to disrupting T. gondii vacuoles upon IFNc-treatment has also been suggested [24]. Similarly, Irgm3 induces T. gondii vacuole vesiculation and fusion with autophagosomes in macrophages activated in vivo [23]. Recently, siRNA targeting of Irga6 was shown to partially revert the IFNc-induced growth inhibition of C. trachomatis, whereas siRNA-mediated knockdown of Irgb6, Irgd, Irgm1, Irgm2 or Irgm3 did not [28]. In support of these data, we uncover Irga6 as a major effector protein in IFNc-induced elimination of C. trachomatis. Despite an accumulation of other IRG members at the C. trachomatis inclusion in Irga62/2 cells, C. trachomatis grow more efficiently than in WT MEFs. Similarly, an analysis of isolated Irga62/2 astrocytes showed a significant loss of resistance to T. gondii, even though Irgb6, Irgd, Irgm2 and Irgm3 still localized to the parasitophorous vacuole [24]. Additionally, C. trachomatis inclusions in Irga62/2 cells do not colocalize with LAMP1 and LC3, confirming an involvement of autophagy in restricting pathogen growth. In T. gondii, it has been proposed that autophagic sequestration follows damage to the vacuolar membrane containing the organism [23], but it is not yet known whether such damage or a molecular modification of the target membrane leads to autophagic uptake. The present study clearly shows that molecular remodelling of the chlamydial inclusion membrane in response to IFNc stimulation promotes fusion with autophagosomes. There is some evidence that GMS subfamily proteins (Irgm1, Irgm2 and Irgm3) are required for the normal function of Irga6 [16]. When overexpressed in cells not stimulated with IFNc, Irga6 and Irgb6 intracellularly mislocalized and aggregated. This mislocalization could be corrected by co-expression of Irgm1, Irgm2 and Irgm3. Another line of evidence shows that IRG proteins accumulate on the T. gondii vacuole in a cooperative manner to regulate anti-parasitic Irga6 function [36]. The assembly of Irga6, Irgb6, Irgd, Irgm2 and Irgm3 on C. trachomatis inclusions in WT MEFs and the lack of Irgm1 colocalization observed in this study is largely consistent with IRGs after infection of murine astrocytes with T. gondii [24]. The function of Irgm1, however, does not necessarily require its direct association with the microbial vacuole. Indeed, Irgm1-deficient mice are susceptible to C. trachomatis [37], T. gondii [38], T. cruzi [39] and M. tuberculosis [19] infection. The role of the other IRGs may be to induce the accumulation of effective Irga6 concentrations and/or its correct positioning for autophagosome remodelling. Recent work has demonstrated an essential role for Atg5, independent of autoph- agy, in trafficking Irga6 to vacuole membranes of T. gondii and subsequent pathogen clearance [40]. Here, we also found recruitment of Irga6 was linked to Atg5, in an autophagy dependent manner, as accumulation of Irga6 at early chlamydial inclusions was blocked in Atg52/2 MEFs. Future experiments will define the importance of these factors in recruitment of Irga6 to the inclusion and its antimicrobial function. The inhibitory effects of IFNc on chlamydial replication have been extensively studied, revealing marked inconsistencies in chlamydial strain susceptibility and antichlamydial effector mechanisms, as demonstrated by discrepancies in the role of IRGs in controlling chlamydial infections. Both Irgm3 and Irgb10 were found to mediate resistance to C. trachomatis in cultures and in systemically infected mice [20]. Irgm2 and Irgb10 have been implicated in C. psittaci resistance in cell cultures and locally infected mice [41]. In agreement with our study, Nelson and co- workers suggested a crucial role for Irga6 in C. trachomatis growth control [28]. In contrast, it was recently shown that Irga62/2 MEFs are not defective, but more efficient in restricting growth of C. trachomatis compared with control IFNc-treated MEFs [37]. Subcellular localization studies only partly agree with our data, Control of Ctr via Irga6 showing Irga6 localization and the absence of Irgm1 localization to inclusions upon IFNc stimulation [20]. Also, Bernstein-Hanley and coworkers detected no Irgm3 at the C. trachomatis inclusion and the bacterium’s growth in systemically infected mice was not affected; it remains unclear why resistance is not affected in Irga62/2 mice. More specific infection of the uterine mucosa by intrauterine inoculation with human chlamydial strains, as previously described [42], might lead to a more coherent outcome. Overall, these studies point to the complexity and diversity of IRGs that participate in host resistance mechanisms. The pleiotropic signaling capabilities and host and tissue specificities of IFNc [43], the genomic differences among chlamydial strains studied [44,45], differences in susceptibility among inbred mouse strains and the inherent experimental variation between labora- tories, may account for these discrepancies. It is indisputable that C. muridarum possesses a very effective mechanism to evade the murine IFNc response, unlike C. trachomatis; however, the underlying mechanism remains largely hypothetical. Nelson and co-workers suggested a gene in the plasticity zone of C. muridarum, which is absent in C. trachomatis, is responsible for avoiding the Irga6-mediated growth inhibition by C. muridarum in murine cells [28]. This gene encodes a relatively large protein with a homology to a clostridial toxin and the Yersinia YopT virulence factor. YopT acts as cysteine protease that can inactivate Rho GTPase by the cleavage of the GTPase and its subsequent release from the membrane. Although indirect, the authors suggested that a C. muridarum hypothetical large toxin inactivates Irga6 by a similar mechanism. In contrast to our work, a recent study demonstrated the transient overexpression of Irgb10 in the absence of IFNc was sufficient to reduce the yield of C. trachomatis, but not C. muridarum [37]. Overexpressed Irgb10 was found associated with C. trachomatis inclusions only. Based on this differential subcellular localization of Irgb10 in infected cells, the authors proposed Irgb10 is recruited to the inclusion to induce bacterial growth blockage. They also suggested that C. muridarum is protected from IFNc-induced immune response by a mechanism that restricts access of Irgb10 to its inclusion. Here we show IFNc- stimulated association of different IRG proteins (Irga6, Irgb6, Irgd, Irgm2 and Irgm3) with inclusions harbouring C. trachomatis, but not C. muridarum. Importantly, Irga6 was found to be the critical effector protein responsible for immune resistance to C. trachomatis, while other IRGs could have cooperative interactions. Cells deficient in Irga6 were highly permissive to C. trachomatis infection, although other IRGs were recruited in response to IFNc. However, C. muridarum inclusions did not associate with any of these IRGs. These results strongly indicate that C. muridarum can prevent, by a yet undefined mechanism, not only the access and/ or the activity of the effector Irga6, but also the localization of the so called ‘co-operative’ IRGs required for the anti-bacterial function of Irga6. Our data indicate that modification of the inclusion is critical to the outcome of the host-parasite interaction; the presence of Irga6 on the inclusion membrane defeats the complex array of processes by which C. trachomatis seeks to delay phagosomal maturation. Further work will now unravel the precise mechanism(s) through which Irga6 promotes IFNc -induced C. trachomatis elimination and C. muridarum uses to escape the murine IFNc-induced response. Methods Chemicals and antibodies RPMI-1640 medium and Dulbecco’s minimal essential medium (DMEM) were purchased from Gibco-Invitrogen (Karlsruhe, PLoS ONE | www.plosone.org 10 February 2009 | Volume 4 | Issue 2 | e4588