The E2-Like Conjugation Enzyme Atg3 Promotes Binding of IRG and Gbp Proteins to Chlamydia- and Toxoplasma-Containing Vacuoles and Host Resistance

Abstract

Cell-autonomous immunity to the bacterial pathogen Chlamydia trachomatis and the protozoan pathogen Toxoplasma gondii is controlled by two families of Interferon (IFN)-inducible GTPases: Immunity Related GTPases (IRGs) and Guanylate binding proteins (Gbps). Members of these two GTPase families associate with pathogen-containing vacuoles (PVs) and solicit antimicrobial resistance pathways specifically to the intracellular site of infection. The proper delivery of IRG and Gbp proteins to PVs requires the autophagy factor Atg5. Atg5 is part of a protein complex that facilitates the transfer of the ubiquitin-like protein Atg8 from the E2-like conjugation enzyme Atg3 to the lipid phosphatidylethanolamine. Here, we show that Atg3 expression, similar to Atg5 expression, is required for IRG and Gbp proteins to dock to PVs. We further demonstrate that expression of a dominant-active, GTP-locked IRG protein variant rescues the PV targeting defect of Atg3- and Atg5-deficient cells, suggesting a possible role for Atg proteins in the activation of IRG proteins. Lastly, we show that IFN-induced cell-autonomous resistance to C. trachomatis infections in mouse cells depends not only on Atg5 and IRG proteins, as previously demonstrated, but also requires the expression of Atg3 and Gbp proteins. These findings provide a foundation for a better understanding of IRG- and Gbp-dependent cell-autonomous resistance and its regulation by Atg proteins.

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

“…IFNγ priming induces GBP-mediated cell-autonomous immunity that results in the degradation of C. trachomatis inside autophagolysosomes (24,27). Because antimicrobial autophagy (also called xenophagy) often depends on host-mediated ubiquitination of invading microbes (28), we asked whether IFNγ priming would lead to the accumulation of ubiquitin on C. trachomatis PVs (referred to henceforth as "inclusions" in agreement with accepted nomenclature).…”

“…Because GBPs mediate IFNγ-induced cell-autonomous resistance to C. trachomatis infections (24,27,40), we next asked whether the diminished targeting of GBPs to PVs in p62 −/− and TRAF6 −/− cells would debilitate the cell-autonomous defense system of the host cell. We found that IFNγ-primed cells lacking either p62 or enzymatically active TRAF6 elicited a less effective cell-autonomous immune response to C. trachomatis (Fig.…”

“…Interference with ATG8 lipidation diminishes GBP and GKS protein recruitment to PVs, demonstrating the importance of these molecules in immunotargeting of PVs (24,25,42,48,(55)(56)(57). Whether and how the ATG8 pathway intersects with the newly discovered IRG-driven ubiquitination pathway will need to be explored further in the future.…”

“…MEFs and African green monkey kidney Vero cells were cultured in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS. C. trachomatis LGV-L2, C. muridarum, and T. gondii tachyzoites of the type I strain RH, the type II strains Pru and ME49 strain, and the type III strain CEP were propagated in Vero cells, as described (19,24). Transgenic T. gondii strains expressing Rop16 and Rop18 were reported previously (18).…”

“…In IRGMdeficient cells, however, GKS proteins enter the active state prematurely, form protein aggregates, mislocalize, and thus fail to bind to PVs (17,21). Although these previous observations help explain how IRGM proteins promote the delivery of GKS proteins to PVs, IRGM proteins also control the subcellular localization of GBPs through an uncharacterized mechanism (6,17,(24)(25)(26).…”