Phosphoinositide 3-Kinase Is Required for Intracellular Listeria monocytogenes Actin-based Motility and Filopod Formation

Abstract

Motile nonmuscle cells concentrate phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) in areas of new actin filament assembly. There is great interest in assessing the in vivo functional significance of these phosphoinositides, and we have used Listeria monocytogenes to explore the contribution of PtdIns(3,4,5)P 3 and PtdIns(4,5)P 2 to its actin-based motility. In Listeria-infected PtK2 cells Aktpleckstrin homology (PH)-green fluorescent protein (GFP) and phospholipase C␦ (PLC␦)-PH-GFP both first concentrate at the front of motile Listeria, subsequently surrounding the bacterium and then concentrating in the actin filament tail. Surprisingly, Listeria ActA mutant strains lacking the putative phosphoinositide binding site are also able to concentrate these probes. Reduction of available PtdIns(3,4,5)P 3 by expression of Akt-PH-GFP and available PtdIns(4,5)P 2 by expression of PLC␦-PH-GFP both significantly slow Listeria actin-based movement. Treatment of cells with the PI 3-kinase inhibitor, LY294002, dissociates Akt-PH but not PLC␦-PH, from the bacterial surface and cell membranes, and results in near complete inhibition of Listeria actin-based motility and filopod formation. Removal of LY294002 results in rapid and full recovery of Akt-PH localization, Listeria actin-based motility, and filopod formation. These findings suggest that PtdIns(4,5)P 2 is concentrated at the surface of Listeria and serves as the substrate for PtdIns(3,4,5)P 3 production, indicating a central role for PI 3-kinases in Listeria intracellular actinbased motility and filopod formation.The onset of actin-based cell motility in nonmuscle cells is signaled by the increased turnover of phosphoinositides, and phosphoinositides are known to interact with and regulate multiple actin-binding proteins, kinases, and guanine nucleotide exchange factors (1, 2). Following exposure to various agonists that stimulate cell migration, plasma membrane PtdIns(4,5)P 2 1 is converted to PtdIns(3,4,5)P 3 , which is then concentrated at the leading edge of the cell membrane. The accumulation of PtdIns(3,4,5)P 3 at these sites is associated with the formation of new actin filaments (3). The mechanisms by which these lipid products induce actin assembly are under active investigation and include the uncapping of actin filaments and stimulation of actin nucleation (2). The shape changes associated with chemotaxis, cell spreading, and phagocytosis are complex and make analysis of rates and directionality of actin assembly difficult. Furthermore, these events involve several steps of receptor-mediated signal transduction. Listeria intracellular infection and movement is a particularly useful model for examining the parameters of in vivo actin assembly, allowing discrete temporal and spatial resolution of actin filament formation. Intracellular Listeria infection of tissue culture cells is accomplished by simply overlaying the bacteria on the surface of host cells. Internalins found on the surface...

LipA, a Tyrosine and Lipid Phosphatase Involved in the Virulence of Listeria monocytogenes

“…During ActA-dependent movement within the cytoplasm, Listeria bacteria accumulate PI(3,4,5)P 3 around their surface (51). The host Akt kinase contains a PH domain that binds PI(3,4)P 2 and PI(3,4,5)P 3 , and a fusion protein of GFP linked to the PH domain of Akt has been reported to concentrate on moving Listeria and the actin-rich tails (51).…”

“…The host Akt kinase contains a PH domain that binds PI(3,4)P 2 and PI(3,4,5)P 3 , and a fusion protein of GFP linked to the PH domain of Akt has been reported to concentrate on moving Listeria and the actin-rich tails (51). We used this construct to investigate Akt-PH recruitment to L. monocytogenes wt and ⌬lipA bacteria.…”

“…Localization of Aktpleckstrin homology-GFP (Akt-PH-GFP) in infected Ptk-2 cells was performed as described previously (51). Ptk-2 cells (3 ϫ 10 6 ) were seeded on coverslips in six-well dishes and incubated for 40 to 48 h. Cells were transfected with 1.2 g plasmid encoding Akt-PH-GFP, using 10 g/ml Lipofectamine reagent (Invitrogen) and 11 g/ml Plus reagent (Invitrogen) for 6 h. Forty-eight hours after transfection, cells were infected.…”

Gelsolin mediates calcium-dependent disassembly ofListeriaactin tails

“…How is Listeria accomplishing this task? Intracellular Listeria is able to attract both PI(4,5)P 2 and PI(3,4,5)P 3 (44), raising the possibility that these phospholipids could serve as substrates for byproducts that locally release intracellular calcium stores. Alternatively, these phosphoinositides could serve as docking sites for a kinase or kinases that activate gelsolin.…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…Examples include the entry of bacteria such as Listeria (1226,1548), Yersinia (1340), enterohepatic E. coli (1348), Salmonella (1544), or viruses such as hepatitis C (1577), HIV (1069), or influenza (472), just to name a few from an ever-increasing list. Another role of PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 has been reported in filopod formation, actin polymerization, and movement of Listeria within the infected cells (1405). PtdIns4P is also utilized by several pathogens to shape their intracellular milieu.…”