The fungal pattern recognition receptor, Dectin-1, and the associated cluster of C-type lectin-like receptors

The mammalian natural killer gene complex (NKC) contains several families of type II transmembrane C-type lectin-like receptors (CLRs) that are best known for their involvement in the detection of virally infected or transformed cells, through the recognition of endogenous (or self) proteinacious ligands. However, certain CLR families within the NKC, particularly those expressed by myeloid cells, recognize structurally diverse ligands and perform a variety of other immune and homoeostatic functions. One such family is the 'Dectin-1 cluster' of CLRs, which includes MICL, CLEC-2, CLEC12B, CLEC9A, CLEC-1, Dectin-1 and LOX-1. Here, we review each of these CLRs, exploring our current understanding of their ligands and functions and highlighting where they have provided new insights into the underlying mechanisms of immunity and homeostasis.     

Host-microbe interactions: innate pattern recognition of fungal pathogens

The recognition of fungi is mediated by germline pattern recognition receptors (PRRs) such as Toll-like receptors and lectin receptors that interact with conserved structures of the microorganisms, the pathogen-associated molecular patterns (PAMPs). Subsequently, PRRs activate intracellular signals that collaborate for the efficient activation of the host defense. The specificity of these responses is achieved through the activation of a particular mosaic of PRRs, that is determined by the available fungal PAMPs and the innate immune cells involved. This will determine a divergence of the final type of reaction, and in this way the innate host defense has the capability to deliver tailored responses to each pathogen.     

Pulmonary surfactant protein A up-regulates activity of the mannose receptor,a pattern recognition receptor expressed on human macrophages

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.     

Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products

Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to natural Borrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent responses were seen using whole Mycobacterium avium and Staphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor in host immune responses to microbial invasion.     

Plant pattern recognition receptor complexes at the plasma membrane

A key feature of innate immunity is the ability to recognize and respond to potential pathogens in a highly sensitive and specific manner. In plants, the activation of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) elicits a defense programme known as PAMP-triggered immunity (PTI). Although only a handful of PAMP-PRR pairs have been defined, all known PRRs are modular transmembrane proteins containing ligand-binding ectodomains. It is becoming clear that PRRs do not act alone but rather function as part of multi-protein complexes at the plasma membrane. Recent studies describing the molecular interactions and protein modifications that occur between PRRs and their regulatory proteins have provided important mechanistic insight into how plants avoid infection and achieve immunity.     

A role for human Sp alpha as a pattern recognition receptor

Human Sp alpha is a soluble protein belonging to group B of the scavenger receptor cysteine-rich (SRCR) superfamily for which little functional information is available. It is expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), and it binds to myelomonocytic and lymphoid cells, which suggests that it may play an important role in the regulation of the innate and adaptive immune systems. In the present study we show that recombinant human Sp alpha (rSp alpha) binds to the surface of several gram-positive and gram-negative bacterial strains. Competition studies indicated that such binding is mediated by the recognition of lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, through nonoverlapping sites on the Sp alpha molecule. The most conserved part of LPS (2-keto-3-deoxyoctulosonic acid and lipid A) was shown to be involved in the recognition by Sp alpha. Bacterial binding studies using the SRCR domain 1 of Sp alpha showed that this domain retains both the LPS and LTA binding activities, indicating that both bacterial interacting sites are retained in a single SRCR domain. Furthermore, rSp alpha induced aggregation of gram-positive and gram-negative bacteria strains. On the other hand, rSp alpha inhibited tumor necrosis factor-alpha secretion by human monocytes stimulated with LPS or LTA. Binding of Sp alpha to conserved components of bacterial surfaces and modulation of the monocyte response indicate that this molecule is an active constituent of the innate immune response of the host.     

细胞质内模式识别受体及其抗病毒免疫研究

先天性免疫监视机制的核心是通过模式识别受体(pattern recognition receptors,PRRs)识别病毒分子诱导抗病毒防御,使宿主免受感染。PRRs表达在不同类型细胞的不同细胞区室,包括细胞膜、内体膜、溶酶体膜和胞质。病毒进入细胞区室后将被一个或多个模式识别受体所识别并激活机体的免疫反应。主要对细胞质内模式识别受体视黄酸诱导基因I样受体(retinoic acid-inducible gene I(RIG-I)-like receptors,RLRs)、核苷酸结合寡聚化结构域样受体(nucleotide-binding oligomerization domain(NOD)-like receptors,NLRs)、DEXDc螺旋酶受体(DLRs)及最近发现的DNA模式识别分子——DAI(DNA-dependent activator of interferonregulatory factors)识别病毒核酸并诱导I型干扰素产生的分子机制作一综述。     

Vitellogenin functions as a multivalent pattern recognition receptor with an opsonic activity

Background

Vitellogenin (Vg), a major reproductive protein, has been associated with infection-resistant response in fish. However, the underlying mechanisms by which Vg is involved in anti-infectious response are not understood.

Methodology/Results

By both protein-microbe interaction analysis and enzyme-linked immunosorbent assay as well as phagocytosis test, we demonstrate for the first time that fish Vg acts as a pattern recognition molecule with multiple specificities that can recognize bacteria as well as fungus rather than self components from fish, and functions as an opsonin that can enhance macrophage phagocytosis.

Conclusions

This study shows that fish Vg plays an integrative function in regulating immunity via its pleiotropic effects on both recognizing pathogen-associated molecular patterns and promoting macrophage phagocytosis. It also supports the notion that factors normally involved in control of female reproduction are associated with immunity in organisms that rely on Vg for oocyte development.     

Genesis of a fungal non-self recognition repertoire

Conspecific allorecognition, the ability for an organism to discriminate its own cells from those of another individual of the same species, has been developed by many organisms. Allorecognition specificities are determined by highly polymorphic genes. The processes by which this extreme polymorphism is generated remain largely unknown. Fungi are able to form heterokaryons by fusion of somatic cells, and somatic non self-recognition is controlled by heterokaryon incompatibility loci (het loci). Herein, we have analyzed the evolutionary features of the het-d and het-e fungal allorecognition genes. In these het genes, allorecognition specificity is determined by a polymorphic WD-repeat domain. We found that het-d and het-e belong to a large gene family with 10 members that all share the WD-repeat domain and show that repeats of all members of the family undergo concerted evolution. It follows that repeat units are constantly exchanged both within and between members of the gene family. As a consequence, high mutation supply in the repeat domain is ensured due to the high total copy number of repeats. We then show that in each repeat four residues located at the protein/protein interaction surface of the WD-repeat domain are under positive diversifying selection. Diversification of het-d and het-e is thus ensured by high mutation supply, followed by reshuffling of the repeats and positive selection for favourable variants. We also propose that RIP, a fungal specific hypermutation process acting specifically on repeated sequences might further enhance mutation supply. The combination of these evolutionary mechanisms constitutes an original process for generating extensive polymorphism at loci that require rapid diversification.     

The therapeutic potential of the humoral pattern recognition molecule PTX3 in chronic lung infection caused by Pseudomonas aeruginosa

Chronic lung infections by Pseudomonas aeruginosa strains are a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Although there is no clear evidence for a primary defect in the immune system of CF patients, the host is generally unable to clear P. aeruginosa from the airways. PTX3 is a soluble pattern recognition receptor that plays nonredundant roles in the innate immune response to fungi, bacteria, and viruses. In particular, PTX3 deficiency is associated with increased susceptibility to P. aeruginosa lung infection. To address the potential therapeutic effect of PTX3 in P. aeruginosa lung infection, we established persistent and progressive infections in mice with the RP73 clinical strain RP73 isolated from a CF patient and treated them with recombinant human PTX3. The results indicated that PTX3 has a potential therapeutic effect in P. aeruginosa chronic lung infection by reducing lung colonization, proinflammatory cytokine levels (CXCL1, CXCL2, CCL2, and IL-1β), and leukocyte recruitment in the airways. In models of acute infections and in in vitro assays, the prophagocytic effect of PTX3 was maintained in C1q-deficient mice and was lost in C3- and Fc common γ-chain-deficient mice, suggesting that facilitated recognition and phagocytosis of pathogens through the interplay between complement and FcγRs are involved in the therapeutic effect mediated by PTX3. These data suggested that PTX3 is a potential therapeutic tool in chronic P. aeruginosa lung infections, such as those seen in CF patients.     

A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D-carboxypeptidase activity

The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-A resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.     

Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor gamma chain to induce innate immune responses

Antigen presenting cells recognize pathogens via pattern recognition receptors (PRR), which upon ligation transduce intracellular signals that can induce innate immune responses. Because some C-type lectin-like receptors (e.g. dectin-1 and DCSIGN) were shown to act as PRR for particular microbes, we considered a similar role for dectin-2. Binding assays using soluble dectin-2 receptors showed the extracellular domain to bind preferentially to hyphal (rather than yeast/conidial) components of Candida albicans, Microsporum audouinii, and Trichophyton rubrum. Selective binding for hyphae was also observed using RAW macrophages expressing dectin-2, the ligation of which by hyphae or cross-linking with dectin-2-specific antibody led to protein tyrosine phosphorylation. Because dectin-2 lacks an intracellular signaling motif, we searched for a signal adaptor that permits it to transduce intracellular signals. First, we found that the Fc receptor gamma (FcRgamma) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcRgamma, activation of NF-kappaB, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor alpha and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcRgamma chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcRgamma to induce innate immune responses.     

Prediction of pattern recognition receptor family using pseudo-amino acid composition

Pattern recognition receptors (PRRs) play a key role in the innate immune response by recognizing pathogen associated molecular patterns derived from a diverse collection of microbial pathogens. PRRs form a superfamily of proteins related to host health and disease. Thus, prediction of PRR family might supply biologically significant information for functional annotation of PRRs and development of novel drugs. In this paper, a computational method is proposed for predicting the families of PRRs. The prediction was performed on the basis of amino acid composition and pseudo-amino acid composition (PseAAC) from primary sequences of proteins using support vector machines. A non-redundant dataset consisted of 332 PRRs in seven families was constructed to do training and testing. It was demonstrated that different families of PRRs were quite closely correlated with amino acid composition as well as PseAAC. In the jackknife test, overall accuracies of amino acid composition-based and PseAAC-based classifiers reached 96.1% and 97.9%, respectively. The results indicate that families of PRRs are predictable with high accuracy. It is anticipated that this computational method might be a powerful tool for the automated assignment of families of PRRs.     

Structural insights into recognition of triple-helical beta-glucans by an insect fungal receptor

The innate ability to detect pathogens is achieved by pattern recognition receptors, which recognize non-self-components such as β1,3-glucan. β1,3-Glucans form a triple-helical structure stabilized by interchain hydrogen bonds. β1,3-Glucan recognition protein (βGRP)/gram-negative bacteria-binding protein 3 (GNBP3), one of the pattern recognition receptors, binds to long, structured β1,3-glucan to initiate innate immune response. However, binding details and how specificity is achieved in such receptors remain important unresolved issues. We solved the crystal structures of the N-terminal β1,3-glucan recognition domain of βGRP/GNBP3 (βGRP-N) in complex with the β1,3-linked glucose hexamer, laminarihexaose. In the crystals, three structured laminarihexaoses simultaneously interact through six glucose residues (two from each chain) with one βGRP-N. The spatial arrangement of the laminarihexaoses bound to βGRP-N is almost identical to that of a β1,3-glucan triple-helical structure. Therefore, our crystallographic structures together with site-directed mutagenesis data provide a structural basis for the unique recognition by such receptors of the triple-helical structure of β1,3-glucan.     

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.     

PEPPeR, a platform for experimental proteomic pattern recognition

Quantitative proteomics holds considerable promise for elucidation of basic biology and for clinical biomarker discovery. However, it has been difficult to fulfill this promise due to over-reliance on identification-based quantitative methods and problems associated with chromatographic separation reproducibility. Here we describe new algorithms termed "Landmark Matching" and "Peak Matching" that greatly reduce these problems. Landmark Matching performs time base-independent propagation of peptide identities onto accurate mass LC-MS features in a way that leverages historical data derived from disparate data acquisition strategies. Peak Matching builds upon Landmark Matching by recognizing identical molecular species across multiple LC-MS experiments in an identity-independent fashion by clustering. We have bundled these algorithms together with other algorithms, data acquisition strategies, and experimental designs to create a Platform for Experimental Proteomic Pattern Recognition (PEPPeR). These developments enable use of established statistical tools previously limited to microarray analysis for treatment of proteomics data. We demonstrate that the proposed platform can be calibrated across 2.5 orders of magnitude and can perform robust quantification of ratios in both simple and complex mixtures with good precision and error characteristics across multiple sample preparations. We also demonstrate de novo marker discovery based on statistical significance of unidentified accurate mass components that changed between two mixtures. These markers were subsequently identified by accurate mass-driven MS/MS acquisition and demonstrated to be contaminant proteins associated with known proteins whose concentrations were designed to change between the two mixtures. These results have provided a real world validation of the platform for marker discovery.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.