Control of Ctr via Irga6 Figure 3. IFNc stimulation induces interaction of lysosomes and autophagosomes with early C. trachomatis inclusions in MEFs. (A) and (B) MEF monolayers were prestimulated for 24 h with 100 U/ml IFNc and then infected with C. trachomatis as described in Figure 2. IFNc untreated control MEFs were similarly infected. (A) Double immunofluorescence labelling 3 h p.i. revealed that IFNc stimulated the association of the lysosomal marker LAMP1 (red) with C. trachomatis (green) inclusions (compare panel 2 with 1). (B) Percentages of colocalization with LAMP1. (C) Inhibition of lysosomal acidification by 100 nM Baf A1 stopped the IFNc-mediated inhibition of C. trachomatis inclusion growth. (D) Baf A1 attenuated the IFNc-induced reduction of C. trachomatis infectivity in MEFs. Infectivity assays were performed as in Figure 1B. Baf A1 led to an increase in the yield of infectious progeny in IFNc-treated MEFs 48 h p.i. (E) and (F) IFNc induces localization of autophagosomes to inclusions. MEFs were transfected for 24 h with the autophagosome membrane marker GFP-LC3 and then exposed to 100 U/ml IFNc for an additional 24 h. Next, cells were infected with C. trachomatis (MOI 5) for 8 h. IFNc-untreated control cells were similarly infected 48 h post-transfection. LC3 (green) localized to bacterial inclusions (red) in response to IFNc stimulation (compare panel 2 with 1 in E). (F) Quantification of GFP-LC3-positive C. trachomatis inclusions in the presence or absence of IFNc. Around 300 bacterial inclusions were examined for LAMP1 or LC3 sequestration. Colocalization expressed as a mean percentage: for each treatment, number of LAMP1 or LC3 +ve inclusions, respectively / total number of inclusions 6100.Scale bars in (A), (C) and (E) represent 2, 20 and 1 mm, respectively. Error bars 6SD, n = 3. doi:10.1371/journal.pone.0004588.g003 Figure 4. IFNc cannot induce lysosomal fusion with early C. trachomatis inclusions to suppress bacterial growth in autophagy- lacking (Atg52/2) MEFs. (A) Normal development of inclusions (green) was observed in Atg52/2 MEFs (red), despite exposure to either 100 U or 200 U IFNc/ml. Knockout cells were IFNc treated, infected, and stained as in Figure 1. (B) Infectivity titration assays onto fresh HeLa cells revealed similar amounts of infectious progeny in IFNc-treated and IFNc-untreated Atg52/2 MEFs. Infectivity expressed as a percentage normalized to control (C) C. trachomatis inclusions avoid interaction with lysosomes in autophagy-defective Atg52/2 MEFs despite IFNc induction. IFNc treated and untreated Atg52/2 MEFs were infected as in Figure 3A and B. Double immunolabeling demonstrated no recruitment of lysosomes to inclusions [bacteria (green) and LAMP1 (red)] (D) Quantification of LAMP1-positive chlamydial inclusions revealed insignificant colocalization rates. Around 300 bacterial inclusions were examined. Colocalization expressed as a mean percentage: for each treatment, number of LAMP1 inclusions / total number of inclusions 6100. Images in (A) were taken under the same magnification, while scale bars in (C) represent 3 mm. Error bars 6SD, n = 3. doi:10.1371/journal.pone.0004588.g004 PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4588