound beads was due primarily to rapid loss of loosely bound beads. Cell spreading on collagen-coated dishes was also inhibited by Msp. Conclusion: Exposure of HGF and Rat2 fibroblasts to the Msp of T. denticola inhibits collagen binding. The effect may be due to a rearrangement of actin filaments in the subcortical area of the cell, which may decrease receptor mobility. Supported by CIHR grants MOP 5619, MOP 36332, CIHR Group Grant, and CIHR Strategic Training Grant.