Listeria hijacks host mitophagy through a novel mitophagy receptor to evade killing

The mammalian immune system has innate and adaptive components, which cooperate to protect the host against microbial infections. The innate immune system consists of functionally distinct 'modules' that evolved to provide different forms of protection against pathogens. It senses pathogens through pattern-recognition receptors, which trigger the activation of antimicrobial defences and stimulate the adaptive immune response. The adaptive immune system, in turn, activates innate effector mechanisms in an antigen-specific manner. The connections between the various immune components are not fully understood, but recent progress brings us closer to an integrated view of the immune system and its function in host defence.

Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria, and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase (Phox) machinery 1 . However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to macrophage bactericidal activity, although the mechanisms linking innate immune signaling to mitochondria for mROS generation remain unclear 2-4 . Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of the TLR signaling adapter tumor necrosis factor receptor-associated factor 6 (TRAF6) to mitochondria where it engages evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), a protein implicated in mitochondrial respiratory chain assembly 5 . Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT and TRAF6 depleted macrophages exhibit decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results therefore reveal a novel pathway linking innate immune signaling to mitochondria, implicate mROS as important components of antibacterial responses, and further establish mitochondria as hubs for innate immune signaling.

Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the CASPASE1 (CASP1) protease 1,2 . Although genetic data support a critical role for inflammasomes in immune defense and inflammatory diseases 3 , the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing C4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin 4,5 and PrgJ 6 , a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homolog of NLRC4, NAIP5 (NLR family, Apoptosis Inhibitory Protein 5), has been implicated in activation of NLRC4 7–11 , but is widely assumed to play only an auxiliary role 1,2 , since NAIP5 is often dispensable for NLRC4 activation 7,8 . However, Naip5 is a member of a small multigene family 12 , raising the possibility of redundancy and functional specialization among Naip genes. Indeed, we show here that different NAIP paralogs dictate the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that NAIP2/NLRC4 physically associates with PrgJ but not flagellin, whereas NAIP5/NLRC4 associates with flagellin but not PrgJ. Taken together, our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP/NLRC4 inflammasomes.