Treponema denticola Msp‐deduced peptide conjugate, P34_BSA, promotes RhoA‐dependent actin stress fiber formation independent of its internalization by fibroblasts

Abstract

P34(BSA), a BSA conjugate of a synthetic 10-mer peptide deduced from Treponema denticola major outer sheath protein (Msp), stabilizes actin filaments in fibroblasts and retards cell motility. We reported previously that it is internalized by cells, binds and bundles actin filaments in vitro, and activates RhoA; yet, its site and mechanism of action were not defined. We have assessed P34(BSA)'s modes of interaction with and signaling to fibroblasts. At 4 degrees C, P34(BSA) was not internalized, but it bound to the plasma membrane and promoted actin stress fiber formation at approximately 80% capacity compared with 37 degrees C controls, casting doubt that cellular uptake is a critical step for its cytoskeleton-stabilizing property. In Rho G-LISA and co-immunoprecipitation assays, P34(BSA) was found to activate RhoA, even at 4 degrees C, to promote its interaction with guanosine nucleotide exchange factor p114RhoGEF. It also caused phosphorylation of cofilin. Upon RhoA inhibition, either by C3 transferase RhoA inhibitor or by transfection with a dominant negative RhoA construct, P34(BSA) did not achieve the stress fiber formation seen with P34(BSA) alone. By inhibiting phosphatidylinositol-3 kinase (PI 3-K) with LY294002, the P34(BSA) effects were completely blocked. Depletion of cholesterol with methyl-beta-cyclodextrin (MbetaCD) partially inhibited P34(BSA) signaling via the plasma membrane to the cytoskeleton. This suggests that multivalent P34(BSA) activation of lipid raft components requires active PI 3-K, and initiates the pathway through a RhoGEF and RhoA, which mediates stress fiber formation in fibroblasts. Hence, P34(BSA) may represent a novel tool to investigate RhoA-dependent processes, such as remodeling filamentous actin in eukaryotic cells.

The Major Outer Membrane Protein of a Periodontopathogen Induces IFN-β and IFN-Stimulated Genes in Monocytes via Lipid Raft and TANK-Binding Kinase 1/IFN Regulatory Factor-3

“…Cholesterol depletion by M␤CD suppressed the ability of P. gingivalis for intracellular persistence and cytokine induction in mouse macrophages (33) and abolished actin polymerization induced by P. gingivalis OM vesicles (32). Lipid raft-dependent internalization was also observed with Treponema denticola Mspdeduced peptide conjugate, P34BSA, which induced actin stress fiber formation through PI3K and RhoA activation, although lipid raft activation by P34BSA, but not lipid raft-dependent internalization, is linked to PI3K and RhoA activation (34).…”

The Effect of First-in-Class Small Molecule RhoA Inhibitor, HL07, on the Phenylephrine-induced Artery Contraction

“…In the study, RhoA activation was determined by the RhoA G-LISA TM kit, the protocol has been validated by previous studies [17,18]. It can accurately and reproducibly measure the change of GTPbound RhoA in HBVSMCs.…”

Treponema denticolainteractions with host proteins

“…Studies by Ellen and coworkers have demonstrated that Msp disrupts the actin cytoskeleton of gingival fibroblasts, with specific effects, including disruption of actin stress fibers (45) and induction of de novo synthesis of subcortical actin filaments (35, 37, 46, 47). A specific peptide sequence within Msp has been shown to promote stress fiber formation (48, 49). Concurrently, and presumably related to this phenomenon, Msp blocks the ability of fibroblasts to regulate intracellular calcium gradients (50), which may contribute to the rather diverse range of morphological responses observed.…”