Control of Ctr via Irga6 Figure 1. IFNc-induced inhibition of C. trachomatis growth in WT MEFs. Host cells were infected for 48 h with C. trachomatis or C. muridarum (MOI 1) and simultaneously treated with 100 U or 200 U/ml IFNc or left untreated (control). (A) and (C) Immunofluorescence micrographs of MEFs infected with C. trachomatis or C. muridarum, respectively, stained with Chlamydia-IMAGEN kit. Chlamydiae (green), Host cells (red). Cytokine treatment resulted in a low number of detectable small inclusions in C. trachomatis infected cells only. Images taken using the same magnification. (B) and (D) Influence of IFNc on development of infectious progeny. The yield of C. trachomatis (B), but not C. muridarum (D), infectious progeny decreased considerably upon IFNc stimulation, Infectivity percentage calculated as follows: IFU/ml estimated for each treated monolayer / IFU/ml of control cells 6100. Infectivity expressed as a percentage of control cells 6standard deviation (SD) from three independent experiments (n = 3). WT, wild type; Ctr, C. trachomatis; Cmur, C. muridarum. doi:10.1371/journal.pone.0004588.g001 MEFs (Figure 4C and D). Thus, fusion of C. trachomatis inclusions with autophagosomes is a prerequisite for lysosomal degradation. Association of Irga6 with early chlamydial inclusions is blocked in Atg52/2 MEFs in response to IFNc To address the role of IFNc-induced IRGs in the regulation of C. trachomatis survival, we examined the intracellular distribution of IRGs at 3 h p.i. in Atg52/2 MEFs. Surprisingly, upon IFNc stimulation most bacterial inclusions colocalized with Irgd (41%), Irgm2 (80%) and Irgm3 (79%) (Figure 5A and B); results comparable to data obtained from IFNc-stimulated WT MEFs (Figure 2). Strikingly, only 2% of inclusions colocalized with Irga6 (Figure 5B), as compared to 83% in IFNc-treated WT MEFs (Figure 2B). As expected, IRG proteins did not colocalize with early inclusions of C. muridarum (Figure S1B). These results strongly suggest an important role for Irga6 in autophagy-mediated control of C. trachomatis infection. Absence of Irga6 stimulates growth of C. trachomatis and induces resistance against IFNc-induced killing To further analyze the role of Irga6 in IFNc-mediated growth restriction of C. trachomatis, we examined Irga6-knockout (Irga62/ 2) MEFs. Untreated Irga62/2 cells infected for 48 h with C. trachomatis generated larger inclusions and yielded a 4 to 5-fold increase in the number of infectious progeny, compared to untreated WT cells (Figure 6A and B). IFNc treatment did not inhibit the growth of inclusions in Irga62/2 cells, but they were bigger than in control untreated WT MEFs (Figure 6A). Surprisingly, infected Irga62/2 MEFs were sensitive to IFNc exposure, which resulted in partial destruction and loss of infected host cells of the monolayer. Therefore, the chlamydial infectivity measured in each sample had to be normalized to the surviving host cells, determined via an LDH release assay (data not shown). This data clearly indicated that IFNc treatment of Irga62/2 cells did not reduce infectivity (Figure 6B). PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4588