Treponema denticola Msp Redistributes Focal Adhesion Proteins in Migrating Fibroblasts

Introduction: Connective tissue healing depends on the ability of fibroblasts to migrate through the extracellular matrix (ECM) and to phagocytose collagen. We reported previously that the major outer sheath protein (Msp) of Treponema denticola (Td) induces cytoskeletal reorganization and diminishes collagen-binding affinity of β1 integrins in fibroblasts. Consequently, Msp may affect focal adhesion actin-binding proteins that are known to complex with integrins. Objective: to determine the effect of Td Msp on the expression and localization of α-actinin, talin and vinculin in fibroblasts migrating to close a wound in cell monolayers. Methods: Msp was prepared from Td 35405. Confluent Rat-2 fibroblasts were pretreated with 20 μg/ml Msp or Msp-free α-MEM vehicle control for 1h. An incision was created in the monolayer using a sterile needle. The cells were incubated for different intervals up to 12 h. Cells were permeabilized, then stained with alexa 488 phalloidin for actin filaments and by indirect immunofluorescence with an alexa 594 conjugate for the actin-binding proteins. Protein localization was recorded in merged images. Fluorescent pixel intensity was measured among cells along the wound edge. Results: There was a significant delay in wound closure (P < 0.05). At early times, Msp-pretreated cells had altered shape and loss of focal distribution of vinculin and talin. Yet, mean pixel intensity along the wound edge was significantly lower in Msp-treated than control cells only for vinculin at 2h (% difference = 37%; P = 0.003). There were no significant differences for vinculin beyond 2h, or for talin (P = 0.053) and α-actinin (P = 0.180) at any time. Conclusions: Exposure of Rat-2 fibroblasts to T. denticola Msp delays cell migration to close wounds. Redistribution of actin-binding proteins, like vinculin or talin, away from discrete foci may contribute to Msp-induced reduction of collagen-binding affinity of integrins. Supported by CIHR MGP-5619