Autophagy Protein Rubicon Mediates Phagocytic NADPH Oxidase Activation in Response to Microbial Infection or TLR Stimulation

Abstract

Summary Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.

Toxoplasma gondii GRA7-Induced TRAF6 Activation Contributes to Host Protective Immunity

“…were isolated from gp91phox, p47phox, TLR2, TLR4, and MyD88 knockout (KO) mice (on the C57BL/6 background) as described previously (22). All animals were maintained in a pathogen-free environment.…”

“…Lentiviral short hairpin RNA (shRNA) produced by transient transfection with packaging plasmids (psPAX2, pMD2.VSV-G; purchased from Addgene) and the target shRNA plasmid DNA (human TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, MyD88, and TRAF6 and mouse TLR11 and TLR12; purchased from Open Biosystems) were cotransfected into 293T cells with Lipofectamine 2000 (Invitrogen catalog no. 11668) as described previously (22). Virus-containing medium was concentrated by ultracentrifugation, titration was determined in 293T cells, and transduction into THP-1 or RAW264.7 cells was carried out as described previously (22).…”

“…11668) as described previously (22). Virus-containing medium was concentrated by ultracentrifugation, titration was determined in 293T cells, and transduction into THP-1 or RAW264.7 cells was carried out as described previously (22). A parallel experiment with a green fluorescent protein-encoding lentivirus (the pGIPZ lentiviral vector; Open Biosystems) indicated that 80% of the cells were successfully transduced by the virus.…”

Inducible Rubicon facilitates viral replication by antagonizing interferon production

“…15,29,41 In one study, Rubicon changed its partner from the Beclin-1 complex to CARD9 in a Dectin-1-stimulation-dependent or RIG-I-stimulationdependent, competitive manner, thereby disassembling the CARD9, BCL10 and MALT1 signaling complex and suppressing NF-κB activation, ultimately stopping PRR-induced inflammatory cytokine production. 15,42 IFN-α is widely used to treat HBV infection because HBV is strongly inhibited by IFNs in some patients, and IFNs are important defenders of the innate immune system.…”

Autophagy proteins are not universally required for phagosome maturation

“…RUBCN is required for LAP 6 and is upregulated in response to TLR2 activation. 29 Thus, the choice of the phagocytic particle also affects the amount of ROS produced and the destiny of the phagosome. It is possible that other positive and negative regulators of the CYBB NADPH oxidase also play a role in LAP.…”