Identification of a vicilin-like major allergen from Prosopis juliflora exhibiting cross- reactivity with legume food allergens.

BACKGROUND: Prosopis juliflora is a clinically relevant allergic sensitizer worldwide and shares cross-reactivity with allergens from several tree pollen and food. The present study aims to purify and immunobiochemically characterize a major allergen from Prosopis pollen. The allergen was further investigated for its cross-reactivity with legume allergens. METHODS: Prosopis extract was fractionated by Q Sepharose and Superdex 75 gel filtration column to purify the allergen. Specific IgE against purified protein was estimated via ELISA and immunoblot. The protein was subjected to mass spectrometric analysis. Glycan characterization was performed by Schiff staining and lectin binding assay followed by deglycosylation studies. The functional activity of the purified protein was evaluated by the basophil activation test. Cross-reactivity was assessed by inhibition studies with legume extracts. RESULTS: A 35 kDa protein was purified and showed 75% IgE reactivity with the patients' sera by ELISA and immunoblot. Glycan characterization of protein demonstrated the presence of terminal glucose and mannose residues. A reduction of 40% and 27% in IgE binding was observed upon chemical and enzymatic deglycosylation of the protein, respectively. The glycoprotein allergen upregulates the expression of CD203c on basophils which was significantly reduced upon deglycosylation, signifying its biological ability to activate the effector cells. The identified protein shared significant homology with Lup an 1 from the lupine bean. Immunoblot inhibition studies of the purified allergen with legume extracts underlined high cross-reactive potential. Complete inhibition was observed with peanut and common bean, while up to 70% inhibition was demonstrated with soy, black gram, chickpea, and lima bean. CONCLUSION: A 35 kDa vicilin-like major allergen was isolated from P. juliflora. The protein possesses glycan moieties crucial for IgE binding and basophil activation. Furthermore, the purified protein shows homology with Lup an 1 and exhibits cross-reactivity with common edible legume proteins.

Predicting the allergenicity of legume proteins using a PBMC gene expression assay.

BACKGROUND: Food proteins differ in their allergenic potential. Currently, there is no predictive and validated bio-assay to evaluate the allergenicity of novel food proteins. The objective of this study was to investigate the potential of a human peripheral blood mononuclear cell (PBMC) gene expression assay to identify biomarkers to predict the allergenicity of legume proteins. RESULTS: PBMCs from healthy donors were exposed to weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine) in three experiments. Possible biomarkers for allergenicity were investigated by exposing PBMCs to a protein pair of weakly (white bean) and strongly allergenic (soybean) 7S globulins in a pilot experiment. Gene expression was measured by RNA-sequencing and differentially expressed genes were selected as biomarkers. 153 genes were identified as having significantly different expression levels to the 7S globulin of white bean compared to soybean. Inclusion of multiple protein pairs from 2S albumins (lupine and peanut) and 7S globulins (white bean and soybean) in a larger study, led to the selection of CCL2, CCL7, and RASD2 as biomarkers to distinguish weakly from strongly allergenic proteins. The relevance of these three biomarkers was confirmed by qPCR when PBMCs were exposed to a larger panel of weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine). CONCLUSIONS: The PBMC gene expression assay can potentially distinguish weakly from strongly allergenic legume proteins within a protein family, though it will be challenging to develop a generic method for all protein families from plant and animal sources. Graded responses within a protein family might be of more value in allergenicity prediction instead of a yes or no classification.

Coconut allergy: Characteristics of reactions and diagnostic predictors in a pediatric tertiary care center.

BACKGROUND: Little is known on the clinical manifestations of coconut allergy. Our knowledge to date is mainly based on case reports. OBJECTIVE: To characterize the allergic reactions to coconut and suggest diagnostic cutoffs for specific immunoglobulin E (sIgE) and skin prick testing (SPT) to predict clinically reactive coconut allergy. METHODS: Methods include retrospective chart review at an urban tertiary care center of patients with positive testing result for coconut. Probability curves were computed by logistic regression for SPT and coconut sIgE. RESULTS: Of 275 records reviewed, 69 patients reported coconut reactions and 206 were sensitized only or nonallergic. The reactions occurred with breastfeeding (n = 2), contact (n = 10), or oral ingestion (n = 57). Approximately 50% of oral ingestion reactions were associated with mild/moderate anaphylaxis. Clinical reactivity vs sensitization was more common in topical coconut users (2-fold) (P = .02). Although not statistically significant, there was a trend toward more coconut allergy vs sensitization in Asian and African American patients. The probability of allergy with positive SPT result was approximately 50% and with sIgE was approximately 60%. At an SPT of 9 mm wheal or sIgE of 58 kU of allergen/L, there is a 95% probability of reaction. Cosensitization with tree nuts, legumes, and seeds was common. Macadamia nut had the strongest correlation with coconut (r = 0.81, P < .001, n = 101). CONCLUSION: Although the rate of reactivity to coconut in sensitized individuals is low, half of the reactions from consumption met the criteria for anaphylaxis. Clinicians should be aware of the spectrum of reactions and diagnostic use of sIgE and SPT.

Transcriptional frameshifts contribute to protein allergenicity.

Transcription infidelity (TI) is a mechanism that increases RNA and protein diversity. We found that single-base omissions (i.e., gaps) occurred at significantly higher rates in the RNA of highly allergenic legumes. Transcripts from peanut, soybean, sesame, and mite allergens contained a higher density of gaps than those of nonallergens. Allergen transcripts translate into proteins with a cationic carboxy terminus depleted in hydrophobic residues. In mice, recombinant TI variants of the peanut allergen Ara h 2, but not the canonical allergen itself, induced, without adjuvant, the production of anaphylactogenic specific IgE (sIgE), binding to linear epitopes on both canonical and TI segments of the TI variants. The removal of cationic proteins from bovine lactoserum markedly reduced its capacity to induce sIgE. In peanut-allergic children, the sIgE reactivity was directed toward both canonical and TI segments of Ara h 2 variants. We discovered 2 peanut allergens, which we believe to be previously unreported, because of their RNA-DNA divergence gap patterns and TI peptide amino acid composition. Finally, we showed that the sIgE of children with IgE-negative milk allergy targeted cationic proteins in lactoserum. We propose that it is not the canonical allergens, but their TI variants, that initiate sIgE isotype switching, while both canonical and TI variants elicit clinical allergic reactions.

Legumes Protease Inhibitors as Biopesticides and Their Defense Mechanisms against Biotic Factors.

Legumes are affected by biotic factors such as insects, molds, bacteria, and viruses. These plants can produce many different molecules in response to the attack of phytopathogens. Protease inhibitors (PIs) are proteins produced by legumes that inhibit the protease activity of phytopathogens. PIs are known to reduce nutrient availability, which diminishes pathogen growth and can lead to the death of the pathogen. PIs are classified according to the specificity of the mechanistic activity of the proteolytic enzymes, with serine and cysteine protease inhibitors being studied the most. Previous investigations have reported the efficacy of these highly stable proteins against diverse biotic factors and the concomitant protective effects in crops, representing a possible replacement of toxic agrochemicals that harm the environment.

A Minimal Genetic Passkey to Unlock Many Legume Doors to Root Nodulation by Rhizobia.

On legume crops, formation of developmentally mature nodules is a prerequisite to efficient nitrogen fixation by populations of rhizobial bacteroids established inside nodule cells. Development of root nodules and concomitant microbial colonisation of plant cells are constrained by sets of recognition signals exchanged by infecting rhizobia and their legume hosts, with much of the specificity of symbiotic interactions being determined by the flavonoid cocktails released by legume roots and the strain-specific nodulation factors (NFs) secreted by rhizobia. Hence, much of Sinorhizobium fredii strain NGR234 symbiotic promiscuity was thought to stem from a family of >80 structurally diverse NFs and associated nodulation keys in the form of secreted effector proteins and rhamnose-rich surface polysaccharides. Here, we show instead that a mini-symbiotic plasmid (pMiniSym2) carrying only the nodABCIJ, nodS and nodD1 genes of NGR234 conferred promiscuous nodulation to ANU265, a derivative strain cured of the large symbiotic plasmid pNGR234a. The ANU265::pMiniSym2 transconjugant triggered nodulation responses on 12 of the 22 legumes we tested. On roots of Macroptilium atropurpureum, Leucaena leucocephala and Vigna unguiculata, ANU265::pMiniSym2 formed mature-like nodule and successfully infected nodule cells. While cowpea and siratro responded to nodule colonisation with defence responses that eventually eliminated bacteria, L. leucocephala formed leghemoglobin-containing mature-like nodules inside which the pMiniSym2 transconjugant established persistent intracellular colonies. This data shows seven nodulation genes of NGR234 suffice to trigger nodule formation on roots of many hosts and to establish chronic infections in Leucaena cells.

Legume Nodules: Massive Infection in the Absence of Defense Induction.

Plants of the legume family host massive intracellular bacterial populations in the tissues of specialized organs, the nodules. In these organs, the bacteria, named rhizobia, can fix atmospheric nitrogen and transfer it to the plant. This special metabolic skill provides to the legumes an advantage when they grow on nitrogen-scarce substrates. While packed with rhizobia, the nodule cells remain alive, metabolically active, and do not develop defense reactions. Here, we review our knowledge on the control of plant immunity during the rhizobia-legume symbiosis. We present the results of an evolutionary process that selected both divergence of microbial-associated molecular motifs and active suppressors of immunity on the rhizobial side and, on the legume side, active mechanisms that contribute to suppression of immunity.

Legume Protein Consumption and the Prevalence of Legume Sensitization.

Sensitization and allergy to legumes can be influenced by different factors, such as exposure, geographical background, and food processing. Sensitization and the allergic response to legumes differs considerably, however, the reason behind this is not yet fully understood. The aim of this study is to investigate if there is a correlation between legume protein consumption and the prevalence of legume sensitization. Furthermore, the association between sensitization to specific peanut allergens and their concentration in peanut is investigated. Legume sensitization data (peanut, soybean, lupin, lentil, and pea) from studies were analyzed in relation to consumption data obtained from national food consumption surveys using the European Food Safety Authority (EFSA), Global Environment Monitoring System (GEMS), and What We Eat in America-Food Commodity Intake Database (WWEIA-FCID) databases. Data were stratified for children <4 years, children 4⁻18 years, and adults. Sufficient data were available for peanut to allow for statistical analysis. Analysis of all age groups together resulted in a low correlation between peanut sensitization and relative peanut consumption (r = 0.407), absolute peanut consumption (r = 0.468), and percentage of peanut consumers (r = 0.243). No correlation was found between relative concentrations of Ara h 1, 2, 3, 6, 7, and 8 in peanut and sensitization to these peanut allergens. The results indicate that the amount of consumption only plays a minor role in the prevalence of sensitization to peanut. Other factors, such as the intrinsic properties of the different proteins, processing, matrix, frequency, timing and route of exposure, and patient factors might play a more substantial role in the prevalence of peanut sensitization.

Investigation of the interaction of allergens of Glycine max with IgE-antibody for designing of peptidomimetics based anti-allergen.

Allergen induced IgE dependent type I hypersensitivity is the main cause of the allergy, which would be a burden on medical setup in coming years. Allergens of Glycine max have been isolated, and their disease relationships are documented. Therefore, it becomes important to investigate the interaction of different allergens of Glycine max with IgE and also screen suitable therapeutics to prevent this interaction. The amino acid sequences of all allergens of Glycine max and their isoallergens have been taken, and 3D structure of allergens (Gly m 3, Gly m 4, Gly m 5, Gly m 6 and Gly m 8) and their isoallergens were generated using Modeller v9.17. The modeled structures were further validated using PSVS, ProSA, RAMPAGE, and PDBsum. HL domain of Fab region of human IgE (PDBID: 2R56) was generated using UCSFchimera. The HL domain was minimized by Schrodinger software using the OPLS_2005 force field. SiteMap identified epitope binding site of the minimized domain. All the predicted epitopes of different allergens were docked to the binding site of HL domain using the Patchdock server. We have also designed a peptidomimetics based inhibitor targeted at interaction interface of Gly m8 and IgE, using in-silico virtual screening, molecular mechanics, and molecular dynamics simulation studies. These studies identified BDE32166344 ((N-(1-{[1-(1-aminocyclopentanecarbonyl)-3-hydroxypyrrolidin-3-yl]methyl}piperidin-4-yl)acetamide) as a peptidomimetics based lead with binding energy of -72.77 kcal/mol. Therefore, the present study investigates the interaction between different Gly m allergens and IgE antibody and identifies peptidomimetics based lead that might be developed as a suitable therapeutics against allergy caused by allergen of Glycine max.

Suppression of Plant Defenses by Herbivorous Mites Is Not Associated with Adaptation to Host Plants.

Some herbivores suppress plant defenses, which may be viewed as a result of the coevolutionary arms race between plants and herbivores. However, this ability is usually studied in a one-herbivore-one-plant system, which hampers comparative studies that could corroborate this hypothesis. Here, we extend this paradigm and ask whether the herbivorous spider-mite Tetranychus evansi, which suppresses the jasmonic-acid pathway in tomato plants, is also able to suppress defenses in other host plants at different phylogenetic distances from tomatoes. We test this using different plants from the Solanales order, namely tomato, jimsonweed, tobacco, and morning glory (three Solanaceae and one Convolvulaceae), and bean plants (Fabales). First, we compare the performance of T. evansi to that of the other two most-commonly found species of the same genus, T. urticae and T. ludeni, on several plants. We found that the performance of T. evansi is higher than that of the other species only on tomato plants. We then showed, by measuring trypsin inhibitor activity and life history traits of conspecific mites on either clean or pre-infested plants, that T. evansi can suppress plant defenses on all plants except tobacco. This study suggests that the suppression of plant defenses may occur on host plants other than those to which herbivores are adapted.

Lupine allergen detecting capability and cross-reactivity of related legumes by ELISA.

Lupine belongs to the genus Lupinus and includes three species commonly consumed by humans. The Lupinus genus is closely related to other legumes, such as peanuts, soya, chickpeas, peas, lentils and beans. However, the consumption of lupine (and related legumes) can cause severe allergenic reactions. Therefore, reliable analytical detection methods are required for the analysis of food samples. In this study three commercially available ELISA test kits were analyzed for the detection capability of three common lupine species, as well as cross-reactivity to related legumes. All three ELISA test kits could detect the lupine species, though with different sensitivities. Cross-reactivity varied for the ELISA test kits and all showed some cross-reactivity to related legume samples analyzed.

Storage molecules from tree nuts, seeds and legumes: relationships and amino acid identity among homologue molecules.

Summary: The families of seed storage proteins, together with profilins, oil-bodies-associated oleosins, and pathogenesis-related (PR) proteins like PR-10 (Bet v 1-like), PR-12 (defensins) and PR-14 (non-specific lipid transfer protein), are the main causes of IgE sensitization to tree nuts, legumes and seeds. All these allergens, with the exclusion of profilins and of PR-10, are heat-stable and possibly responsible for fatal or almost fatal adverse reactions to such foods. In this short review, we will discuss the relationship and amino acid identities among some of the seed storage homologue molecules identified to date from tree nuts, seeds and legumes.

Allergy to Peanut, Soybean, and Other Legumes: Recent Advances in Allergen Characterization, Stability to Processing and IgE Cross-Reactivity.

Peanut and soybean are members of the Leguminosae family. They are two of the eight foods that account for the most significant food allergies in the United States and Europe. Allergic reactions to other legume species can be of importance in other regions of the world. The major allergens from peanut and soybean have been extensively analyzed and members of new protein families identified as potential marker allergens for symptom severity. Important recent advances concerning their molecular properties or clinical relevance have been made. Therefore, there is increasing interest in the characterization of allergens from other legume species such as lupine, lentil, chickpea, green bean, or pea. As legumes are mainly consumed after thermal processing, knowledge about the effect of such processing on the allergenicity of legumes has increased during the last years. In the present review, recent advances in the identification of allergens from peanut, soybean, lupine, and other legume species are summarized and discussed. An overview of the most recently described effects of thermal processing on the allergenic properties of legumes is provided and the potential IgE cross-reactivity among members of the Leguminosae family is discussed.

Cross-reactivity profiles of legumes and tree nuts using the xMAP® multiplex food allergen detection assay.

The homology between proteins in legumes and tree nuts makes it common for individuals with food allergies to be allergic to multiple legumes and tree nuts. This propensity for allergenic and antigenic cross-reactivity means that commonly employed commercial immunodiagnostic assays (e.g., dipsticks) for the detection of food allergens may not always accurately detect, identify, and quantitate legumes and tree nuts unless additional orthogonal analytical methods or secondary measures of analysis are employed. The xMAP® Multiplex Food Allergen Detection Assay (FADA) was used to determine the cross-reactivity patterns and the utility of multi-antibody antigenic profiling to distinguish between legumes and tree nuts. Pure legumes and tree nuts extracted using buffered detergent displayed a high level of cross-reactivity that decreased upon dilution or by using a buffer (UD buffer) designed to increase the stringency of binding conditions and reduce the occurrence of false positives due to plant-derived lectins. Testing for unexpected food allergens or the screening for multiple food allergens often involves not knowing the identity of the allergen present, its concentration, or the degree of modification during processing. As such, the analytical response measured may represent multiple antigens of varying antigenicity (cross-reactivity). This problem of multiple potential analytes is usually unresolved and the focus becomes the primary analyte, the antigen the antibody was raised against, or quantitative interpretation of the content of the analytical sample problematic. The alternative solution offered here to this problem is the use of an antigenic profile as generated by the xMAP FADA using multiple antibodies (bead sets). By comparing the antigenic profile to standards, the allergen may be identified along with an estimate of the concentration present. Cluster analysis of the xMAP FADA data was also performed and agreed with the known phylogeny of the legumes and tree nuts being analyzed. Graphical abstract The use of cluster analysis to compare the multi-antigen profiles of food allergens.

Resistance to Bean common mosaic necrosis virus Conferred by the bc-1 Gene Affects Systemic Spread of the Virus in Common Bean.

Bean common mosaic necrosis virus (BCMNV) isolates belong to two pathogroups (PG), PG-III and PG-VI, which are distinguished in common bean due to the inability of the PG-III isolates of BCMNV to overcome the two recessive resistance alleles bc-1 and bc-12. The biological and molecular basis of this distinction between PG-III and PG-VI isolates of BCMNV is not known. Here, three isolates of BCMNV were typed biologically on a set of 12 bean differentials and molecularly through whole-genome sequencing. Two isolates (1755b and TN1a) were assigned to PG-VI and one isolate (NL8-CA) was assigned to PG-III. Isolate NL8-CA (PG-III) induced only local necrosis on inoculated leaves in 'Top Crop' and 'Jubila' bean harboring the I gene and the bc-1 allele, whereas isolates TN1, TN1a, and 1755b (all PG-VI) induced rapid whole-plant necrosis (WPN) in Top Crop 7 to 14 days postinoculation, and severe systemic necrosis but not WPN in Jubila 3 to 5 weeks postinoculation. In 'Redland Greenleaf C' expressing bc-1 and 'Redland Greenleaf B' expressing bc-12 alleles, isolate NL8-CA was able to systemically infect only a small proportion of upper uninoculated leaves (less than 13 and 3%, respectively). The whole genomes of isolates 1755b, TN1a, and NL8-CA were sequenced and sequence analysis revealed that, despite the overall high nucleotide sequence identity between PG-III and PG-VI isolates (approximately 96%), two areas of the BCMNV genome in the P1/HC-Pro and HC-Pro/P3 cistrons appeared to be more divergent between these two pathotypes of BCMNV. The data suggest that the phenotypic differences among PG-III and PG-VI isolates of BCMNV in common bean cultivars from host resistance groups 2, 3, and 9 carrying bc-1 alleles were related to the impaired systemic movement of the PG-III isolates to the upper, uninoculated leaves, and also suggest a role of the recessive bc-1 gene in interfering with systemic spread of BCMNV.

Phenotypical characterization of peanut allergic children with differences in cross-allergy to tree nuts and other legumes.

BACKGROUND: Peanut allergy in children is often associated with allergies to tree nuts and/or legumes. The aim of this study was to analyze in cluster a cohort of children allergic to peanuts and assessed for cross-reactivity to nuts and legumes and to identify different phenotypes. METHODS: We included retrospectively 317 children with peanut allergy evaluated at the Allergy Unit of the Saint Vincent Hospital of Lille in the last 12 years. A complete workup for peanut allergy and nuts and legumes was carried out for each patient. A hierarchical agglomerative clustering method was used to search for clusters of individuals in the evaluated cohort. RESULTS: Cross-allergy to TN and/or other legumes was identified in 137 patients (43.2%), atopic dermatitis being a major risk factor (adjusted OR = 16 [95% CI: 7.4-37]; p < 0.001). Three phenotypes emerged from cluster analysis. Cluster 1 (72 patients) is characterized by high level of rAra h 2, low threshold reactive doses for peanut and high proportion of asthma; Cluster 2 (93 patients) is characterized by high threshold reactive doses for peanut and the lowest proportion of cross-allergy to TN and/or legumes; Cluster 3 (152 patients) has a high risk of cross-allergy to TN and/or legumes and most patients suffer from eczema. CONCLUSIONS: The three phenotypes highlighted by this study could be useful to identify children with high risk of cross-allergic reaction to TNs and legumes early after PA diagnosis.

The Role of Plant Innate Immunity in the Legume-Rhizobium Symbiosis.

A classic view of the evolution of mutualism is that it derives from a pathogenic relationship that attenuated over time to a situation in which both partners can benefit. If this is the case for rhizobia, then one might uncover features of the symbiosis that reflect this earlier pathogenic state. For example, as with plant pathogens, it is now generally assumed that rhizobia actively suppress the host immune response to allow infection and symbiosis establishment. Likewise, the host has retained mechanisms to control the nutrient supply to the symbionts and the number of nodules so that they do not become too burdensome. The open question is whether such events are strictly ancillary to the central symbiotic nodulation factor signaling pathway or are essential for rhizobial host infection. Subsequent to these early infection events, plant immune responses can also be induced inside nodules and likely play a role in, for example, nodule senescence. Thus, a balanced regulation of innate immunity is likely required throughout rhizobial infection, symbiotic establishment, and maintenance. In this review, we discuss the significance of plant immune responses in the regulation of symbiotic associations with rhizobia, as well as rhizobial evasion of the host immune system.

Genetic alteration of UDP-rhamnose metabolism in Botrytis cinerea leads to the accumulation of UDP-KDG that adversely affects development and pathogenicity.

Botrytis cinerea is a model plant-pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)-glucose, the major fungal wall nucleotide sugar precursor, to UDP-rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose-containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP-rhamnose pathway intermediate UDP-4-keto-6-deoxy-glucose (UDP-KDG) in hyphae of the Δbcer strain. UDP-KDG could not be detected in hyphae of the wild-type strain, indicating fast conversion to UDP-rhamnose by the BcEr enzyme. The correlation between high UDP-KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP-KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP-KDG may lead to the development of new antifungal drugs.