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IFN-gamma-inducible Irga6 mediates host resistance against Chlamydia trachomatis via autophagy.

Al-Zeer MA, Al-Younes HM, Braun PR, Zerrahn J, Meyer TF - PLoS ONE (2009)

Bottom Line: Irgm2, Irgm3 and Irgd still localize to inclusions in IFNgamma-induced Atg5-/- cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance.Irga6-deficient (Irga6-/-) MEFs, in which chlamydial growth is enhanced, do not respond to IFNgamma 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

ABSTRACT
Chlamydial infection of the host cell induces Gamma interferon (IFNgamma), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNgamma-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 IFNgamma-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNgamma, 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 Atg5-/- MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNgamma-induced Atg5-/- cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6-/-) MEFs, in which chlamydial growth is enhanced, do not respond to IFNgamma 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.

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Absence of Irga6 enhances C. trachomatis replication and resistance against IFNγ-induced growth inhibition.(A) Deletion of Irga6 promotes intracellular growth of the bacterial inclusion. Unstimulated WT and Irga6-knockout MEFs were infected for 48 h (MOI 1). Micrographs show an increased inclusion size in Irga6-knockout cells (panel 3) compared to WT MEFs (panel 1). IFNγ (100 U/ml) stimulation did not suppress chlamydial growth in Irga6-deficient MEFs (panel 4) as in IFNγ-induced WT MEFs (panel 2). (B) Infectivity of bacteria in Irga6-knockout MEFs is 4-fold higher than in WT MEFs and is unaffected by IFNγ induction. Results depicted as mean percentage normalized to control. (C) Lack of appreciable lysosomal fusion with C. trachomatis inclusions in IFNγ-stimulated Irga6−/− MEFs (panel 4) unlike that in stimulated WT MEFs (panel 2). Cells were stimulated, infected and stained for bacteria (green) and LAMP1 (red) as in Figure 3A. Untreated WT and Irga6-knockout monolayers (panels 1 and 3, respectively) served as controls. (D) IFNγ did not considerably increase rates of LAMP1 localization to C. trachomatis inclusions in Irga6−/− MEFs, compared with that in WT cells. Around 300 bacterial inclusions were examined Colocalization expressed as a mean percentage: for each treatment, number of LAMP1 +ve inclusions / total number of inclusions ×100. Scale bar in (A) and (C) represent 40 and 4 µm, respectively. Error bars ±SD, n = 3.
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pone-0004588-g006: Absence of Irga6 enhances C. trachomatis replication and resistance against IFNγ-induced growth inhibition.(A) Deletion of Irga6 promotes intracellular growth of the bacterial inclusion. Unstimulated WT and Irga6-knockout MEFs were infected for 48 h (MOI 1). Micrographs show an increased inclusion size in Irga6-knockout cells (panel 3) compared to WT MEFs (panel 1). IFNγ (100 U/ml) stimulation did not suppress chlamydial growth in Irga6-deficient MEFs (panel 4) as in IFNγ-induced WT MEFs (panel 2). (B) Infectivity of bacteria in Irga6-knockout MEFs is 4-fold higher than in WT MEFs and is unaffected by IFNγ induction. Results depicted as mean percentage normalized to control. (C) Lack of appreciable lysosomal fusion with C. trachomatis inclusions in IFNγ-stimulated Irga6−/− MEFs (panel 4) unlike that in stimulated WT MEFs (panel 2). Cells were stimulated, infected and stained for bacteria (green) and LAMP1 (red) as in Figure 3A. Untreated WT and Irga6-knockout monolayers (panels 1 and 3, respectively) served as controls. (D) IFNγ did not considerably increase rates of LAMP1 localization to C. trachomatis inclusions in Irga6−/− MEFs, compared with that in WT cells. Around 300 bacterial inclusions were examined Colocalization expressed as a mean percentage: for each treatment, number of LAMP1 +ve inclusions / total number of inclusions ×100. Scale bar in (A) and (C) represent 40 and 4 µm, respectively. Error bars ±SD, n = 3.

Mentions: To further analyze the role of Irga6 in IFNγ-mediated growth restriction of C. trachomatis, we examined Irga6-knockout (Irga6−/−) MEFs. Untreated Irga6−/− 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). IFNγ treatment did not inhibit the growth of inclusions in Irga6−/− cells, but they were bigger than in control untreated WT MEFs (Figure 6A). Surprisingly, infected Irga6−/− MEFs were sensitive to IFNγ 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 IFNγ treatment of Irga6−/− cells did not reduce infectivity (Figure 6B).


IFN-gamma-inducible Irga6 mediates host resistance against Chlamydia trachomatis via autophagy.

Al-Zeer MA, Al-Younes HM, Braun PR, Zerrahn J, Meyer TF - PLoS ONE (2009)

Absence of Irga6 enhances C. trachomatis replication and resistance against IFNγ-induced growth inhibition.(A) Deletion of Irga6 promotes intracellular growth of the bacterial inclusion. Unstimulated WT and Irga6-knockout MEFs were infected for 48 h (MOI 1). Micrographs show an increased inclusion size in Irga6-knockout cells (panel 3) compared to WT MEFs (panel 1). IFNγ (100 U/ml) stimulation did not suppress chlamydial growth in Irga6-deficient MEFs (panel 4) as in IFNγ-induced WT MEFs (panel 2). (B) Infectivity of bacteria in Irga6-knockout MEFs is 4-fold higher than in WT MEFs and is unaffected by IFNγ induction. Results depicted as mean percentage normalized to control. (C) Lack of appreciable lysosomal fusion with C. trachomatis inclusions in IFNγ-stimulated Irga6−/− MEFs (panel 4) unlike that in stimulated WT MEFs (panel 2). Cells were stimulated, infected and stained for bacteria (green) and LAMP1 (red) as in Figure 3A. Untreated WT and Irga6-knockout monolayers (panels 1 and 3, respectively) served as controls. (D) IFNγ did not considerably increase rates of LAMP1 localization to C. trachomatis inclusions in Irga6−/− MEFs, compared with that in WT cells. Around 300 bacterial inclusions were examined Colocalization expressed as a mean percentage: for each treatment, number of LAMP1 +ve inclusions / total number of inclusions ×100. Scale bar in (A) and (C) represent 40 and 4 µm, respectively. Error bars ±SD, n = 3.
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pone-0004588-g006: Absence of Irga6 enhances C. trachomatis replication and resistance against IFNγ-induced growth inhibition.(A) Deletion of Irga6 promotes intracellular growth of the bacterial inclusion. Unstimulated WT and Irga6-knockout MEFs were infected for 48 h (MOI 1). Micrographs show an increased inclusion size in Irga6-knockout cells (panel 3) compared to WT MEFs (panel 1). IFNγ (100 U/ml) stimulation did not suppress chlamydial growth in Irga6-deficient MEFs (panel 4) as in IFNγ-induced WT MEFs (panel 2). (B) Infectivity of bacteria in Irga6-knockout MEFs is 4-fold higher than in WT MEFs and is unaffected by IFNγ induction. Results depicted as mean percentage normalized to control. (C) Lack of appreciable lysosomal fusion with C. trachomatis inclusions in IFNγ-stimulated Irga6−/− MEFs (panel 4) unlike that in stimulated WT MEFs (panel 2). Cells were stimulated, infected and stained for bacteria (green) and LAMP1 (red) as in Figure 3A. Untreated WT and Irga6-knockout monolayers (panels 1 and 3, respectively) served as controls. (D) IFNγ did not considerably increase rates of LAMP1 localization to C. trachomatis inclusions in Irga6−/− MEFs, compared with that in WT cells. Around 300 bacterial inclusions were examined Colocalization expressed as a mean percentage: for each treatment, number of LAMP1 +ve inclusions / total number of inclusions ×100. Scale bar in (A) and (C) represent 40 and 4 µm, respectively. Error bars ±SD, n = 3.
Mentions: To further analyze the role of Irga6 in IFNγ-mediated growth restriction of C. trachomatis, we examined Irga6-knockout (Irga6−/−) MEFs. Untreated Irga6−/− 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). IFNγ treatment did not inhibit the growth of inclusions in Irga6−/− cells, but they were bigger than in control untreated WT MEFs (Figure 6A). Surprisingly, infected Irga6−/− MEFs were sensitive to IFNγ 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 IFNγ treatment of Irga6−/− cells did not reduce infectivity (Figure 6B).

Bottom Line: Irgm2, Irgm3 and Irgd still localize to inclusions in IFNgamma-induced Atg5-/- cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance.Irga6-deficient (Irga6-/-) MEFs, in which chlamydial growth is enhanced, do not respond to IFNgamma 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

ABSTRACT
Chlamydial infection of the host cell induces Gamma interferon (IFNgamma), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNgamma-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 IFNgamma-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNgamma, 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 Atg5-/- MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNgamma-induced Atg5-/- cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6-/-) MEFs, in which chlamydial growth is enhanced, do not respond to IFNgamma 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.

Show MeSH
Related in: MedlinePlus