Ivan Tattoli

NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-κB and JNK pathways by inducing reactive oxygen species production

NOD‐like receptors (NLRs) are a family of intracellular sensors of microbial‐ or danger‐associated molecular patterns. Here, we report the identification of NLRX1, which is a new member of the NLR family that localizes to the mitochondria. NLRX1 alone failed to trigger most of the common signalling pathways, including nuclear factor‐κB (NF)‐κB‐ and type I interferon‐dependent cascades, but could potently trigger the generation of reactive oxygen species (ROS). Importantly, NLRX1 synergistically potentiated ROS production induced by tumour necrosis factor α, Shigella infection and double‐stranded RNA, resulting in amplified NF‐κB‐dependent and JUN amino‐terminal kinases‐dependent signalling. Together, these results identify NLRX1 as a NLR that contributes to the link between ROS generation at the mitochondria and innate immune responses.

Amino Acid Starvation Induced by Invasive Bacterial Pathogens Triggers an Innate Host Defense Program

Autophagy, which targets cellular constituents for degradation, is normally inhibited in metabolically replete cells by the metabolic checkpoint kinase mTOR. Although autophagic degradation of invasive bacteria has emerged as a critical host defense mechanism, the signals that induce autophagy upon bacterial infection remain unclear. We find that infection of epithelial cells with Shigella and Salmonella triggers acute intracellular amino acid (AA) starvation due to host membrane damage. Pathogen-induced AA starvation caused downregulation of mTOR activity, resulting in the induction of autophagy. In Salmonella-infected cells, membrane integrity and cytosolic AA levels rapidly normalized, favoring mTOR reactivation at the surface of the Salmonella-containing vacuole and bacterial escape from autophagy. In addition, bacteria-induced AA starvation activated the GCN2 kinase, eukaryotic initiation factor 2α, and the transcription factor ATF3-dependent integrated stress response and transcriptional reprogramming. Thus, AA starvation induced by bacterial pathogens is sensed by the host to trigger protective innate immune and stress responses.

Mitochondria in innate immunity

Mitochondria are cellular organelles involved in host‐cell metabolic processes and the control of programmed cell death. A direct link between mitochondria and innate immune signalling was first highlighted with the identification of MAVS—a crucial adaptor for RIGI‐like receptor signalling—as a mitochondria‐anchored protein. Recently, other innate immune molecules, such as NLRX1, TRAF6, NLRP3 and IRGM have been functionally associated with mitochondria. Furthermore, mitochondrial alarmins—such as mitochondrial DNA and formyl peptides—can be released by damaged mitochondria and trigger inflammation. Therefore, mitochondria emerge as a fundamental hub for innate immune signalling.

pH-dependent internalization of muramyl peptides from early endosomes enables Nod1 and Nod2 signaling

Nod1 and Nod2 are members of the Nod-like receptor family that detect intracellular bacterial peptidoglycan-derived muramyl peptides. The biological effects of muramyl peptides have been described for over three decades, but the mechanism underlying their internalization to the cytosol remains unclear. Using the human epithelial cell line HEK293T as a model system, we demonstrate here that Nod1-activating ligands entered cells through endocytosis, most likely by the clathrin-coated pit pathway, as internalization was dynamin-dependent but not inhibited by methyl-β-cyclodextrin. In the endocytic pathway, the cytosolic internalization of Nod1 ligands was pH-dependent, occurred prior to the acidification mediated by the vacuolar ATPase, and was optimal at pH ranging from 5.5 to 6. Similarly, the Nod2 ligand MDP was internalized into host cytosol through a similar pathway with optimal pH for internalization ranging from 5.5 to 6.5. Moreover, Nod1-activating muramyl peptides likely required processing by endosomal enzymes, prior to transport into the cytosol, suggesting the existence of a sterically gated endosomal transporter for Nod1 ligands. In support for this, we identified a role for SLC15A4, an oligopeptide transporter expressed in early endosomes, in Nod1-dependent NF-κB signaling. Interestingly, SLC15A4 expression was also up-regulated in colonic biopsies from patients with inflammatory bowel disease, a disorder associated with mutations in Nod1 and Nod2. Together, our results shed light on the mechanisms by which muramyl peptides get access to the host cytosol, where they are detected by Nod1 and Nod2, and might have implications for the understanding of human diseases, such as inflammatory bowel disease.

Nod-like proteins in inflammation and disease.

The field of innate immunity has undergone an enormous upheaval during the last decade. The discovery of different groups of proteins, called pattern recognition molecules (PRMs), which detect microbial components, so‐called pathogen‐associated molecular patterns (PAMPs) and trigger protective responses, had a huge impact on the understanding of innate immune responses. Among the PRMs, the intracellular Nod‐like receptors (NLRs) have recently been identified as key mediators of inflammatory and immune responses. The NLR family is divided into subfamilies on the basis of their different signal transduction domains, and recent studies have highlighted the role of certain NLRs, including Nod1, Nod2, Nalp3, Ipaf and Naip5, in the detection of intracellular microbes and possibly ‘danger signals’. In this review, we summarize the current knowledge on the function of these proteins in immunity and inflammation, with a focus on their participation in different disease pathologies. Copyright

Shigella Induces Mitochondrial Dysfunction and Cell Death in Nonmyleoid Cells

Shigella rapidly kills myeloid cells via a caspase-1 inflammasome-dependent cell death mechanism. However, despite a critical role for nonmyeloid cells in the physiopathology of Shigella infection, the mechanism by which Shigella kills nonmyeloid cells remains uncharacterized. Here we demonstrate that, in nonmyeloid cells, Shigella infection induces loss of mitochondrial inner membrane potential, mitochondrial damage, and necrotic cell death through a pathway dependent on Bnip3 and cyclophilin D, two molecules implicated in the host oxidative stress responses. This mitochondrial cell death mechanism was potently counterbalanced by a Nod1-dependent Rip2/IKKbeta/NF-kappaB signaling pathway activated by the pathogen in the first hours of infection. Our results suggest that in nonmyeloid cells, oxidative stress pathways and signaling triggered by an intracellular bacterial pathogen are tightly linked and demonstrate the existence of specific Shigella-induced prodeath and prosurvival pathways converging at the mitochondria to control a necrotic cell death program.

An N-terminal addressing sequence targets NLRX1 to the mitochondrial matrix

NLRX1 is the only member of the Nod-like receptor (NLR) family that is targeted to the mitochondria, and its overexpression induces the generation of reactive oxygen species (ROS), thus impacting on NFκB- and JNK-dependent signaling cascades. In addition, NLRX1 has been shown to interact with MAVS (also known as IPS-1, VISA and Cardif) at the mitochondrial outer membrane and to modulate antiviral responses. Here we report that NLRX1 has a functional leader sequence and fully translocates to the mitochondrial matrix via a mechanism requiring the mitochondrial inner-membrane potential, ΔΨm. Importantly, we failed to detect NLRX1 at the mitochondrial outer membrane. We also show that the leader sequence of NLRX1 is removed, which generates a mature protein lacking the first 39 amino acids through a maturation process that is common for mitochondrial-matrix proteins. Finally, we identified UQCRC2, a matrix-facing protein of the respiratory chain complex III, as an NLRX1-interacting molecule, thus providing a molecular basis for the role of NLRX1 in ROS generation. These results provide the first identification of a protein belonging to the NLR family that is targeted to the mitochondrial matrix.

The Nodosome: Nod1 and Nod2 control bacterial infections and inflammation

Toll-like receptors (TLRs) and the nucleotide-binding domain, leucine rich repeat containing family (or Nod-like receptors, NLRs) are two important families of microbial sensors that are membrane-associated and cytosolic molecules, respectively. The Nod proteins Nod1 and Nod2 are two NLR family members that trigger immune defense in response to bacterial peptidoglycan. Nod proteins fight off bacterial infections by stimulating proinflammatory signaling and cytokine networks and by inducing antimicrobial effectors, such as nitric oxide and antimicrobial peptides. Nod1 is also critically implicated in shaping adaptive immune responses towards bacterial-derived constituents. In addition, recent evidence has demonstrated that mutations in Nod1 and Nod2 are associated with a number of human inflammatory disorders, including Crohn’s disease, Blau syndrome, early-onset sarcoidosis, and atopic diseases. Together, Nod1 and Nod2 represent central players in the control of immune responses to bacterial infections and inflammation.

Enhancement of Reactive Oxygen Species Production and Chlamydial Infection by the Mitochondrial Nod-like Family Member NLRX1

Chlamydia trachomatis infections cause severe and irreversible damage that can lead to infertility and blindness in both males and females. Following infection of epithelial cells, Chlamydia induces production of reactive oxygen species (ROS). Unconventionally, Chlamydiae use ROS to their advantage by activating caspase-1, which contributes to chlamydial growth. NLRX1, a member of the Nod-like receptor family that translocates to the mitochondria, can augment ROS production from the mitochondria following Shigella flexneri infections. However, in general, ROS can also be produced by membrane-bound NADPH oxidases. Given the importance of ROS-induced caspase-1 activation in growth of the chlamydial vacuole, we investigated the sources of ROS production in epithelial cells following infection with C. trachomatis. In this study, we provide evidence that basal levels of ROS are generated during chlamydial infection by NADPH oxidase, but ROS levels, regardless of their source, are enhanced by an NLRX1-dependent mechanism. Significantly, the presence of NLRX1 is required for optimal chlamydial growth.

The role of mitochondria in cellular defense against microbial infection

Mitochondria have been long recognized for their key role in the modulation of cell death pathways. Thus, it is therefore not surprising that this organelle represents a recurrent target for pathogenic microbes, aiming to manipulate the fate of the infected host cell. More recently, mitochondria have been shown to serve as a crucial platform for innate immune signaling, as illustrated by the identification of MAVS (also known as IPS-1, VISA and Cardif), NLRX1 and STING as mitochondrial proteins. This review discusses the tight interplay between microbial infection, innate immune signaling and mitochondria.