Control of Ctr via Irga6 Figure 7. Irga6 plays a role in the regulation of the host autophagic machinery. (A–C) Anti-LC3 immunoblot analysis of total lysates from uninfected WT, Atg52/2 and Irga62/2 MEFs or from cultures infected for indicated time points. Some uninfected cell cultures were exposed to 100 nM Rapa for 3 h or to 100 U/ml IFNc for 32 h. Other monolayers were pretreated with IFNc for 24 h prior to infection and then infected in the presence of chemicals. 1–7 indicate the different treatments. Host b-actin was used to control equal loading of proteins. (A) Autophagy is induced by Rapa and IFNc in WT cells, while infection alone does not stimulate autophagy, as indicated by the amount of LC3-II. (B) Defective autophagy in Atg52/2 MEFs was observed, indicated by absence of LC3 processing (C) Abolishment of Irga6 apparently impaired autophagy induction. Only very low amounts of cellular LC3 (LC3-II) can be detected in Irga6-knockout cells, compared to those in WT MEFs. (D)–(E) Quantification of GFP-LC3- vacoules under different conditions in WT (D) and Irga62/2 MEFs (E) treated as indicated for (A)–(C), 24 h after transfection. Around 100 cells were examined by immunofluorescence microscopy. Data normalized against control. Data show average numbers per cell within a set cytoplasmic area. Error bars 6SD, n = 3. doi:10.1371/journal.pone.0004588.g007 remodels a classically non-fusogenic intracellular pathogen vacuole, stimulating fusion with autophagosomes and directing the intruder to the lysosomal compartment for destruction. Autophagy reroutes cytoplasmic material and organelles internalized into autophagosomes to lysosomes, culminating in the formation of autolysosomes and degradation of their cargo [30,32,33]. Similarly, this process can induce pathogen eradica- tion. Here we showed IFNc stimulates localization of LC3 and the lysosomal marker LAMP1 to C. trachomatis, but not C. muridarum, inclusions in WT MEFs. Despite the presence of IFNc, autophagosome-deficient MEFs were highly permissive to C. trachomatis replication in a compartment disconnected from lysosomes, confirming an involvement of autophagy that leads to lysosomal degradation of the bacteria. Similarly, a study in macrophages showed that IFNc stimulates recruitment of LC3 and LAMP1 to the M. tuberculosis compartment and induces autophagy to inhibit bacterial viability [13]. In addition, the induction of structures carrying LC3 close to disrupting T. gondii vacuoles in IFN-c-induced astrocytes has been reported [24]. IFNc therefore represents a novel means to counteract the non- fusogenicity of the C. trachomatis inclusion by remodelling it into a compartment with autophagic characteristics to prompt fusion with lysosomes for degradation. In contrast to our results, a previous study implied that IFNc- mediated suppression of C. trachomatis in MECs was not the result of fusion with lysosomes, but due to a reduction of lipid trafficking to inclusions [28]. This discrepancy can be explained by the fact that our LAMP1 analysis was done at 3 h p.i., a time at which most C. trachomatis early inclusions strongly colocalized with LAMP1. In contrast, Nelson and coworkers examined LAMP1 colocalization to 24 h-old inclusions, which probably represent the 20% of C. trachomatis inclusions that might partially have survived the IFNc-mediated immunity. Nevertheless, we cannot dismiss a role for lipid and nutrient, or indeed other vacuolar trafficking in the IFNc-mediated suppression of Chlamydial growth. For instance, it has been shown that Irga6 interacts with the microtubule-binding protein hook3; therefore, it is tempting to consider that Irga6 participates in the modulation of intracellular membrane-dependent processes [34,46] like vesicular trafficking and interactions with the pathogen-containing vacuolar mem- brane components. Several studies have implicated the IRG family of proteins in growth regulation of intracellular pathogens [13,14,19,35]. For instance, Irga6 is required for resistance against T. gondii in cultured murine astrocytes by activating vacuole vesiculation [24]. A role for Irga6 in the induction of structures carrying LC3 close PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4588