Control of Ctr via Irga6 Figure 2. IFNc triggers accumulation of Irga6, Irgd, Irgm2 and Irgm3 at early inclusions of C. trachomatis. (A) Double immunofluorescence labelling of IRGs and C. trachomatis in MEFs stimulated for 24 h with 100 U/ml IFNc and then infected for 3 h with C. trachomatis (MOI 5). IFNc untreated MEFs were similarly infected. Upon IFNc induction Irga6, Irgd, Irgm2 and Irgm3 localized to inclusions. Irgb6 colocalized strongly with inclusions in treated as well as untreated cells, whereas Irgm1 localization was minimal. (B) For quantification, around 300 bacterial inclusions were examined for each IRG in cytokine-treated or untreated MEFs. Colocalization expressed as a mean percentage: for each treatment, number of IRG +ve inclusions / total number of inclusions 6100. Error bars 6SD, n = 3. Scale bar represents 4 mm. doi:10.1371/journal.pone.0004588.g002 Similar to our data from WT and Atg52/2 MEFs (Figure 2 and 5, respectively), IFNc treatment of Irga62/2 MEFs at 3 h p.i. increased the association of Irgd, Irgm2 and Irgm3 with early bacterial inclusions (Figure S3A and S3B). As expected, no signal for Irga6 was detected in IFNc-exposed or unexposed Irga6- deficient MEFs (Figure S3A). In contrast, no IRG proteins localized to C. muridarum inclusions (Figure S1C). Next, we investigated whether enhanced propagation of C. trachomatis in Irga62/2 MEFs was connected with a lack of autolysosomal features of inclusions. Indeed, IFNc treatment of Irga62/2 MEFs did not induce an association of LAMP1 with chlamydial inclusions (Figure 6C and D). Numbers of bacteria colocalizing with LAMP1 in IFNc induced Irga62/2 cells were comparable to untreated WT MEFs (Figure 6C and D). Consistently, only approximately 1% of bacterial inclusions colocalized with GFP-LC3 in Irga62/2 cells. These results indicate that IFNc cannot prevent C. trachomatis development in cells lacking Irga6. Thus, our data suggest a critical role for Irga6 in provoking anti-chlamydial effects. In contrast, despite their noticeable association with bacterial inclusions, Irgd, Irgm2 and Irgm3 do not play a critical role in anti-chlamydial defense. The loss of Irga6 renders cells incapable of capturing C. trachomatis in an autolysosome, thus enabling pathogen survival. Irga6 is important in the induction of autophagy independent of C. trachomatis infection Is IFNc capable of activating autophagy in MEFs and is Irga6 required for IFNc-induced autophagy? To answer these questions, we used immunoblots to monitor the formation of early autophagosomal precursors and newly formed autophagosomes by following changes in LC3 expression. LC3 exists in two forms: the cytosolic LC3-I form, which has a molecular weight of approximately 18 kDa and the membrane-bound LC3-II form, with a molecular weight of 16 kDa [30]. LC3-II is bound to the PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4588