Control of Ctr via Irga6 Figure 6. Absence of Irga6 enhances C. trachomatis replication and resistance against IFNc-induced growth inhibition. (A) Deletion of Irga6 promotes intracellular growth of the bacterial inclusion. Unstimulated WT and Irga6-knockout MEFs were infected for 48 h (MOI 1). Micrographs show an increased inclusion size in Irga6-knockout cells (panel 3) compared to WT MEFs (panel 1). IFNc (100 U/ml) stimulation did not suppress chlamydial growth in Irga6-deficient MEFs (panel 4) as in IFNc-induced WT MEFs (panel 2). (B) Infectivity of bacteria in Irga6-knockout MEFs is 4-fold higher than in WT MEFs and is unaffected by IFNc induction. Results depicted as mean percentage normalized to control. (C) Lack of appreciable lysosomal fusion with C. trachomatis inclusions in IFNc-stimulated Irga62/2 MEFs (panel 4) unlike that in stimulated WT MEFs (panel 2). Cells were stimulated, infected and stained for bacteria (green) and LAMP1 (red) as in Figure 3A. Untreated WT and Irga6-knockout monolayers (panels 1 and 3, respectively) served as controls. (D) IFNc did not considerably increase rates of LAMP1 localization to C. trachomatis inclusions in Irga62/2 MEFs, compared with that in WT cells. Around 300 bacterial inclusions were examined Colocalization expressed as a mean percentage: for each treatment, number of LAMP1 +ve inclusions / total number of inclusions 6100. Scale bar in (A) and (C) represent 40 and 4 mm, respectively. Error bars 6SD, n = 3. doi:10.1371/journal.pone.0004588.g006 In summary, our data clearly demonstrates the ability of IFNc to induce autophagy in nonphagocytic MEFs. More importantly, low LC3 expression and small numbers of autophagosomes in Irga62/2 cells indicate a role for Irga6 in the regulation of the IFNc-induced autophagic pathway. Our results reveal Irga6 as an important factor in assigning chlamydial inclusions for degrada- tion via autophagy. Discussion Here we elucidate the mechanisms underlying IFNc/IRGs- induced immunity to C. trachomatis in murine cells, demonstrating a pivotal role for Irga6 in mediating host resistance to infection via the induction of autophagy. We show that C. trachomatis, but not C. muridarum, inclusions strongly colocalized with 4 IRGs (Irga6, Irgd, Irgm2 and Irgm3) in response to IFNc, whereas Irgb6 colocalized in the presence or absence of the cytokine. IFNc also induced accumulation of the autophagosomal membrane marker LC3, and the lysosomal component LAMP1, suggesting a rerouting of C. trachomatis phagosomes to autolysosomes. However, autophagy- deficient MEFs (Atg52/2) and WT cells with an impairment in lysosomal acidification surrendered to infection. While Irgb6, Irgd, Irgm2 and Irgm3 still localized to chlamydial inclusions in IFNc- induced Atg52/2 cells, Irga6 localization was disrupted, indicating a pivotal role for Irga6 in resistance to the microbe. Strikingly, Irga6-deficient MEFs (Irga62/2), in which chlamydial growth is enhanced compared to WT MEFs, showed no response to IFNc even though all the major IRG proteins studied still localized to inclusions. Thus, Irga6 constitutes a critical resistance factor against C. trachomatis infection in IFNc-induced MEFs that PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4588