Fraser Soares

Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry

Autophagy is emerging as a crucial defense mechanism against bacteria, but the host intracellular sensors responsible for inducing autophagy in response to bacterial infection remain unknown. Here we demonstrated that the intracellular sensors Nod1 and Nod2 are critical for the autophagic response to invasive bacteria. By a mechanism independent of the adaptor RIP2 and transcription factor NF-κB, Nod1 and Nod2 recruited the autophagy protein ATG16L1 to the plasma membrane at the bacterial entry site. In cells homozygous for the Crohns disease–associated NOD2 frameshift mutation, mutant Nod2 failed to recruit ATG16L1 to the plasma membrane and wrapping of invading bacteria by autophagosomes was impaired. Our results link bacterial sensing by Nod proteins to the induction of autophagy and provide a functional link between Nod2 and ATG16L1, which are encoded by two of the most important genes associated with Crohns disease.

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.

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.

NOD-LIKE RECEPTORS: VERSATILE CYTOSOLIC SENTINELS

Nucleotide binding oligomerization domain (NOD)-like receptors are cytoplasmic pattern-recognition receptors that together with RIG-I-like receptor (retinoic acid-inducible gene 1), Toll-like receptor (TLR), and C-type lectin families make up the innate pathogen pattern recognition system. There are 22 members of NLRs in humans, 34 in mice, and even a larger number in some invertebrates like sea urchins, which contain more than 200 receptors. Although initially described to respond to intracellular pathogens, NLRs have been shown to play important roles in distinct biological processes ranging from regulation of antigen presentation, sensing metabolic changes in the cell, modulation of inflammation, embryo development, cell death, and differentiation of the adaptive immune response. The diversity among NLR receptors is derived from ligand specificity conferred by the leucine-rich repeats and an NH2-terminal effector domain that triggers the activation of different biological pathways. Here, we describe NLR genes associated with different biological processes and the molecular mechanisms underlying their function. Furthermore, we discuss mutations in NLR genes that have been associated with human diseases.

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.

NLRX1 does not inhibit MAVS-dependent antiviral signalling.

NLRX1 is a member of the Nod-like receptor family of intracellular sensors of microbial- and danger-associated molecular patterns. NLRX1 has a N-terminal mitochondrial addressing sequence that localizes the protein to the mitochondrial matrix. Recently, conflicting reports have been presented with regard to the putative implication of NLRX1 as a negative regulator of MAVS-dependent cytosolic antiviral responses. Here, we generated a new NLRX1 knockout mouse strain and observed that bone marrow-derived macrophages and murine embryonic fibroblasts from NLRX1-deficient mice displayed normal antiviral and inflammatory responses following Sendai virus infection. Importantly, wild type and NLRX1-deficient mice exhibited unaltered antiviral and inflammatory gene expression following intranasal challenge with influenza A virus or i.p. injection of Poly (I:C). Together, our results demonstrate that NLRX1 does not participate in the negative regulation of MAVS-dependent antiviral responses.

Peptidoglycan LD-Carboxypeptidase Pgp2 Influences Campylobacter jejuni Helical Cell Shape and Pathogenic Properties, and Provides the Substrate for the DL-Carboxypeptidase Pgp1

Background: C. jejuni helical shape is important to pathogenesis. Results: Deletion of pgp2 results in loss of C. jejuni helical shape and change in peptidoglycan structure and pathogenic properties. Conclusion: Pgp2 is a ld-carboxypeptidase cleaving peptidoglycan tetrapeptides to tripeptides. Significance: Characterization of enzymes involved in C. jejuni peptidoglycan and cell shape maintenance is crucial to the understanding of fundamental properties of this organism. Despite the importance of Campylobacter jejuni as a pathogen, little is known about the fundamental aspects of its peptidoglycan (PG) structure and factors modulating its helical morphology. A PG dl-carboxypeptidase Pgp1 essential for maintenance of C. jejuni helical shape was recently identified. Bioinformatic analysis revealed the CJJ81176_0915 gene product as co-occurring with Pgp1 in several organisms. Deletion of cjj81176_0915 (renamed pgp2) resulted in straight morphology, representing the second C. jejuni gene affecting cell shape. The PG structure of a Δpgp2 mutant showed an increase in tetrapeptide-containing muropeptides and a complete absence of tripeptides, consistent with ld-carboxypeptidase activity, which was confirmed biochemically. PG analysis of a Δpgp1Δpgp2 double mutant demonstrated that Pgp2 activity is required to generate the tripeptide substrate for Pgp1. Loss of pgp2 affected several pathogenic properties; the deletion strain was defective for motility in semisolid agar, biofilm formation, and fluorescence on calcofluor white. Δpgp2 PG also caused decreased stimulation of the human nucleotide-binding oligomerization domain 1 (Nod1) proinflammatory mediator in comparison with wild type, as expected from the reduction in muropeptide tripeptides (the primary Nod1 agonist) in the mutant; however, these changes did not alter the ability of the Δpgp2 mutant strain to survive within human epithelial cells or to elicit secretion of IL-8 from epithelial cells after infection. The pgp2 mutant also showed significantly reduced fitness in a chick colonization model. Collectively, these analyses enhance our understanding of C. jejuni PG maturation and help to clarify how PG structure and cell shape impact pathogenic attributes.

The mitochondrial protein NLRX1 controls the balance between extrinsic and intrinsic apoptosis.

Background: NLRX1 is a member of the Nod-like receptor family that localizes to the mitochondrial matrix and whose function remains poorly understood. Results: NLRX1 plays a key role in regulating apoptotic cell death and is important in the control of tumorigenesis. Conclusion: NLRX1 regulates the balance between extrinsic and intrinsic apoptosis in cancer cells. Significance: Targeting NLRX1 function could be an interesting strategy against cancer. NLRX1 is a mitochondrial Nod-like receptor (NLR) protein whose function remains enigmatic. Here, we observed that NLRX1 expression was glucose-regulated and blunted by SV40 transformation. In transformed but not primary murine embryonic fibroblasts, NLRX1 expression mediated resistance to an extrinsic apoptotic signal, whereas conferring susceptibility to intrinsic apoptotic signals, such as glycolysis inhibition, increased cytosolic calcium and endoplasmic reticulum stress. In a murine model of colorectal cancer induced by azoxymethane, NLRX1−/− mice developed fewer tumors than wild type mice. In contrast, in a colitis-associated cancer model combining azoxymethane and dextran sulfate sodium, NLRX1−/− mice developed a more severe pathology likely due to the increased sensitivity to dextran sulfate sodium colitis. Together, these results identify NLRX1 as a critical mitochondrial protein implicated in the regulation of apoptosis in cancer cells. The unique capacity of NLRX1 to regulate the cellular sensitivity toward intrinsic versus extrinsic apoptotic signals suggests a critical role for this protein in numerous physiological processes and pathological conditions.