IFN-c-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy Munir A. Al-Zeer1., Hesham M. Al-Younes3., Peter R. Braun1, Jens Zerrahn2, Thomas F. Meyer1* 1 Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany, 2 Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany, 3 Department of Biological Sciences, Faculty of Sciences, University of Jordan, Amman, Jordan Abstract Chlamydial infection of the host cell induces Gamma interferon (IFNc), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNc-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen’s elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNc-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNc, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg52/2 MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNc- induced Atg52/2 cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga62/2) MEFs, in which chlamydial growth is enhanced, do not respond to IFNc even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction. Citation: Al-Zeer MA, Al-Younes HM, Braun PR, Zerrahn J, Meyer TF (2009) IFN-c-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy. PLoS ONE 4(2): e4588. doi:10.1371/journal.pone.0004588 Editor: Georg Ha¨cker, Technical University Munich, Germany Received January 6, 2009; Accepted January 26, 2009; Published February 26, 2009 Copyright: 2009 Al-Zeer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part through the Fonds der Chemischen Industrie to T.F.M., by a doctoral fellowship grant of the German Academic Exchange Service (DAAD) to M.A.Z. and a visiting scientist grant of the Deutsche Forschungsgemeinschaft (DFG) to H.A.Y. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tfm@mpiib-berlin.mpg.de . These authors contributed equally to this work. Introduction Chlamydiae are obligate intracellular pathogens that cause a variety of infections in humans and animals. Chlamydia trachomatis is a primarily human pathogen associated with common sexually transmitted diseases and trachoma [1]. Chlamydiae undergo a unique biphasic developmental cycle that alternates between extracellular infectious, metabolically inert elementary bodies (EBs) and the intracellular non-infectious, metabolically active, multiplying reticulate bodies (RBs) [2,3]. Bacteria enter the host cell and survive within a membrane-bound vacuole, termed the inclusion, in which they ensure their successful propagation by avoiding fusion with lysosomes [4,5]. Non-fusogenity with lysosomes is controlled by the mode of cellular uptake [6,7] and chlamydial protein factors [8,9]. Gamma interferon (IFNc) plays a central role in innate immunity against intracellular pathogens. It induces the expression of more than 1,200 genes, a number of which include effectors that function to eradicate pathogens from host cells. Their activation leads to a depletion of the tryptophan (Trp) pool [10], production of toxic nitric oxide [11], deprivation of intracellular iron pools [12] and induction of host autophagy [13,14]. Among the genes highly induced by IFNc are the immunity-related GTPases (IRGs; also known as small p47 GTPases), reviewed in [1,15–18]. Recent work has indicated the involvement of several mouse IRG proteins in the growth regulation of pathogens within IFNc- induced host cells: for instance, Irgm1 stimulates IFNc-induced control of Mycobacterium tuberculosis and Trypanosoma cruzi in macrophages [19] and Irgm3 regulates IFNc-induced control of C. trachomatis in fibroblasts [20]. Irgm3-, Irgd- and Irgm1-knockout mice displayed reduced resistance to several bacterial and protozoan pathogens despite an immune response and IFNc production [21,22]. This strong correlation between loss of resistance in intact mice and loss of IFNc-induced control in cultured host cells suggests eliminating pathogens from host cells is a major function of these proteins. IRG proteins localize predominantly to the endoplasmic reticulum (ER) (Irga6 and Irgm3), the Golgi (Irga6, Irgm1 and Irgm2), the plasma membrane and in nascent pathogen-containing vacuoles or phagosomes [reviewed in 16–18]. The localization of IRGs to pathogen-containing vacuoles in host cells suggests that they restrict pathogen growth by vacuole processing. IRGs have been shown to drive vacuole acidification and fusion with lysosomes in M. tuberculosis [19], to disrupt the vacuolar membrane in Toxoplasma gondii [23,24] and to eliminate mycobacteria containing vacuoles through regulating IFNc-induced autophagy [13,14]. Autophagy is an evolutionary conserved lysosomal degradation pathway that maintains cellular homeostasis and selectively PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4588