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Multicellular organisms activate immunity upon recognition of pathogen-associated molecular patterns (PAMPs). Chitin is the major component of fungal cell walls, and chitin oligosaccharides act as PAMPs in plant and mammalian cells. Microbial pathogens deliver effector proteins to suppress PAMP-triggered host immunity and to establish infection. Here, we show that the LysM domain-containing effector protein Ecp6 of the fungal plant pathogen Cladosporium fulvum mediates virulence through perturbation of chitin-triggered host immunity. During infection, Ecp6 sequesters chitin oligosaccharides that are released from the cell walls of invading hyphae to prevent elicitation of host immunity. This may represent a common strategy of host immune suppression by fungal pathogens, because LysM effectors are widely conserved in the fungal kingdom.

Cladosporium cladosporioides causes asthma and superficial and deep infections, mostly in immunodeficient individuals and animals. This study aimed to investigate whether C. cladosporioides spores can enter the lungs through pulmonary circulation and influence pulmonary immune response. We intravenously injected mice with C. cladosporioides spore suspension and conducted several assays on the lungs. Pulmonary hemorrhage symptoms and congestion were most severe on days 1, 2, and 3 post-inoculation (PI). Extensive inflammatory cell infiltration occurred throughout the period of infection. More spores and hyphae colonizing the lungs were detected on days 1, 2, and 3 PI, and fewer spores and hyphae were observed within 21 d of infection. Numerous macrophages, dendritic cells, and neutrophils were observed on day 5 PI, along with upregulation of CD54, an intercellular adhesion molecule. Th1 and Th2 cells increased after infection; specifically, Th2 cells increased considerably on day 5 PI. These results suggest that days 2 and 5 PI represent the inflammatory peak in the lungs and that the Th2 and Th1 signaling pathways are potentially involved in pulmonary immune responses. In conclusion, the further adaptive immune responses played important roles in establishing effective pulmonary immunity against C. cladosporioides systemic infections based on innate immune responses.

Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc)6] at 1.95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc)6 binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc)6 molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc)6 positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6 enabled the elucidation of the precise topography and amino acid composition of Avr4's ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4's stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family.

While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex. The ability to launch an immune response is not unique to animals. Plants have also evolved the ability to detect molecules present on the surface of pathogens such as fungi. These molecular signatures are known as pathogen-associated molecular patterns (PAMPs), and they are detected by specialized receptors on the surface of plant cells. Chitin, the main structural component of the cell wall in fungi, is one example of a PAMP. Many species of plants are able to detect chitin using receptors that contain sequences of amino acids called lysin motifs. Previous work in the model plant Arabidopsis has shown that chitin binds to a single lysin motif within each plant receptor. However, just as plants have evolved the ability to recognize PAMPs, so fungi have evolved ways to outwit plants. They have developed small molecules called effector proteins that bind to PAMPs, in effect hiding them from the plant receptors. The tomato fungus Cladosporium fulvum, for example, secretes an effector protein called Ecp6, which contains lysin motifs just like those in the plant receptors. By binding chitin fragments, Ecp6 helps the fungus to avoid detection by its host plant. Now, Sánchez-Vallet et al. present the high resolution crystal structure of Ecp6 and reveal the mechanism by which it outcompetes the plant’s own chitin receptors. In the presence of chitin, two lysin binding motifs within the Ecp6 protein combine to produce a binding site with ultrahigh affinity for chitin. This can outcompete the plant receptors, which use only a single lysin domain to bind the fungal protein. As well as providing a molecular explanation for how certain fungi manage to evade the immune response in plants, the work of Sánchez-Vallet et al. offers an unusual example of convergent evolution, in which two evolutionarily distant organisms have evolved the ability to recognize the same molecule through structurally diverse proteins.

is an opportunistic fungal pathogen responsible for a spectrum of clinical manifestations. Dendritic cells recognize pathogen-associated molecular patterns of via two main receptor families, Toll-like receptors (TLRs) and C-type lectin receptors (CLR). Here, the importance of TLR and CLR signaling in the regulation of T-helper cell type 2 (Th2) responses was analyzed using a mouse model based on the transfer of bone marrow-derived dendritic cells (BMDCs) pulsed with conidia. BMDCs were generated from mice deficient in either MyD88 or MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1). Both the MyD88 and MALT1 signaling pathway in BMDCs contributed to the production of inflammatory cytokines induced by conidia. Mice sensitized with MyD88 BMDCs pulsed with conidia showed an exacerbated allergic inflammation, with stronger eosinophil recruitment in the BAL and higher Th2 cytokine production compared with mice sensitized with wild-type or MALT1 BMDCs. This exacerbation was not observed when MyD88 BMDCs were pulsed with , a nonpathogenic mold. A lack of TLR2 signaling recapitulated the exacerbation of the Th2 response observed in the absence of MyD88 signaling, whereas TLR2 agonist dampened the response induced with and conidia. IL-10 production by BMDCs in response to was dependent on the expression of TLR2 and MyD88. IL-10 BMDCs exacerbated, whereas MyD88 BMDCs supplemented with exogenous IL-10 decreased the allergic pulmonary inflammation. These results indicate that TLR2/MyD88-specific recognition of PAMPs from conidia can upregulate IL-10 production and downregulate lung eosinophilia and the development of a Th2 response.

Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants.

Background: The identification of the factors associated with severe asthma may shed some light on its etiology and on the mechanisms of its development. We aimed to describe asthma severity using the Global Initiative for Asthma (GINA) classification and to investigate its determinants in a cross-sectional, population-based sample in Europe. Methods: In the European Community Respiratory Health Survey II (1999–2002), 1,241 adults with asthma were identified. Severity was assessed using the 2002 GINA classification (intermittent, mild persistent, moderate persistent, severe persistent) and it was related to potential determinants by a multinomial logistic model, using the intermittent group as the reference category for relative risk ratios. Results: About 30% of asthmatic subjects were affected by moderate-to-severe asthma. Sensitization to Cladosporium was associated with a more than 5-fold greater risk of having (mild, moderate or severe) persistent asthma than intermittent asthma. Persistent asthma was positively associated with sensitization to house dust mite, nonseasonal asthma, an older age at asthma onset, and chronic cough and phlegm. Sensitization to cat increased the risk of severe asthma only. Smoking was more strongly associated with asthma severity in men, while rhinitis was more strongly associated with asthma severity in women. Conclusions: One third of the asthmatic population have moderate-to-severe asthma. Sensitization to perennial indoor allergens, particularly Cladosporium, is strongly associated with asthma severity. The role of smoking and rhinitis in determining asthma severity may differ between the sexes, and it should be further investigated.

Background:Cladosporium is an important allergenic fungus worldwide. We report here a major allergen of C. cladosporioides.Methods: Major C. cladosporioides allergens were characterized by immunoblotting, N-terminal amino acid sequencing, protein purification and cDNA cloning. Results: Seventy-four sera (38%) from 197 bronchial asthmatic patients demonstrated IgE binding against C. cladosporioides extracts. Among these 74 sera, 41 (55%) and 38 (51%) showed IgE binding against a 36- and a 20-kDa protein of C. cladosporioides, respectively. Both IgE-reacting components reacted with FUM20, a monoclonal antibody against fungal serine proteases. N-terminal amino acid sequencing results suggest that they are vacuolar serine proteases, and the 20-kDa component is possibly a degraded product of the 36-kDa allergen. A corresponding 5′-truncated 1,425-bp cDNA fragment was isolated. The mature protein after N-terminal processing starts with an N-terminal serine that is the ninth residue encoded by the 5′-truncated cDNA. The protein sequence deduced shares 69–72% sequence identity with Penicillium vacuolar serine proteases and was designated as Cla c 9. The purified 36-kDa Cla c 9 allergen showed proteolytic activity with peptide Z-Ala-Ala-Leu-pNA as substrate. IgE cross-reactivity was detected between the purified Cla c 9 and serine protease allergens from Aspergillusfumigatus and Penicillium chrysogenum.Conclusion: We identified a vacuolar serine protease as a major allergen of C. cladosporioides (Cla c 9) and a major pan-allergen of prevalent airborne fungi. IgE cross-reactivity among these highly conserved serine protease pan-fungal allergens was also detectable.

Abstract Objective: To assess whether the severity of asthma is associated with sensitisation to airborne moulds rather than to other seasonal or perennial allergens. Design: Multicentre epidemiological survey in 30 centres. Setting: European Community respiratory health survey. Participants: 1132 adults aged 20-44 years with current asthma and with skin prick test results. Main outcome measure: Severity of asthma according to score based on forced expiratory volume in one second, number of asthma attacks, hospital admissions for breathing problems, and use of corticosteroids in past 12 months. Results: The frequency of sensitisation to moulds (Alternaria alternata or Cladosporium herbarum, or both) increased significantly with increasing asthma severity (odds ratio 2.34 (95% confidence interval 1.56 to 3.52) for either for severe v mild asthma). This association existed in all of the study areas (gathered into regions), although there were differences in the frequency of sensitisation. There was no association between asthma severity and sensitisation to pollens or cats. Sensitisation to Dermatophagoides pteronyssinus was also positively associated with severity. In multivariable logistic regressions including sensitisation to moulds, pollens, D pteronyssinus, and cats simultaneously, the odds ratios for sensitisation to moulds were 1.48 (0.97 to 2.26) for moderate v mild asthma and 2.16 (1.37 to 3.35) for severe v mild asthma (P<0.001 for trend). Conclusions: Sensitisation to moulds is a powerful risk factor for severe asthma in adults. This should be taken into account in primary prevention, management, and patients' education.

Alternaria and Cladosporium, known as the most allergenic spores were first collected by means of Durham gravimetric sampler from Eskisehir atmosphere from January 1, 2000 to December 31, 2001. The daily, monthly and annual variations in spores/cm² of Cladosporium and Alternaria were recorded. During this period, a total of 10.231 spores belonging to Cladosporium and Alternaria genera were recorded. Of these spores, 5,103 were identified in 2000 and 5,128 in 2001. While 63.09% of the total spores were those of Cladosporium, 36.91% were of Alternaria. Relationships between airborne fungal spore presence and meteorological conditions were statistically investigated. A Shapiro-Wilk test revealed that the airborne Cladosporium and Alternaria spores differed from a normal distribution. Thus, a Friedmann test was performed followed by a Pearson Correlation Analysis. The effects of rainfall, temperature and wind speed on Cladosporium and Alternaria numbers were non-significant according to the sites and months (p > 0.05), but the effects of relative humidity on Cladosporium and Alternaria numbers were significant (p < 0.01). Spore concentrations reached to their highest levels in May 2001.