disposes of intracellular pathogens [13,25,26]. Not only intracel- lular pathogens residing in the cytosol, but also pathogens residing in membranous or intravacuolar compartments are sequestered into autophagosomes for degradation in autolysosomes, as shown for T. gondii [23,27]. Chlamydiae exhibit a wide range of host tropism that has been linked to differences in immune responses elicited by IFNc [28]. In human cells, IFNc can effectively suppress growth of C. trachomatis and C. muridarum by activating indolamine dioxygenase, which deprives both of essential Trp. However, in murine genital epithelial cells (MECs), IFNc can restrict growth of C. trachomatis, but not C. muridarum [28]. Growth inhibition of C. trachomatis in mouse cells is Trp depletion independent and is largely attributed to the IFNc-inducible IRGs. Little is known about the cellular functions of mouse IRGs in host resistance against Chlamydia species. A role for Irga6 in controlling pathogen growth upon IFNc stimulation in MECs was demonstrated by RNA silencing of Irga6, which led to increased C. trachomatis survival [28]. However, the major effector mechanism(s) by which IRGs control chlamyd- ial infection remain elusive. Here we investigated the immunoprotective role of IRGs in C. trachomatis infection of murine cells and in the IFNc-insensitive mouse strain C. muridarum. We show that C. trachomatis growth is arrested by the development of early inclusions with autolysosomal features. In contrast, C. muridarum inclusions remained segregated from lysosomes and autophagosomes. Subcellular analysis re- vealed that C. trachomatis inclusions sequestered Irga6, Irgd, Irgm2 and Irgm3 in response to IFNc blocking chlamydial growth. However, autophagy-deficient cells tolerated C. trachomatis infec- tion despite an accumulation of Irgd, Irgm2 and Irgm3 at inclusions. Strikingly, Irga6 did not associate with inclusions in autophagy-deficient cells. In addition, Irga62/2 MEFs did not respond to IFNc and did not restrict C. trachomatis growth, although Irgd, Irgm2 and Irgm3 localized to inclusions. Thus, our data indicate that Irga6 modifies the inclusion membrane to mediate fusion with autophagosomes as a mechanism to dispose of C. trachomatis. Results IFNc negatively affects the growth of C. trachomatis, but not of C. muridarum in MEFs IFNc is a critical mediator for controlling chlamydial infection. To assess the effect of IFNc on chlamydial growth in mouse embryonic fibroblasts (MEFs), cells were infected with C. trachomatis LGV L2 or C. muridarum at a multiplicity of infection (MOI) of 1 for 2 h. Following 48 h incubation in the presence of IFNc, cells were immunostained and analyzed by microscopy. IFNc treat- ment resulted in much smaller and substantially reduced numbers of C. trachomatis inclusions (Figure 1A). Similar results were obtained from the pretreated cells with IFNc. Furthermore, a reduction of infectious C. trachomatis EBs of more than 70% as compared to non-treated infected MEFs (Figure 1B) was found. In contrast, IFNc affected neither inclusion development nor formation of infectious progeny of C. muridarum (Figure 1C and D). Thus, distinct chlamydial species exhibit differential sensitiv- ities to IFNc in host resistance: Growth of C. trachomatis in MEFs is arrested in response to IFNc, whereas growth of C. muridarum is not affected in response to IFNc. Irga6, Irgd, Irgm2 and Irgm3 interact specifically with early C. trachomatis inclusions in response to IFNc Since IFNc can upregulate IRG expression in C. trachomatis infected murine cells [28], we investigated the involvement of Control of Ctr via Irga6 IRGs in IFNc-mediated inhibition of chlamydial growth. Un- treated and IFNc-pretreated MEFs were infected with C. trachomatis or C. muridarum (MOI 5) in the presence of IFNc. Three hours post infection (h p.i.) cells were processed for indirect immunofluorescence to analyze colocalization of early chlamydial inclusions with IRGs (Figure 2A). Upon IFNc treatment C. trachomatis inclusions colocalized to a high degree with IRGs: Irga6 (83%), Irgd (49%), Irgm2 (74%) and Irgm3 (87%). Surprisingly, Irgb6 also colocalized with inclusions in both treated (63%) and untreated cells, whereas Irgm1 colocalization was minimal (Figure 2B). However, no localization of IRGs to C. muridarum inclusions was detected (Figure S1A). Thus, IFNc stimulation leads to a specific modification of C. trachomatis inclusions by recruiting four different IRGs. IFNc stimulates C. trachomatis elimination by lysosomal fusion with early inclusions We next investigated whether IRG-positive C. trachomatis inclusions are directed to lysosomes for degradation. First we monitored colocalization of inclusions with the lysosomal marker LAMP1 at 3 h p.i. In control, untreated MEFs, only 20% of inclusions colocalized with LAMP1. In contrast, LAMP1 coloca- lization in inclusions of IFNc-treated cells was increased 4-fold (Figure 3A and B). Consistent with an ability to grow in the presence of IFNc, no LAMP1 colocalization with C. muridarum inclusions was observed in untreated or IFNc-treated cells (Figure S2A and B). To confirm that IFNc-induced phagosome-lysosome fusion eliminates C. trachomatis, we studied the effect of inhibiting lysosomal acidification with bafilomycin A1 (Baf A1), a specific vacuolar H+-ATPase inhibitor. Growth of C. trachomatis inclusions was rescued and formation of infectious EBs increased upon Baf treatment (Figure 3C and D). These data clearly indicate that lysosomal fusion elicited by IFNc eliminates chlamydial inclusions. IFNc-induced autophagy is required for the elimination of C. trachomatis IFNc can induce autophagy to eliminate intracellular pathogens [13,14]. We hypothesized that IFNc induces the interaction of C. trachomatis inclusions with autophagosomes to reroute the intruder to the lysosomal compartment for destruction; therefore, we transfected MEFs with the autophagosome-associated marker GFP-LC3, a GFP fusion with the microtubule-associated protein light chain 3 (LC3). Then, MEFs were either infected with C. trachomatis or C. muridarum in the presence or absence of IFNc. In contrast to IFNc-untreated cells, GFP-LC3 colocalized strongly to C. trachomatis inclusions (74%) in IFNc treated cells (Figure 3E and F). However, minimal GFP-LC3 colocalization was observed in C. muridarum inclusions (Figure S2C and D). Taken together with our LAMP1 data, these findings clearly suggest the nature of C. trachomatis inclusions upon IFNc stimulation is autolysosomal. To further support our hypothesis, we used Atg5-deficient (Atg52/2) MEFs (Atg, autophagy-related). Atg5 is a crucial factor in autophagosome formation as its deletion prevents their appearance [29]. We infected Atg52/2 MEFs with either C. trachomatis or C. muridarum (MOI 1) for 48 h in the presence or absence of IFNc. IFNc treatment did not affect chlamydial growth as inclusion size and recovery of infectious progeny in these cells were comparable to those of untreated knockout cells (Figure 4A and B) and WT MEFs. The completion of chlamydial develop- ment in cells devoid of autophagy shows that host cell resistance to chlamydial inclusions depends on their fusion with autophago- somes. Furthermore, subcellular analysis of cultures at 3 h p.i. with LAMP1 revealed that lysosomal fusion is blocked in Atg52/2 PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4588