Listeria monocytogenes infection of P388D1 macrophages results in a biphasic NF-kB (RelAyp50) activation induced by lipoteichoic acid and bacterial phospholipases and mediated by IkBa and IkBb degradation

As previously reported, Listeria monocytogenes infection of P388D1 macrophages results in a rapid induction of NF-kB DNA-binding activity. Here we show that this induction of NF-kB activity occurs in a biphasic mode: first, a transient, IkBa degradation-dependent phase of activity, also induced by the nonvirulent species Listeria innocua, which is mediated by binding of the bacteria to the macrophage, or by adding Listeria-derived lipoteichoic acid to the macrophage; the second persistent phase of activation is only markedly induced when the bacteria enter the cytoplasm of the host cell and express the virulence genes plcA and plcB, encoding two phospholipases. We suggest that products of the enzymatic activity of phospholipases directly interfere with host cell signal transduction pathways, thus leading to persistent NF-kB activation via persistent IkBb degradation. The transcription factor NF-kB, a pleiotropic mediator of inducible and tissue-specific gene control, is involved in the transcription of a variety of genes, many of which are activated during the immune response. NF-kB belongs to the Rel family of transcriptional activator proteins. The major form of this transcription factor is a heterodimer composed of a 50-kDa (NF-kB1 or p50) protein and a 65-kDa RelA DNA-binding protein, which is sequestered in the cytoplasm by association with inhibitory proteins of the IkB family (1, 2). The most intensively studied inhibitor of the Rel protein dimers is IkBa. Stimulation of cells with different inducers lead to hyperphosphorylation of IkBa, which in turn results in polyubiquitination. Subsequent degradation of IkBa by the proteasome allows NF-kB to be released from this inactive complex (3–6). Recently it was shown that IkBa and IkBb are mediators of either transient (IkBa) or persistent (IkBb) NF-kB activation in response to stimulators like tumor necrosis factor a and lipopolysaccharide, respectively (7). Free NF-kB enters the nucleus, where it binds to its target sequences and activates transcription (1). Listeria monocytogenes, a Gram-positive bacterium and a causative agent of food-borne septicemia and meningitis, has been widely used as a model system of facultative intracellular bacteria (8, 9). L. monocytogenes can invade, survive, and replicate within nonprofessional phagocytic cells as well as in macrophages of different origin. It has been conclusively shown that the extracellular protein listeriolysin O (LLO) is absolutely required for intracellular survival and replication (10, 11). The gene encoding LLO (12) is part of a gene cluster which includes genes encoding a phosphatidylinositol-specific phospholipase C (plcA; PI-PLC) (13, 14), a metalloprotease (mpl) (15, 16), a protein necessary for induction of actin polymerization (actA) (17, 18), and a broad spectrum specificity phospholipase called phosphatidylcholinespecific phospholipase C (plcB; PC-PLC) (19). These genes, as well as the internalin family genes (inlA, inlB, and inlC) (20, 21), which are not part of the virulence gene cluster are under the control of the transcriptional activator PrfA encoded by the prfA gene (22). Recently it was shown that heat-killed Staphylococcus aureus, invasive Shigella flexneri, and pneumococcal cell walls induce NF-kB (RelAyp50)-like activities in murine macrophages, HeLa cells, and human macrophages, respectively (23–25). We have reported on the rapid generation of nuclear NF-kB complexes in the macrophage-like cell line P388D1 after infection with a virulent L. monocytogenes strain (26). In the present study we show that L. monocytogenes infection of P388D1 macrophages results in a biphasic NF-kB activation in which the first transient phase is induced by lipoteichoic acid (LTA) and paralleled by IkBa decrease. The second persistent phase is mediated by the expression of the PrfA-dependent listerial phospholipases PIPLC and PC-PLC and is paralleled by IkBb degradation. MATERIALS AND METHODS Bacteria, Mammalian Cell Culture, and Infection. Listeria used for cell culture infections were grown in brain heart infusion broth (Difco) at 37°C with aeration. Mid-log phase cultures were washed twice in PBS and stored at 280°C. The bacterial strains used for infection are shown in Table 1. P388D1 macrophages were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine (all from GIBCO). Macrophages were seeded 48 h prior to infection in tissue culture plates (Greiner, Nurtingen, Germany) at 5 3 106 cellsyplate. Twenty-four hours before infection the medium containing only 0.5% fetal calf serum was added. The macrophages were infected with Listeria to give a multiplicity of infection of 50 bacteria per eukaryotic cell and incubated for 40 min. They were washed with PBS and incubated further in medium containing gentamicin (50 mgyml) to kill extracellular bacteria and prevent reinfection. Preparation of Cytoplasmic and Nuclear Protein Extracts. At the indicated time points following infection, cytoplasmic and nuclear protein extracts were prepared essentially as described (26, 27). The washed macrophages were scraped from the culture plate and collected by centrifugation. Pellets were resuspended in 0.4 ml of hypotonic lysis buffer containing 10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). After 15 min on ice, Nonidet P-40 was added to give a final concentration of 0.5%, and the cells were vortex mixed for 10 sec. The cells were centrifuged (30 sec, 4°C, 12,000 3 g). The supernatant (5cytoplasmic extract) was frozen immediately and the nuclear pellets The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y949394-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: EMSA, electrophoretic mobility shift assay; LTA, lipoteichoic acid; pi, postinfection; PC-PLC, phosphatidylcholinespecific phospholipase C; PI-PLC, phosphatidylinositol-specific phospholipase C; LLO, listeriolysin O. ‡To whom reprint requests should be addressed.