Identification of Drosophila Yin and PEPT2 as Evolutionarily Conserved Phagosome-associated Muramyl Dipeptide Transporters

NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-κB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-κB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.     

Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors: A NEW MECHANISM FOR SENSING PATHOGENS*

Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.     

Studies of the pattern recognition molecule H-ficolin: specificity and purification

Ficolins are pattern recognition molecules of the innate immune system. H-ficolin is found in plasma associated with mannan-binding lectin-associated serine proteases (MASPs). When H-ficolin binds to microorganisms the MASPs are activated, which in turn activate the complement system. H-ficolin is the most abundant ficolin in humans, yet its ligand binding characteristics and biological role remain obscure. We examined the binding of H-ficolin to Aerococcus viridans as well as to a more defined artificial target, i.e. acetylated bovine serum albumin. A strict dependence for calcium ions and inhibition at high NaCl concentration was found. The binding to acetylated bovine serum albumin was inhibited by acetylsalicylic acid and sodium acetate as well as by N-acetylated glucosamine and galactosamine (GlcNAc and GalNAc) and glycine (GlyNAc). The binding to A. viridans was sensitive to the same compounds, but, importantly, higher concentrations were needed for inhibition. N-Acetylated cysteine was also inhibitory, but this inhibition was parallel with reduction in the oligomerization of H-ficolin and thus represents structural changes of the molecule. Based on our findings, we developed a procedure for the purification of H-ficolin from serum, involving PEG precipitation, affinity chromatography on Sepharose derivatized with acetylated serum albumin, ion exchange chromatography, and gel permeation chromatography. The purified H-ficolin was observed to elute at 700 kDa, similar to what we find for H-ficolin in whole serum. MASP-2 was co-purified with H-ficolin, and the purified H-ficolin·MASP-2 complex could activate complement as measured by cleavage of complement factor C4. This study extends our knowledge of the specificity of this pattern recognition molecule, and the purified product will enable further studies.     

Multiligand specificity of pathogen-associated molecular pattern-binding site in peptidoglycan recognition protein

The peptidoglycan recognition protein PGRP-S is an innate immunity molecule that specifically interacts with microbial peptidoglycans and other pathogen-associated molecular patterns. We report here two structures of the unique tetrameric camel PGRP-S (CPGRP-S) complexed with (i) muramyl dipeptide (MDP) at 2.5 Å resolution and (ii) GlcNAc and β-maltose at 1.7Å resolution. The binding studies carried out using surface plasmon resonance indicated that CPGRP-S binds to MDP with a dissociation constant of 10⁻⁷ m, whereas the binding affinities for GlcNAc and β-maltose separately are in the range of 10⁻⁴ m to 10⁻⁵ m, whereas the dissociation constant for the mixture of GlcNAc and maltose was estimated to be 10⁻⁶ m. The data from bacterial suspension culture experiments showed a significant inhibition of the growth of Staphylococcus aureus cells when CPGRP-S was added to culture medium. The ELISA experiment showed that the amount of MDP-induced production of TNF-α and IL-6 decreased considerably after the introduction of CPGRP-S. The crystal structure determinations of (i) a binary complex with MDP and (ii) a ternary complex with GlcNAc and β-maltose revealed that MDP, GlcNAc, and β-maltose bound to CPGRP-S in the ligand binding cleft, which is situated at the interface of molecules C and D of the homotetramer formed by four protein molecules A, B, C, and D. In the binary complex, the muramyl moiety of MDP is observed at the C-D interface, whereas the peptide chain protrudes into the center of tetramer. In the ternary complex, GlcNAc and β-maltose occupy distinct non-overlapping positions belonging to different subsites.     

Molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-I (RIG-I)

RIG-I is a major innate immune sensor for viral infection, triggering an interferon (IFN)-mediated antiviral response upon cytosolic detection of viral RNA. Double-strandedness and 5'-terminal triphosphates were identified as motifs required to elicit optimal immunological signaling. However, very little is known about the response dynamics of the RIG-I pathway, which is crucial for the ability of the cell to react to diverse classes of viral RNA while maintaining self-tolerance. In the present study, we addressed the molecular mechanism of RIG-I signal detection and its translation into pathway activation. By employing highly quantitative methods, we could establish the length of the double-stranded RNA (dsRNA) to be the most critical determinant of response strength. Size exclusion chromatography and direct visualization in scanning force microscopy suggested that this was due to cooperative oligomerization of RIG-I along dsRNA. The initiation efficiency of this oligomerization process critically depended on the presence of high affinity motifs, like a 5'-triphosphate. It is noteworthy that for dsRNA longer than 200 bp, internal initiation could effectively compensate for a lack of terminal triphosphates. In summary, our data demonstrate a very flexible response behavior of the RIG-I pathway, in which sensing and integration of at least two distinct signals, initiation efficiency and double strand length, allow the host cell to mount an antiviral response that is tightly adjusted to the type of the detected signal, such as viral genomes, replication intermediates, or small by-products.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

Identification and Characterization of a Chitin-binding Protein Purified from Coelomic Fluid of the Lugworm Arenicola marina Defining a Novel Protein Sequence Family

We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm A. marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass of 27 and 31 kDa in the reduced state. The N-terminal amino acid sequences were identical in these two bands. In the unreduced state, a complex band pattern was observed with bands from 35 kDa to more than 200 kDa. Two different full-length clones encoding polypeptides of 241 and 243 amino acids, respectively, were isolated from a coelomocyte cDNA library. The two clones, designated AML-1a and AML-1b, were 92% identical at the protein level and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a in the reduced state lacked a 27 kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed that AML-1 is expressed by coelomocytes in the nephridium and in round cells in the epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components.     

Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR^N143Q lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR^N143Q in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.     

Dectin-1 Activation Controls Maturation of β-1,3-Glucan-containing Phagosomes

Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.     

NLRP7, a nucleotide oligomerization domain-like receptor protein, is required for normal cytokine secretion and co-localizes with Golgi and the microtubule-organizing center

A hydatidiform mole (HM) is a human pregnancy with hyperproliferative placenta and abnormal embryonic development. Mutations in NLRP7, a member of the nucleotide oligomerization domain-like receptor family of proteins with roles in inflammation and apoptosis, are responsible for recurrent HMs. However, little is known about the functional role of NLRP7. Here, we demonstrate that peripheral blood mononuclear cells from patients with NLRP7 mutations and rare variants secrete low levels of IL-1β and TNF in response to LPS. We show that the cells from patients, carrying mutations or rare variants, have variable levels of increased intracellular pro-IL-1β indicating that normal NLRP7 down-regulates pro-IL-1β synthesis in response to LPS. Using transient transfections, we confirm the role of normal NLRP7 in inhibiting pro-IL-1β and demonstrate that this inhibitory function is abolished by protein-truncating mutations after the Pyrin domain. Within peripheral blood mononuclear cells, NLRP7 co-localizes with the Golgi and the microtubule-organizing center and is associated with microtubules. This suggests that NLRP7 mutations may affect cytokine secretion by interfering, directly or indirectly, with their trafficking. We propose that the impaired cytokine trafficking and secretion caused by NLRP7 defects makes the patients tolerant to the growth of these earlier arrested conceptions with no fetal vessels and that the retention of these conceptions until the end of the first trimester contribute to the molar phenotype. Our data will impact our understanding of postmolar choriocarcinomas, the only allograft non-self tumors that are able to invade maternal tissues.     

Bacterial Pathogen-associated Molecular Patterns Stimulate Biological Activity of Orthopaedic Wear Particles by Activating Cognate Toll-like Receptors

Aseptic loosening of orthopaedic implants is induced by wear particles generated from the polymeric and metallic components of the implants. Substantial evidence suggests that activation of Toll-like receptors (TLRs) may contribute to the biological activity of the wear particles. Although pathogen-associated molecular patterns (PAMPs) produced by Gram-positive bacteria are likely to be more common in patients with aseptic loosening, prior studies have focused on LPS, a TLR4-specific PAMP produced by Gram-negative bacteria. Here we show that both TLR2 and TLR4 contribute to the biological activity of titanium particles with adherent bacterial debris. In addition, lipoteichoic acid, a PAMP produced by Gram-positive bacteria that activates TLR2, can, like LPS, adhere to the particles and increase their biological activity, and the increased biological activity requires the presence of the cognate TLR. Moreover, three lines of evidence support the conclusion that TLR activation requires bacterially derived PAMPs and that endogenously produced alarmins are not sufficient. First, neither TLR2 nor TLR4 contribute to the activity of “endotoxin-free” particles as would be expected if alarmins are sufficient to activate the TLRs. Second, noncognate TLRs do not contribute to the activity of particles with adherent LPS or lipoteichoic acid as would be expected if alarmins are sufficient to activate the TLRs. Third, polymyxin B, which inactivates LPS, blocks the activity of particles with adherent LPS. These results support the hypothesis that PAMPs produced by low levels of bacterial colonization may contribute to aseptic loosening of orthopaedic implants.     

Mitochondria and the Lectin Pathway of Complement

Mitochondria, the powerhouses of our cells, are remnants of a eubacterial endosymbiont. Notwithstanding the evolutionary time that has passed since the initial endosymbiotic event, mitochondria have retained many hallmarks of their eubacterial origin. Recent studies have indicated that during perturbations of normal homeostasis, such as following acute trauma leading to massive necrosis and release of mitochondria, the immune system might mistake symbiont for enemy and initiate an inappropriate immune response. The innate immune system is the first line of defense against invading microbial pathogens, and as such is the primary suspect in the recognition of mitochondria-derived danger-associated molecular patterns and initiation of an aberrant response. Conversely, innate immune mechanisms are also central to noninflammatory clearance of innocuous agents. Here we investigated the role of a central humoral component of innate immunity, the lectin pathway of complement, in recognition of mitochondria in vitro and in vivo. We found that the soluble pattern recognition molecules, mannan-binding lectin (MBL), L-ficolin, and M-ficolin, were able to recognize mitochondria. Furthermore, MBL in complex with MBL-associated serine protease 2 (MASP-2) was able to activate the lectin pathway and deposit C4 onto mitochondria, suggesting that these molecules are involved either in homeostatic clearance of mitochondria or in induction of untoward inflammatory reactions. We found that following mitochondrial challenge, C3 was consumed in vivo in the absence of overt inflammation, indicating a potential role of complement in noninflammatory clearance of mitochondria. Thus, we report here the first indication of involvement of the lectin pathway in mitochondrial immune handling.     

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.     

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H₂O₂ generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H₂O₂ generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹⁷⁸, Ser¹⁸³, Ser²¹², and Thr³⁵¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹⁷⁸ and Ser¹⁸³ in K1 flagellin prevent epitope recognition in rice.     

Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells

Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells.     

Recognition of pathogenic microbes by the Drosophila phagocytic pattern recognition receptor Eater

Non-opsonic phagocytosis is a primordial form of pathogen recognition that is mediated by the direct interaction of phagocytic receptors with microbial surfaces. In the fruit fly Drosophila melanogaster, the EGF-like repeat containing scavenger receptor Eater is expressed by phagocytes and is required to survive infections with gram-positive and gram-negative bacteria. However, the mechanisms by which this receptor recognizes different types of bacteria are poorly understood. To address this problem, we generated a soluble, Fc-tagged receptor variant of Eater comprising the N-terminal 199 amino acids including four EGF-like repeats. We first established that Eater-Fc displayed specific binding to broad yet distinct classes of heat- or ethanol-inactivated microbes and behaved similarly to the membrane-bound, full-length Eater receptor. We then used Eater-Fc as a tool to probe Eater binding to the surface of live bacteria. Eater-Fc bound equally well to naive or inactivated Staphylococcus aureus or Enterococcus faecalis, suggesting that in vivo, Eater directly targets live gram-positive bacteria, enabling their phagocytic clearance and destruction. By contrast, Eater-Fc was unable to interact with live, naive gram-negative bacteria (Escherichia coli, Serratia marcescens, and Pseudomonas aeruginosa). For these bacteria, Eater-Fc binding required membrane-disrupting treatments. Furthermore, we found that cecropin A, a cationic, membrane-disrupting antimicrobial peptide, could promote Eater-Fc binding to live E. coli, even at sublethal concentrations. These results suggest a previously unrecognized mechanism by which antimicrobial peptides cooperate with phagocytic receptors to extend the range of microbes that can be targeted by a single, germline-encoded receptor.