Environmental parameters, and not phylogeny, determine the composition of extracellular polymeric substances in microbial mats from extreme environments.

The ability to establish biofilms is a key trait for microorganisms growing in extreme environments. The extracellular polymeric substances (EPS) present in biofilms provide not only surface attachment, but also protection against all kinds of environmental stressors, including desiccation, salinity, temperature or heavy metal pollution. The acquisition of suitable biofilm characteristics might thus be an important process mediating the adaptation of microorganisms to novel environmental conditions. In this work we have characterized the EPS of 20 phylogenetically diverse biofilms collected in situ from five contrasting extreme environments, including two geothermal areas (Copahue, Argentina; Seltun, Iceland), two cold areas (Pastoruri glacier, Peru; Byers Peninsula, Antarctica) and one extremely acidic river (Río Tinto, Spain). Biofilms were subjected to biochemical characterization, glycan profiling and immunoprofiling with an antibody microarray. Our results showed that environmental conditions strongly influence biofilm characteristics, with microorganisms from the same environment achieving similar EPS compositions regardless of the phylogeny of their main species. The concentration of some monosaccharides in the EPS could be related to environmental conditions such as temperature or heavy metal toxicity, suggesting that in some cases stress resistance can be mediated by specific sugars. Overall, our results highlight the existence of conserved EPS compositional patterns for each extreme environment, which could in turn be exploited to engineer ecological adaptations in genetically modified microorganisms.

Deciphering the prokaryotic community and metabolisms in South African deep-mine biofilms through antibody microarrays and graph theory.

In the South African deep mines, a variety of biofilms growing in mine corridor walls as water seeps from intersections or from fractures represents excellent proxies for deep-subsurface environments. However, they may be greatly affected by the oxygen inputs through the galleries of mining activities. As a consequence, the interaction between the anaerobic water coming out from the walls with the oxygen inputs creates new conditions that support rich microbial communities. The inherent difficulties for sampling these delicate habitats, together with transport and storage conditions may alter the community features and composition. Therefore, the development of in situ monitoring methods would be desirable for quick evaluation of the microbial community. In this work, we report the usefulness of an antibody-microarray (EMChip66) immunoassay for a quick check of the microbial diversity of biofilms located at 1.3 km below surface within the Beatrix deep gold mine (South Africa). In addition, a deconvolution method, previously described and used for environmental monitoring, based on graph theory and applied on antibody cross-reactivity was used to interpret the immunoassay results. The results were corroborated and further expanded by 16S rRNA gene sequencing analysis. Both culture-independent techniques coincided in detecting features related to aerobic sulfur-oxidizers, aerobic chemoorganotrophic Alphaproteobacteria and metanotrophic Gammaproteobacteria. 16S rRNA gene sequencing detected phylotypes related to nitrate-reducers and anaerobic sulfur-oxidizers, whereas the EMChip66 detected immunological features from methanogens and sulfate-reducers. The results reveal a diverse microbial community with syntrophic metabolisms both anaerobic (fermentation, methanogenesis, sulphate and nitrate reduction) and aerobic (methanotrophy, sulphur oxidation). The presence of oxygen-scavenging microbes might indicate that the system is modified by the artificial oxygen inputs from the mine galleries.

Occupational Rhinoconjunctivitis due to Maize in a Snack Processor: A Cross-Reactivity Study Between Lipid Transfer Proteins From Different Cereals and Peach.

We report the case of a snack processor who developed occupational rhinoconjunctivitis due to maize brand exposure during the extrusion process, and who experienced abdominal pain upon drinking beer. The allergens implicated and the cross-reactivity between non-specific lipid transfer proteins (LTPs) from different cereals and peach were investigated. Skin prick tests and specific IgE to cereal flours, pulmonary functions tests and specific conjunctival and inhalation challenges to maize extract were performed. In vitro studies included IgE immunoblotting and ELISA inhibition assays. Skin prick tests with maize flour, maize brand and wheat flour extracts were positive, whereas serum specific IgE was positive only to maize flour. Specific inhalation challenge (SIC) to maize flour did not elicit an asthmatic reaction; however, conjunctival challenge test with the same extract was positive. Patient's serum recognized IgE-binding bands in the maize and beer extracts corresponding to LTPs. In the ELISA inhibition assays, a significant degree of allergenic cross-reactivity was found between maize and beer LTPs, whereas no cross-reactivity was observed between maize LTP and wheat and peach LTPs.

Comparison of specific IgE antibodies to wheat component allergens in two phenotypes of wheat allergy.

Specific IgE to gliadin was proposed as a marker for wheat dependent exercise induced anaphylaxis, while Tri a 14 was found to induce IgE response in baker's asthma. We evaluated whether these components could be used for discriminating phenotypes of wheat allergy. Twenty-nine patients who were wheat-induced anaphylaxis and/or urticaria (n=21, group I) and baker's asthma (n=8, group II) were enrolled. The prevalence of serum specific IgE to Tri a 14 was higher in group II (25%) than in group I (4.8%), while the serum specific IgE to gliadin was significantly higher in group I (70%) than in group II (12.5%). The cutoff value for predicting the baker's asthma using the ratio of serum specific IgE to Tri a 14 to gliadin was 742.8 optical density×1,000/(kU/L) with high sensitivity and specificity. These findings suggest that Tri a 14/gliadin may be a potential marker for predicting baker's asthma.

Antigenic proteins involved in occupational rhinitis and asthma caused by obeche wood (Triplochiton scleroxylon).

BACKGROUND: Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. OBJECTIVE: To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma MATERIALS AND METHODS: An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. RESULTS: Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA-), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases. CONCLUSIONS: Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens.

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray.

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

Computational study of ligand binding in lipid transfer proteins: Structures, interfaces, and free energies of protein-lipid complexes.

Plant nonspecific lipid transfer proteins (nsLTPs) bind a wide variety of lipids, which allows them to perform disparate functions. Recent reports on their multifunctionality in plant growth processes have posed new questions on the versatile binding abilities of these proteins. The lack of binding specificity has been customarily explained in qualitative terms on the basis of a supposed structural flexibility and nonspecificity of hydrophobic protein-ligand interactions. We present here a computational study of protein-ligand complexes formed between five nsLTPs and seven lipids bound in two different ways in every receptor protein. After optimizing geometries in molecular dynamics calculations, we computed Poisson-Boltzmann electrostatic potentials, solvation energies, properties of the protein-ligand interfaces, and estimates of binding free energies of the resulting complexes. Our results provide the first quantitative information on the ligand abilities of nsLTPs, shed new light into protein-lipid interactions, and reveal new features which supplement commonly held assumptions on their lack of binding specificity.

A mutant of the major melon allergen, Cuc m 2, with reduced IgE binding capacity is a good candidate for specific immunotherapy.

Hypoallergenic mutants with reduced IgE-binding capacity but which show a similar T-cell response to the corresponding natural allergen are ideal tools for immunotherapy, for preventing a possible anaphylactic shock. An IgE conformational epitope has been identified in Cuc m 2, the major allergen and profilin from melon. Since this epitope is highly conserved in most pollen profilins, it may contribute to an explanation of cross-reactivity between pollen and food profilins. Mutants (Mut 1 and Mut 2) were generated by changing specific residues of the Cuc m 2 epitope to alanine, produced in Escherichia coli, and purified by chromatographic methods. Mut 1 showed a slight reduction in IgE binding but an allergenic activity that was similar to recombinant Cuc m 2, as measured by basophil activation test (BAT) and skin prick test (SPT). By contrast, Mut 2 displayed a substantial reduction in IgE-binding capacity (57%) and positive responses, as determined by BAT (33%) and SPT (50%), when compared to those of rCuc m 2. However, the T-cell proliferation and cytokine production induced by Mut 2 and rCuc m 2 were similar. Thus, this mutant represent potential candidate for immunotherapy of profilin allergies.

LocaPep: localization of epitopes on protein surfaces using peptides from phage display libraries.

The use of peptides from a phage display library selected by binding to a given antibody is a widespread technique to probe epitopes of antigenic proteins. However, the identification of interaction sites mimicked by these peptides on the antigen surface is a difficult task. LocaPep is a computer program developed to localize epitopes using a new clusters algorithm that focuses on protein surface properties. The program is constructed with the aim of providing a flexible computational tool for predicting the location of epitopes on protein structures. As a first set of testing results, the localization of epitope regions in eight different antigenic proteins for which experimental data on their antibody interactions exist is correctly identified by LocaPep. These results represent a disparate sample of biologically different systems. The program is freely available at http://atenea.montes.upm.es.

Sensitization profiles to purified plant food allergens among pediatric patients with allergy to banana.

Banana fruit allergy is well known, but neither immunoglobulin E recognition patterns to purified plant food allergens nor true prevalences of putative banana allergens have been established. This study aimed to characterize ß-1,3-glucanase and thaumatin-like protein (TLP) as banana allergens, testing them, together with other plant food allergens, in 51 children with allergic reactions after banana ingestion and both positive specific IgE and skin prick test (SPT) to banana. Banana ß-1,3-glucanase and TLP were isolated and characterized. Both banana allergens, together with kiwifruit TLP Act d 2, avocado class I chitinase Pers a 1, palm pollen profilin Pho d 2 and peach fruit lipid transfer protein (LTP) Pru p 3, were tested by in vitro and in vivo assays. Banana ß-1,3-glucanase (Mus a 5) was glycosylated, whereas banana TLP (Mus a 4) was not, in contrast with its homologous kiwi allergen Act d 2. Specific IgE to both banana allergens, as well as to peach Pru p 3, was found in over 70% of sera from banana-allergic children, and Mus a 4 and Pru p 3 provoked positive SPT responses in 6 of the 12 tested patients, whereas Mus a 5 in only one of them. Both peptidic epitopes and cross-reactive carbohydrate determinants were involved in the IgE-binding to Mus a 5, whereas cross-reactivity between Mus a 4 and Act d 2 was only based on common IgE protein epitopes. Profilin Pho d 2 elicited a relevant proportion of positive responses on in vitro (41%) and in vivo (58%) tests. Therefore, Mus a 4 and LTP behave as major banana allergens in the study population, and profilin seems to be also a relevant allergen. Mus a 5 is an equivocal allergenic protein, showing high IgE-binding to its attached complex glycan, and low in vivo potency.

Prebiotic synthesis of protobiopolymers under alkaline ocean conditions.

Clasically, prebiotic chemistry has focused on the production and identification of simple organic molecules, many of them forming part of "intractable polymers" named tholins. In a previous work, we demonstrated that in experiments using an external energy source and inorganic carbon the aqueous aerosols improved the formation of hydrophilic tholins. Herein, we elucidate the role of pH (from 4 to 12) in prebiotic experiments using saline aqueous aerosols, spark discharges and an atmosphere containing CH(4). At all values of pH, the saline aqueous aerosols increased the production of a significant variety of carboxylic acids that could have been present in a primitive Krebs cycle. Moreover, the study for the first time of hydrophilic tholins by 2-D electrophoresis revealed that these are formed by a set of unexpected heavy polymeric species. The initial alkaline conditions significantly increased both the apparent molecular weight of polymeric species up to 80 kDa and their diversity. We propose the term of protobiopolymers to denote those polymeric species fractionated by 2-D electrophoresis since these are formed by biomolecules present in living systems and show diversity in length as well as in functional groups. Thus, aerosols formed in simulated alkaline ocean conditions could provide an optimal medium for the formation of the primeval materials that could be precursors to the emergence of life.

Anaphylaxis to wheat flour-derived foodstuffs and the lipid transfer protein syndrome: a potential role of wheat lipid transfer protein Tri a 14.

BACKGROUND: Food allergy to wheat-derived foodstuffs is on the rise. Tri a 14, a wheat flour lipid transfer protein (LTP) allergen, has been described as a major allergen associated with baker's asthma and wheat food allergy. Cross-reactivity among LTP allergens leads to the so-called 'LTP syndrome'. METHODS: Eight adult patients showing anaphylaxis after ingestion of wheat-derived foodstuffs were selected. A homemade wheat extract, purified natural (n) and recombinant (r) Tri a 14, and peach fruit and Artemisia pollen LTP allergens Pru p 3 and Art v 3 were subjected to skin prick test, specific IgE determination (ELISA) and IgE immunodetection assays. RESULTS: All tests were positive in the 8 selected patients with the homemade extract. Positive skin prick test responses to nTri a 14, Pru p 3 and Art v 3 were found in 5/8, 6/8 and 4/4 patients, respectively. Specific IgE determined by ELISA assays was detected in 6 to nTri a 14 and rTri a 14, in 4 to Pru p 3 and in 3 to Art v 3 out of 8 individual sera tested, whereas all these sera showed IgE binding to nTri a 14 and Pru p 3 in immunodetection after SDS-PAGE separation. CONCLUSIONS: Tri a 14 seems to be a relevant allergen in patients with anaphylaxis after ingestion of wheat flour foodstuffs, according to in vitro and in vivo results. Clinical history of the analyzed patients, together with sensitization to peach Pru p 3 and Artemisia pollen Art v 3, suggests that 6 of them suffer from LTP syndrome.

Salt-soluble proteins from wheat-derived foodstuffs show lower allergenic potency than those from raw flour.

Salt-soluble proteins from wheat flour have been described as main allergens associated with both baker's asthma and food allergy. However, most studies have used raw flour as starting material, thus not considering potential changes in allergenic properties induced by the heat treatment and other industrial processing to produce wheat-derived foodstuffs. Salt extracts from different commercial wheat-derived products were obtained and their allergenic properties investigated by IgE-immunodetection, ELISA assays, and skin prick test. The IgE-binding capacity of salt-soluble proteins from commercial breads and cooked pastas was reduced around 50% compared with that of raw flour, the reduction being less dramatic in noncooked pastas and biscuits. Several wheat-derived foodstuffs showed major IgE-binding components of 20 and 35 kDa, identified as avenin-like and globulin proteins, respectively. These proteins, as well as most flour and bread salt-soluble proteins, were hydrolyzed when subjected to simulated gastrointestinal digestion. However, the digested products still exhibited a residual IgE-binding capacity. Therefore, processing of wheat flour to obtain derived foodstuffs decreases the IgE binding-capacity of the major salt-soluble wheat proteins. Moreover, simulated gastric fluid digestion further inactivates some heat-resistant IgE-binding proteins.

Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens.

BACKGROUND: Baker's asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. METHODS: Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. RESULTS: Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with baker's asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. CONCLUSIONS: A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

Mimotope mapping as a complementary strategy to define allergen IgE-epitopes: peach Pru p 3 allergen as a model.

Lipid transfer proteins (LTPs) are the major allergens of Rosaceae fruits in the Mediterranean area. Pru p 3, the LTP and major allergen of peach, is a suitable model for studying food allergy and amino acid sequences related with its IgE-binding capacity. In this work, we sought to map IgE mimotopes on the structure of Pru p 3, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms (OAS-pool or SYS-pool), and used for screening of a random dodecapeptide phage display library. Positive clones were further confirmed by ELISA assays testing individual sera from each pool. Three-dimensional modelling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surface. Twenty-one phage clones were selected using Pru p 3-specific IgE, 9 of which were chosen using OAS-specific IgE while the other 12 were selected with systemic-specific IgE. Peptide alignments revealed consensus sequences for each pool: L37 R39 T40 P42 D43 R44 A46 P70 S76 P78 Y79 for OAS-IgE, and N35 N36 L37 R39 T40 D43 A46 S76 I77 P78 for systemic-IgE. These 2 consensus sequences were mapped on the same surface of Pru p 3, corresponding to the helix 2-loop-helix 3 region and part of the non-structured C-terminal coil. Thus, 2 relevant conformational IgE-binding regions of Pru p 3 were identified using a random peptide phage display library. Mimotopes can be used to study the interaction between allergens and IgE, and to accelerate the process to design new vaccines and new immunotherapy strategies.

Rye flour allergens: an emerging role in baker's asthma.

BACKGROUND: Exposure to wheat flour is usually considered the most important cause of baker's asthma. However, other flours frequently used in bakeries may play an emerging role as relevant allergens causing occupational asthma. AIMS OF STUDY: We report on two cases of baker's asthma mainly caused by exposure to rye flour. The profile of allergen sensitization to cereal flour was investigated. METHODS: Two bakery workers suffering from rhinoconjunctivitis and asthma symptoms at work underwent an in vivo study (skin prick tests and bronchial allergen challenge) and in vitro study (total serum IgE, specific serum IgE and immunoblotting). RESULTS: Specific inhalation challenge with wheat flour did not elicit an asthmatic reaction, however both patients showed an early asthmatic reaction with the rye flour challenge. Rye flour-immunoblotting showed IgE-binding bands around 12-15 kDa, that correspond to rye flour enzymatic inhibitors which were not present in the wheat flour immunoblot. CONCLUSIONS: Both bakers had developed occupational asthma to rye flour (confirmed by specific inhalation challenge test). Rye flour allergens (enzymatic inhibitors) are important allergens that should be considered in the diagnosis of baker's asthma.

Wheat lipid transfer protein is a major allergen associated with baker's asthma.

BACKGROUND: Baker's asthma is a frequent occupational allergic disorder mainly caused by inhalation of cereal flours. Lipid transfer proteins (LTPs) constitute a family of plant food panallergens, but their role as inhalant and wheat allergens is still unclear. OBJECTIVE: We sought to explore the involvement of wheat LTPs in baker's asthma caused by wheat flour sensitization. METHODS: Forty patients with occupational asthma caused by wheat flour inhalation were studied. Wheat LTP, Tri a 14, was purified by using a 2-step chromatographic protocol and characterized by N-terminal amino acid sequencing and 3-dimensional modeling. Its reactivity was confirmed by means of IgE immunodetection, ELISA and ELISA-inhibition assays, and skin prick tests. RESULTS: Specific IgE to Tri a 14 was found in 60% of 40 individual sera from patients with baker's asthma, and the purified allergen elicited positive skin prick test reactions in 62% of 24 of these patients. Tri a 14 and peach LTP, Pru p 3, showed a sequence identity of 45%, but the low cross-reactivity between both allergens detected in several individual sera reflected great differences in their 3-dimensional IgE-binding regions. CONCLUSIONS: Wheat LTP is a major inhalant allergen associated with baker's asthma caused by wheat flour sensitization. Poor cross-reactivity with its peach homolog was found in some patients. CLINICAL IMPLICATIONS: LTPs can be considered relevant inhalant allergens linked to respiratory disorders. LTP from wheat (Tri a 14) can be used as a helpful tool for the diagnosis of baker's asthma.