Germany). Cycloheximide was obtained from Calbiochem (Darmstadt, Germany). IFNc was purchased from Strathmann Biotec Gmbh. (Hamburg, Germany). Bafilomycin A1 (Baf A1) was purchased from Calbiochem. Rapamycin (Rapa) was obtained from Sirolimus LC Laboratories (Massachusetts, USA). Goat antibodies raised against mouse Irgb6 (clone A-20), Irgm2 (clone L-15), Irgm3 (clone Y-16), and Irgm1 (clone A-19), in addition to the rabbit antibody against mouse Irgd (clone M-85), were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Other serological reagents used were: monoclonal anti- mouse Irga6 antibody raised in mice (clone 10D7 [46]), rat anti- mouse lysosomal associated membrane protein 1 (LAMP1) (clone 1D4B; Abcam, Cambridge, UK), monoclonal antibody to light chain 3 (LC3) (clone 5F10; purchased from nanoTools, Teningen, Germany), mouse monoclonal anti-human b-actin (Sigma-Al- drich), rabbit polyclonal anti-Chlamydia genus-specific antibody (Milan Analytica AG, La Roche, Switzerland), and mouse monoclonal anti-C. trachomatis hsp60 (Alexis, Loerrach, Germany) and LPS (clone CF6J12, Abcam, Cambridge, UK). The IMAGEN kit for detection of Chlamydia was from DAKO (Hamburg, Germany). Secondary labeled antibodies for immunofluorescence and Western analyses were purchased from Molecular Probes- MoBiTech (Goettingen, Germany), Dianova (Hamburg, Ger- many), BD Biosciences-Pharmingen (Heidelberg, Germany), or Amersham (NJ, USA). Chlamydia trachomatis and C. muridarum propagation and murine cell cultures C. trachomatis Lymphogranuloma venereum (LGV) serovar L2 and C. muridarum, a generous gift from Konrad Sachse (Friedrich- Loeffler-Institut, Federal Research Institute for Animal Health, Jena, Germany) were routinely propagated in HeLa cells grown in RPMI-1640 medium supplemented with glutamine and 5% fetal bovine serum (FBS). Chlamydia culturing, preparation of EB stock, and estimation of inclusion forming units (IFU)/ml were conducted as previously described [47]. Wild type (WT) MEFs and autophagy-deficient (Atg5-knockout) MEFs were generously provided by Noburo Mizushima (Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo Japan). Irga6-lacking MEFs were generated in-house (see below). MEFs were grown in DMEM provided with 10% FBS, 200 mM glutamine and 1 mM sodium pyruvate, and incubated at 37uC and 5% CO2 in a humidified tissue culture chamber. Infection of MEFs MEFs were seeded onto 6-well-plates and incubated overnight to allow adherence. Host cells were then infected with either C. trachomatis or C. muridarum diluted in cell growth medium at an MOI of 1 and incubated at 35uC and 5% CO2. Two hours post infection (p.i.) cells were washed and loaded with fresh medium containing 100 or 200 U/ml IFNc. Control infected cells were not treated with the cytokine. Cell cultures were then incubated as above for 48 h. Infectivity titration assays Formation of infectious C. trachomatis and C. muridarum progeny in a secondary infection was assessed as described elsewhere [47]. Briefly, infected cells were mechanically destructed using glass beads. Lysates were serially diluted in IM and inoculated onto HeLa cells for 2 h. Cells were then washed and further incubated for 24 h in RPMI medium with 5% FBS and 1 mg/ml cycloheximide. Chlamydial inclusions were fixed with methanol and then immunostained with IMAGEN Chlamydia kit. Subse- Control of Ctr via Irga6 quently, inclusions were visualized and counted using immuno- fluorescence microscopy (Nikon), and infectivity of progeny was expressed as IFU/ml. Generation of Irga6-deficient MEFs MEFs were prepared as described previously [48]. Briefly, embryos generated from Irga62/26C57BL/6 and Irga62/ 26Irga62/2 crosses were isolated on day 13.5 of development. Placenta, membranes, visceral tissues and the head were removed from embryos and the remaining tissue was minced and trypsinized to produce single cells. Single cells were passaged twice in DMEM containing 10% FBS and then stored in liquid nitrogen for later use. Transfection of host cells MEFs were seeded onto coverslips in 12-well-plates and incubated overnight at 37uC and 5% CO2 to allow adherence. MEFs were then washed once and transfected with 1 mg/ml plasmid DNA encoding pEGFP-rat LC3 (a kind gift from Tamotsu Yoshimori, Department of Cellular Regulation, Re- search Institute for Microbial Diseases, Osaka University, Osaka, Japan), using Lipofectamine 2000 (Gibco-Invitrogen) according to manufacturer’s instructions. One day post-transfection, cells were stimulated for 24 h with 100 U/ml IFNc or left unstimulated (control cells) and incubated in a humidified cell culture incubator. Next, transfected cells were infected with C. trachomatis or C. muridarum at a MOI of 5 and incubated for time periods as indicated at 35uC and 5% CO2, before processing for confocal microscopy and Western blotting. For colocalization studies MEFs were stained 3 h p.i. (MOI 5), facilitating the detection of a large number of intracellular bacteria before eradication in response to IFNc. Fluorescence confocal microscopy MEFs were plated onto coverslips in 12-well-plates at a density of 26105 cells per well and incubated overnight. Next, cells were treated with 100 U/ml IFNc for 24 h before infection or left untreated as a control. MEFs were then inoculated with C. trachomatis at a MOI of 5 and incubated at 35uC and 5% CO2. Two hours p.i., cells were washed, provided with fresh medium with or without IFNc, and incubated as before. At indicated time points, cells were fixed and permeabilized with ice-cold methanol for 5 min at room temperature (RT). In some experiments, cells were fixed for 25 min with 4% paraformaldehyde at RT, permeabilized with either 0.2% saponin, 0.3% Triton-1006 or digitonin and blocked using 2.5% BSA. Cells were sequentially incubated for 1 h at RT with primary and secondary antibodies. Then, coverslips were mounted onto glass slides using Mowiol and examined by Leica TCS-SP laser scanning confocal microscope equipped with a krypton/argon laser. Photomicrographs were processed using Adobe Photoshop 6.0 (Adobe Systems) and Microsoft Power-Point. Immunoblotting MEFs were stimulated with IFNc, as indicated above, or with the classical autophagy inducer Rapa at a concentration of 100 nM for 2 h. In some experiments, unstimulated and IFNc- stimulated cells were infected with C. trachomatis or C. muridarum for either 3 or 8 h. Cells were washed with cold PBS, and lysed for 30 min on ice in Triton-1006 lysis buffer (20 mM Tris-HCl; pH 7.6, 150 mM NaCl, 1% Triton-100X), containing 2 mM PMSF and complete protease inhibitor cocktail (Roche). The lysates were then centrifuged at 8,0006g for 10 min. Equal PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4588