Discriminating symbiosis and immunity signals by receptor competition in rice.

Plants encounter various microbes in nature and must respond appropriately to symbiotic or pathogenic ones. In rice, the receptor-like kinase OsCERK1 is involved in recognizing both symbiotic and immune signals. However, how these opposing signals are discerned via OsCERK1 remains unknown. Here, we found that receptor competition enables the discrimination of symbiosis and immunity signals in rice. On the one hand, the symbiotic receptor OsMYR1 and its short-length chitooligosaccharide ligand inhibit complex formation between OsCERK1 and OsCEBiP and suppress OsCERK1 phosphorylating the downstream substrate OsGEF1, which reduces the sensitivity of rice to microbe-associated molecular patterns. Indeed, OsMYR1 overexpression lines are more susceptible to the fungal pathogen Magnaporthe oryzae, whereas Osmyr1 mutants show higher resistance. On the other hand, OsCEBiP can bind OsCERK1 and thus block OsMYR1-OsCERK1 heteromer formation. Consistently, the Oscebip mutant displayed a higher rate of mycorrhizal colonization at early stages of infection. Our results indicate that OsMYR1 and OsCEBiP receptors compete for OsCERK1 to determine the outcome of symbiosis and immunity signals.

Effectors with chitinase activity (EWCAs), a family of conserved, secreted fungal chitinases that suppress chitin-triggered immunity.

In plants, chitin-triggered immunity is one of the first lines of defense against fungi, but phytopathogenic fungi have developed different strategies to prevent the recognition of chitin. Obligate biotrophs such as powdery mildew fungi suppress the activation of host responses; however, little is known about how these fungi subvert the immunity elicited by chitin. During epiphytic growth, the cucurbit powdery mildew fungus Podosphaera xanthii expresses a family of candidate effector genes comprising nine members with an unknown function. In this work, we examine the role of these candidates in the infection of melon (Cucumis melo L.) plants, using gene expression analysis, RNAi silencing assays, protein modeling and protein-ligand predictions, enzymatic assays, and protein localization studies. Our results show that these proteins are chitinases that are released at pathogen penetration sites to break down immunogenic chitin oligomers, thus preventing the activation of chitin-triggered immunity. In addition, these effectors, designated effectors with chitinase activity (EWCAs), are widely distributed in pathogenic fungi. Our findings reveal a mechanism by which fungi suppress plant immunity and reinforce the idea that preventing the perception of chitin by the host is mandatory for survival and development of fungi in plant environments.

A haustorial-expressed lytic polysaccharide monooxygenase from the cucurbit powdery mildew pathogen Podosphaera xanthii contributes to the suppression of chitin-triggered immunity.

Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a limiting factor of crop productivity. The lifestyle of this fungus is determined by the development of specialized parasitic structures inside epidermal cells, termed haustoria, that are responsible for the acquisition of nutrients and the release of effectors. A typical function of fungal effectors is the manipulation of host immunity, for example the suppression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Chitin is a major component of fungal cell walls, and chitin oligosaccharides are well-known PAMP elicitors. In this work, we examined the role of PHEC27213, the most highly expressed, haustorium-specific effector candidate of P. xanthii. According to different computational predictions, the protein folding of PHEC27213 was similar to that of lytic polysaccharide monooxygenases (LPMOs) and included a conserved histidine brace; however, PHEC27213 had low sequence similarity with LPMO proteins and displayed a putative chitin-binding domain that was different from the canonical carbohydrate-binding module. Binding and enzymatic assays demonstrated that PHEC27213 was able to bind and catalyse colloidal chitin, as well as chitooligosaccharides, acting as an LPMO. Furthermore, RNAi silencing experiments showed the potential of this protein to prevent the activation of chitin-triggered immunity. Moreover, proteins with similar features were found in other haustorium-forming fungal pathogens. Our results suggest that this protein is a new fungal LPMO that catalyses chitooligosaccharides, thus contributing to the suppression of plant immunity during haustorium development. To our knowledge, this is the first mechanism identified in the haustorium to suppress chitin signalling.

Chitin induces steroid-resistant airway inflammation and airway hyperresponsiveness in mice.

BACKGROUND: Previous reports have shown that pathogen-associated patterns (PAMPs) induce the production of interleukin (IL)-1ß in macrophages. Moreover, studies using mouse models also suggest that chitin, which acts as a PAMP, induces adjuvant effects and eosinophilic infiltration in the lung. Thus, we investigated the effects of inhaled chitin in mouse models. METHODS: We developed mouse models of inhaled chitin particle-induced airway inflammation and steroid-resistant ovalbumin (OVA)-induced airway inflammation. Some experimental groups of mice were treated additionally with dexamethasone (DEX). Murine alveolar macrophages (AMs), which were purified from bronchoalveolar lavage (BAL) fluids, were incubated with chitin, and treated with or without DEX. RESULTS: The numbers of total cells, AMs, lymphocytes, eosinophils, and neutrophils among BAL-derived cells, as well as the IL-1ß levels in BAL fluids and the numbers of IL-1ß-positive cells in lung, were significantly increased by chitin stimulation. Airway hyperresponsiveness (AHR) was aggravated in mice of the chitin inflammation model compared to control animals. The production of IL-1ß was significantly increased in murine AMs by chitin treatment, but DEX administration did not inhibit this chitin-induced IL-1ß production. Furthermore, in mouse models, DEX treatment inhibited the OVA-induced airway inflammation and AHR but not the airway inflammation and AHR induced by chitin or the combination of OVA and chitin. CONCLUSIONS: These results suggest that inhaled chitin induces airway inflammation, AHR, and the production of IL-1ß. Furthermore, our findings demonstrate for the first time that inhaled chitin induces steroid-resistant airway inflammation and AHR. Inhaled chitin may contribute to features of steroid-resistant asthma.

Genome-wide association analysis of the strength of the MAMP-elicited defense response and resistance to target leaf spot in sorghum.

Plants have the capacity to respond to conserved molecular features known as microbe-associated molecular patterns (MAMPs). The goal of this work was to assess variation in the MAMP response in sorghum, to map loci associated with this variation, and to investigate possible connections with variation in quantitative disease resistance. Using an assay that measures the production of reactive oxygen species, we assessed variation in the MAMP response in a sorghum association mapping population known as the sorghum conversion population (SCP). We identified consistent variation for the response to chitin and flg22-an epitope of flagellin. We identified two SNP loci associated with variation in the flg22 response and one with the chitin response. We also assessed resistance to Target Leaf Spot (TLS) disease caused by the necrotrophic fungus Bipolaris cookei in the SCP. We identified one strong association on chromosome 5 near a previously characterized disease resistance gene. A moderately significant correlation was observed between stronger flg22 response and lower TLS resistance. Possible reasons for this are discussed.

Chitin: A "Hidden Figure" in the Fungal Cell Wall.

Chitin and chitosan are two related polysaccharides that provide important structural stability to fungal cell walls. Often embedded deeply within the cell wall structure, these molecules anchor other components at the cell surface. Chitin-directed organization of the cell wall layers allows the fungal cell to effectively monitor and interact with the external environment. For fungal pathogens, this interaction includes maintaining cellular strategies to avoid excessive detection by the host innate immune system. In turn, mammalian and plant hosts have developed their own strategies to process fungal chitin, resulting in chitin fragments of varying molecular size. The size-dependent differences in the immune activation behaviors of variably sized chitin molecules help to explain how chitin and related chitooligomers can both inhibit and activate host immunity. Moreover, chitin and chitosan have recently been exploited for many biomedical applications, including targeted drug delivery and vaccine development.

Cross-Microbial Protection via Priming a Conserved Immune Co-Receptor through Juxtamembrane Phosphorylation in Plants.

Living organisms can be primed for potentiated responses to recurring stresses based on prior experience. However, the molecular basis of immune priming remains elusive in plants that lack adaptive immunity. Here, we report that bacterial challenges can prepare plants for fungal attacks by inducing juxtamembrane phosphorylation of CERK1, the co-receptor indispensable for signaling in response to the fungal elicitor chitin. This phosphorylation is mediated by BAK1, a co-receptor for signaling in response to multiple elicitors. BAK1 interacts with CERK1, and loss of BAK1 reduces priming phosphorylation of CERK1. Juxtamembrane phosphomimetic mutations of CERK1 confer accelerated chitin responses and fortified fungal resistance without triggering constitutive immunity, whereas juxtamembrane phosphodeficient mutations diminish bacteria-induced protection against fungal infection. These findings reveal that crosstalk between cell-surface immune co-receptors can prime defense and demonstrate that juxtamembrane phosphorylation of plant receptor-like kinases can occur independent of kinase activation to place the protein into a prime state.

Fungal Chitin Reduces Platelet Activation Mediated via TLR8 Stimulation.

Platelets play an important role in the innate immune response. During candidaemia, circulating fungal polysaccharides, including chitin, are released into the bloodstream and can interact with platelets and induce modulation of platelet activities. However, the role of circulating chitin in platelet modulation has not been investigated. The aims of the present study were to assess the effect of fungal chitin on activation, adhesion, aggregation and receptor expression of platelets and their impact on the host defense against Candida albicans. Platelets pre-treated with different concentrations of chitin (10-400 µg/mL) extracted from C. albicans were analyzed in terms of activation, Toll-like receptor (TLR) expression, aggregation and adhesion to C. albicans. Chitin treatment reduced platelet adhesion to C. albicans and neutrophils. P-selectin expression was significantly decreased in platelets challenged with chitin. Aggregation and intracellular Ca2+ influx were also decreased in platelets. TLR8 mRNA and proteins were expressed in platelets pre-treated with chitin when compared to untreated platelets. Overall, chitin purified from C. albicans reduced the adhesion, activation and aggregation of platelets mediated via TLR8 stimulation by decreasing intracellular Ca2+ influx and P-selectin expression.

Secretion of Phospholipase Dδ Functions as a Regulatory Mechanism in Plant Innate Immunity.

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.

Co-administration of chitin micro-particle and Leishmania antigen proposed a new immune adjuvant against experimental leishmaniasis.

AIMS: We investigated the protective effect of chitin micro-particle (CMP) as an adjuvant against Leishmania infection in BALB/c mice. METHODS: Mice were immunized subcutaneously with soluble Leishmania antigen (SLA) plus CMP (100 µg SLA + 100 µg CMP/100 µL) as the test group. Three weeks after the last immunization, test and control groups were infected by Leishmania major (L major). Eight weeks post-infection, evaluation of parasites load in lymph nodes was performed using limiting dilution assay. Then, the spleen cell cytokine response (TNF-α, IFN-γ, IL-4, IL-10, IL-17 and IL-27) to SLA among vaccinated and nonvaccinated groups was investigated using ELISA. Serum levels of IgG1 and IgG2a were measured as well. RESULTS: The SLA plus CMP group demonstrated the protection. The responses included reduced lesion formation and lower parasite load. Also, in comparison with control group higher levels of IFN-γ and, IL-10 in the culture of spleen cells, and lower levels of IgG1 in sera were seen in SLA plus CMP group. CONCLUSION: The data supported the possibility of using CMP as a suitable adjuvant in Leishmania vaccination.

Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure.

In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.

Maize-Produced Ag2 as a Subunit Vaccine for Valley Fever.

Coccidioides is the causative agent of San Joaquin Valley fever, a fungal disease prevalent in the semiarid regions of the Americas. Efforts to develop a fungal vaccine over the last 2 decades were unsuccessful. A candidate antigen, Antigen 2 (Ag2), is notoriously difficult to express in Escherichia coli, and this study sought to accumulate the antigen at high levels in maize. Transformed maize lines accumulated recombinant Ag2 at levels >1 g/kg. Mice immunized with this antigen and challenged with live Coccidioides arthroconidia showed a reduction in the fungal load when Ag2 derived from either E. coli or maize was loaded into glucan chitin particles. A fusion of Ag2 to dendritic cell carrier peptide (DCpep) induced a T-helper type 17 response in the spleen when orally delivered, indicative of a protective immune response. The maize production platform and the glucan chitin particle adjuvant system show promise for development of a Coccidioides vaccine, but further testing is needed to fully assess the optimal method of administration.

Chitin Prevalence and Function in Bacteria, Fungi and Protists.

Chitin is an important structural polysaccharide, which supports and organizes extracellular matrices in a variety of taxonomic groups including bacteria, fungi, protists, and animals. Additionally, chitin has been recognized as a molecule that is required for Rhizobia-legume symbiosis and involved in arbuscular mycorrhizal signaling in the symbiotic interaction between terrestrial plants and fungi. Moreover, it serves as a unique molecular pattern in the plant defense system against pathogenic fungi and parasites, and in the innate and adaptive immune response of mammals and humans. In this review, we will focus on the prevalence and structural function of chitin in bacteria, fungi, and protists, with a particular focus on the evolution of chitin synthases and the function of chitin oligosaccharides as a signaling molecule in symbiosis and immunity.

Immune Responses of Mammals and Plants to Chitin-Containing Pathogens.

Chitin-containing organisms, such as fungi and arthropods, use chitin as a structural component to protect themselves from harsh environmental conditions. Hosts such as mammals and plants, however, sense chitin to initiate innate and adaptive immunity and exclude chitin-containing organisms. A number of protein factors are then expressed, and several signaling pathways are triggered. In this chapter, we focus on the responses and signal transduction pathways that are activated in mammals and plants upon invasion by chitin-containing organisms. As host chitinases play important roles in the glycolytic processing of chitin, which is then recognized by pattern-recognition receptors, we also pay special attention to the chitinases that are involved in immune recognition.

Reciprocal Inhibition of Adiponectin and Innate Lung Immune Responses to Chitin and Aspergillus fumigatus.

Chitin is a structural biopolymer found in numerous organisms, including pathogenic fungi, and recognized as an immune-stimulating pathogen associated molecular pattern by pattern recognition molecules of the host immune system. However, programming and regulation of lung innate immunity to chitin inhalation in the context of inhalation of fungal pathogens such as Aspergillus fumigatus is complex and our understanding incomplete. Here we report that the systemic metabolism-regulating cytokine adiponectin is decreased in the lungs and serum of mice after chitin inhalation, with a concomitant decrease in surface expression of the adiponectin receptor AdipoR1 on lung leukocytes. Constitutive lung expression of acidic mammalian chitinase resulted in decreased inflammatory cytokine gene expression and neutrophil recruitment, but did not significantly affect lung adiponectin transcription. Exogenous recombinant adiponectin specifically dampened airway chitin-mediated eosinophil recruitment, while adiponectin deficiency resulted in increased airway eosinophils. The presence of adiponectin also resulted in decreased CCL11-mediated migration of bone marrow-derived eosinophils. In contrast to purified chitin, aspiration of viable conidia from the high chitin-expressing A. fumigatus isolate Af5517 resulted in increased neutrophil recruitment and inflammatory cytokine gene expression in adiponectin-deficient mice, while no significant changes were observed in response to the isolate Af293. Our results identify a novel role for the adiponectin pathway in inhibition of lung inflammatory responses to chitin and A. fumigatus inhalation.

Coating With Chitooligosaccharides Enhances the Cytokine Induction of Listeria ivanovii-Based Vaccine Strain.

Listeria ivanovi (LI) is an available live bacterial vaccine vector. This work attempted to coat LI-based vaccine candidates (LI-Rv0129c) with chitooligosaccharides (COSs) as an adjuvant to enhance the cellular immune responses induced. COS-bacteria composite was achieved by mixing the bacteria suspension with equal volume of COS solution, and this process accompanied with the increase of bacteria superficial zeta potential and formation of special superficial configurations. COS coating improved the ratio swallowed by the macrophage-like RAW264.7 cells from 0.54% to 2.88% (p < 0.001). In vivo, the COS-coated LI-Rv0129c strain did elicit significantly higher specific CD4+ IFN-γ, CD4+ TNF-α or CD8+ IFN-γ secretion (0.91%, 1.00%, 0.30%, respectively) than naked LI-Rv0129c (0.32%, 0.38%, 0.07%, respectively) (p < 0.01). These results demonstrated that COS is a promising adjuvant to enhance the protective cellular immune responses induced by LI-based vaccine strains. Our work provided a notion for developing adjuvant for Listeria and other bacterial vector-based vaccines.

Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota.

Mammalian gut microbiota are integral to host health. However, how this association began remains unclear. We show that in basal chordates the gut space is radially compartmentalized into a luminal part where food microbes pass and an almost axenic peripheral part, defined by membranous delamination of the gut epithelium. While this membrane, framed with chitin nanofibers, structurally resembles invertebrate peritrophic membranes, proteome supports its affinity to mammalian mucus layers, where gut microbiota colonize. In ray-finned fish, intestines harbor indigenous microbes, but chitinous membranes segregate these luminal microbes from the surrounding mucus layer. These data suggest that chitin-based barrier immunity is an ancient system, the loss of which, at least in mammals, provided mucus layers as a novel niche for microbial colonization. These findings provide a missing link for intestinal immune systems in animals, revealing disparate mucosal environment in model organisms and highlighting the loss of a proven system as innovation.

Diagnostic utility of serological biomarkers in patients with Crohn's disease: A case-control study.

The aim of this study was to examine the expression of serological markers in patients with inflammatory bowel disease in China, and determine the diagnostic utility of serological markers, individually and in combination, for the diagnosis and differential diagnosis of Crohn's disease (CD).Serum samples were obtained from 160 participants in Eastern China. Among the participants, 98 were diagnosed with CD, 33 had ulcerative colitis (UC), and 29 were healthy controls (HC). The serum samples were tested for the presence of antibodies against outer membrane porin C (anti-OmpC), Pseudomonas fluorescens bacterial sequence I2 (anti-I2), anti-laminarin (anti-L), anti-chitin (anti-C), anti-chitobioside carbohydrate antibody (ACCA), anti-laminaribioside carbohydrate antibody (ALCA), anti-mannobioside carbohydrate antibody (AMCA), and anti-Saccharomyces cerevisiae antibody (ASCA) by indirect enzyme-linked immunosorbent assay (ELISA).Individually, anti-C, anti-L, ASCA-IgG, and ALCA lacked diagnostic value in the differentiation of CD. ASCA-IgA remained the most accurate marker for the diagnosis of CD, with an area under the curve (AUC) of 0.77; however, its sensitivity and specificity were both lower than 75%. Among the combinations of the 5 markers with significant diagnosing ability for CD, combinations with any 2 of the 3 markers, ASCA IgA, AMCA, and ACCA positive, provided the best accuracy in the diagnosis and differential diagnosis of CD (sensitivity and specificity both above 75%) and had the highest Youden index.Serological antibodies, when considered in combination, have remarkable value in the diagnosis and differential diagnosis of CD. Especially, the combination of any 2 of the 3 markers, ASCA-IgA, AMCA, ACCA positive, appears to be optimal.

Chitin and Its Effects on Inflammatory and Immune Responses.

Chitin, a potential allergy-promoting pathogen-associated molecular pattern (PAMP), is a linear polymer composed of N-acetylglucosamine residues which are linked by ß-(1,4)-glycosidic bonds. Mammalians are potential hosts for chitin-containing protozoa, fungi, arthropods, and nematodes; however, mammalians themselves do not synthetize chitin and thus it is considered as a potential target for recognition by mammalian immune system. Chitin is sensed primarily in the lungs or gut where it activates a variety of innate (eosinophils, macrophages) and adaptive immune cells (IL-4/IL-13 expressing T helper type-2 lymphocytes). Chitin induces cytokine production, leukocyte recruitment, and alternative macrophage activation. Intranasal or intraperitoneal administration of chitin (varying in size, degree of acetylation and purity) to mice has been applied as a routine approach to investigate chitin's priming effects on innate and adaptive immunity. Structural chitin present in microorganisms is actively degraded by host true chitinases, including acidic mammalian chitinases and chitotriosidase into smaller fragments that can be sensed by mammalian receptors such as FIBCD1, NKR-P1, and RegIIIc. Immune recognition of chitin also involves pattern recognition receptors, mainly via TLR-2 and Dectin-1, to activate immune cells to induce cytokine production and creation of an immune network that results in inflammatory and allergic responses. In this review, we will focus on various immunological aspects of the interaction between chitin and host immune system such as sensing, interactions with immune cells, chitinases as chitin degrading enzymes, and immunologic applications of chitin.