Abstract
The major outer sheath protein (Msp) of Treponema denticola inhibits neutrophil polarization and directed chemotaxis together with actin dynamics in vitro in response to the chemoattractant N-formyl-methionine-leucine-phenylanine (fMLP). Msp disorients chemotaxis through inhibition of a Rac1-dependent signaling pathway, but the upstream mechanisms are unknown. We challenged murine bone marrow neutrophils with enriched native Msp to determine the role of phospholipid modifying enzymes in chemotaxis and actin assembly downstream of fMLP-stimulation. Msp modulated cellular phosphoinositide levels through inhibition of phosphatidylinositol 3-kinase (PI3-kinase) together with activation of the lipid phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Impaired phosphatidylinositol[(3,4,5)]-triphosphate (PIP3) levels prevented recruitment and activation of the downstream mediator Akt. Release of the actin capping proteins gelsolin and CapZ in response to fMLP was also inhibited by Msp exposure. Chemical inhibition of PTEN restored PIP3 signaling, as measured by Akt activation, Rac1 activation, actin uncapping, neutrophil polarization and chemotaxis in response to fMLP-stimulation, even in the presence of Msp. Transduction with active Rac1 also restored fMLP-mediated actin uncapping, suggesting that Msp acts at the level of PIP3 in the hierarchical feedback loop of PIP3 and Rac1 activation. Taken together, Msp alters the phosphoinositide balance in neutrophils, impairing the cell “compass”, which leads to inhibition of downstream chemotactic events.
Highlights
Neutrophils are key rapid-response cells of the innate immune system that are recruited to sites of infection to eradicate pathogens [1]
major outer sheath protein (Msp) treatment alone increased PTEN activity by approximately 40%. Together these results indicate that Msp is able to stimulate PTEN activity in neutrophils and that exposure to Msp leads to PIP3 to PIP2 conversion in both resting and fMLP-activated cells
Signaling and regulatory pathways that include plasma membrane-associated phosphoinositides, lipid kinases, and lipid phosphatases are crucial for determining the actin dynamics, polarization, and directional migration events that drive neutrophil chemotaxis
Summary
Neutrophils are key rapid-response cells of the innate immune system that are recruited to sites of infection to eradicate pathogens [1]. Neutrophils migrate in a directed manner in response to chemoattractants such as cytokines, complement peptides, bacterial products and chemicals including peptides bearing the N-formyl group (N-formyl-methionine-leucine-phenylanine, fMLP). Key to chemotaxis is the ability of neutrophils to polarize, with formation of a leading edge (lamellipodium) toward the chemoattractant and a trailing edge (uropod). Movement of the cell is mediated by dynamic protrusion and retraction of the lamellipodium and uropod, respectively, driven by pathways that regulate remodeling of the actin cytoskeleton [2,3]. Polarization and chemotaxis require asymetrical distribution of molecules within the cell; including the accumulation of phosphatidylinositol[(3,4,5)]-triphosphate (PtdIns [(3,4,5)]P3, PIP3) at the leading edge [4,5]. Proper spatial localization and accumulation of PIP3 has been proposed to act as an internal cellular ‘‘compass’’, leading to efficient directional migration [6,7,8]
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