Expression of Alt a 1 allergen from Alternaria alternata in the yeast Yarrowia lipolytica

Allergies affect almost 25% of the population in industrialized countries. Alternaria alternata is known to be a significant source of aeroallergens and sensitization to this mold is a risk factor for the development of wheezing, asthma, and atopic dermatitis. Diagnosis and treatment of allergies requires the production of large amounts of pure and well defined protein. Yarrowia lipolytica, a non-pathogenic ascomycete able to secrete high levels of enzymes that can grow in inexpensive substrates, has been considered a useful host for heterologous gene expression. In the present work, we have developed two vectors for expressing Alt a 1, the most relevant A. alternata allergen, in Y. lipolytica. One vector is autosomal and one is integrative. With both systems, rAlt a 1 was secreted into the culture medium. The immunological characteristics of the purified recombinant allergen were determined by IgE-blot using sera from 42 A. alternata-allergic patients. We have carried out ELISA-inhibition experiments using sera from four patients to compare the IgE-binding capacity of natural and recombinant allergens. Our results show that Y. lipolytica is able to produce a recombinant Alt a 1 which is immunochemically equivalent to the natural counterpart and could be used for immunotherapy and diagnostics.     

Particle size distribution of the major Alternaria alternata allergen,Alt a 1, derived from airborne spores and subspore fragments

Fungal fragments are abundant immunoreactive bioaerosols that may outnumber the concentrations of intact spores in the air. To investigate the importance of Alternaria fragments as sources of allergens compared to Alternaria spores, we determined the levels of Alternaria spores and Alt a 1 (the major allergen in Alternaria alternata spores) collected on filters within three fractions of particulate matter (PM) of different aerodynamic diameter: (1) PM_>10, (diameter>10 μm); (2) PM_2.5-10 (2.5–10μm); (3) PM_2.5 (0.12–2.5 μm). The airborne particles were collected using a three stage high-volume ChemVol cascade impactor during the Alternaria sporulation season in Poznań, Poland (30 d between 6 July and 22 September 2016). The quantification of Alt a 1 was performed using the enzyme-linked immunosorbent assay. High concentrations of Alt a 1 were recorded during warm and dry d characterized by high sunshine duration, lack of clouds and high dew point values. Atmospheric concentrations of Alternaria spores correlated significantly (r = 0.930, p < 0.001) with Alt a 1 levels. The highest Alt a 1 was recorded in PM_2.5-10 (66.8 % of total Alt a 1), while the lowest in PM_2.5 (<1.0 %). Significantly more Alt a 1 per spore (>30 %) was observed in PM_2.5-10 than in PM_>10. This Alt a 1 excess may be derived from sources other than spores, e.g. hyphal fragments. Overall, in outdoor air the major source of Alt a 1 are intact Alternaria spores, but the impact of other fungal fragments (hyphal parts, broken spores, conidiophores) cannot be neglected, as they may increase the total atmospheric Alt a 1 concentration.     

Alt a 1 allergen homologs from Alternaria and related taxa: analysis of phylogenetic content and secondary structure

A gene for the Alternaria major allergen, Alt a 1, was amplified from 52 species of Alternaria and related genera, and sequence information was used for phylogenetic study. Alt a 1 gene sequences evolved 3.8 times faster and contained 3.5 times more parsimony-informative sites than glyceraldehyde-3-phosphate dehydrogenase (gpd) sequences. Analyses of Alt a 1 gene and gpd exon sequences strongly supported grouping of Alternaria spp. and related taxa into several species-groups described in previous studies, especially the infectoria, alternata, porri, brassicicola, and radicina species-groups and the Embellisia group. The sonchi species-group was newly suggested in this study. Monophyly of the Nimbya group was moderately supported, and monophyly of the Ulocladium group was weakly supported. Relationships among species-groups and among closely related species of the same species-group were not fully resolved. However, higher resolution could be obtained using Alt a 1 sequences or a combined dataset than using gpd sequences alone. Despite high levels of variation in amino acid sequences, results of in silico prediction of protein secondary structure for Alt a 1 demonstrated a high degree of structural similarity for most of the species suggesting a conservation of function.     

IgE and IgG binding of peptides expressed from fragments of cDNA encoding the major house dust mite allergen Der p I

Large peptides expressed from cDNA fragments of a clone encoding the mite allergen Der p I were able to bind IgE and IgG in sera from allergic individuals. The binding was found for peptides from sequences throughout the molecule, with at least five regions, comprising residues 1-56, 53-99, 98-140, 166-194, and 188-222. The only limitation was that more than 30 amino acid residues were required for consistent binding. Each of seven sera examined showed a different profile of antibody binding to the peptides. For the most part the pattern of IgE and IgG binding to the peptides for each serum was similar, demonstrating a concordant repertoire. In 5/7 sera, however, IgG bound to some peptides which had little or no IgE binding activity, thus showing more diverse specificities. It is suggested that some divergence of repertoire can develop during the maturation of the B cell response.     

Alt a 1 from Alternaria interacts with PR5 thaumatin-like proteins

Alt a 1 is a protein found in Alternaria alternata spores related to virulence and pathogenicity and considered to be responsible for chronic asthma in children. We found that spores of Alternaria inoculated on the outer surface of kiwifruits did not develop hyphae. Nevertheless, the expression of Alt a 1 gene was upregulated, and the protein was detected in the pulp where it co-localized with kiwi PR5. Pull-down assays demonstrated experimentally that the two proteins interact in such a way that Alt a 1 inhibits the enzymatic activity of PR5. These results are relevant not only for plant defense, but also for human health as patients with chronic asthma could suffer from an allergic reaction when they eat fruit contaminated with Alternaria.     

Alternaria alternata, a new pathotype pathogenic to aphids

A fungus, identified as Alternaria alternata, was isolated from dying or dead aphids and proved to be pathogenic. It was isolated from different parts of Greece from aphid specimens on cultivated plants, ornamentals and weeds. In the laboratory, disease development started with the germination of spores on the insect integument and the subsequent growth of mycelium. The fungus formed apical and intercalary, globose or lobate appressoria which were firmly attached onto the host exoskeleton and facilitated entrance of the mycelium into the insect body. Under favorable conditions of temperature (15–35 °C) and relative humidity (100%), infected aphids died in 2–4 days. A characteristic brown discoloration accompanied the death of the insects. Both mycelial growth and sporulation were profuse on dead specimens. The pathogen infected all 26 aphid species tested but was unable to infect other insects (Drosophila melanogaster and Ceratitis capitata) or aphid host plants. There were significant differences in mortality rate among aphid species only during the first two days after inoculation. It is suggested that A. alternata may be a good candidate to be exploited for the biological control of aphids.     

Tenuazonic Acid, a Toxin Produced by Alternaria alternata

Fifty-seven of 87 isolates of Alternaria alternata (Fr) Keissler grown on autoclaved, moist corn-rice substrate and fed to rats were lethal. The major toxin produced was isolated and characterized as tenuazonic acid. Twenty of 23 toxigenic Alternaria isolates examined produced tenuazonic acid. No tenuazonic acid could be detected in either of the field samples of sorghum or blackeyed peas, which were heavily invaded by Alternaria.     

Mutagenicity of stemphyltoxin III, a metabolite of Alternaria alternata

Some common decay organisms of vegetables and ripened fruits are Alternaria species. Even fruits and vegetables kept under refrigeration can be spoiled by Alternaria species because the mold grows at low temperatures. Alternaria alternata is commonly found in grain in areas with a high incidence of esophageal cancer. Three metabolites, altertoxins I, II, and III, have been isolated from A. alternata and have hydroxyperylenequinone structures. Although other perylenequinone metabolites such as stemphyperylenol and stemphyltoxins I, II, III, and IV, have been isolated from Stemphylium botryosum var. lactucum, a plant pathogen and mold, we isolated and identified stemphyltoxin III from A. alternata. This metabolite was tested for mutagenicity in the Ames Salmonella typhimurium plate incorporation assay with and without Aroclor 1254-induced rat S-9 metabolic activation. A positive response was noted with and without metabolic activation in S. typhimurium TA98 and TA1537, and there was a marginal response in strain TA100.     

Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination.

Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross-reacts with most grass-, corn-, and monocot-derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28-32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild-type allergen and group 1 allergens from other grass species. Moreover, peptide-induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild-type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE- mediated allergies.     

Mapping of conformational IgE epitopes with peptide-specific monoclonal antibodies reveals simultaneous binding of different IgE antibodies to a surface patch on the major birch pollen allergen, Bet v 1

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients' IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients' IgE binding to Bet v 1 (52-75%) were obtained with mAbs specific for two peptides comprising aa 29-58 (P2) and aa 73-103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.     

Mutagenicity of stemphyltoxin III, a metabolite of Alternaria alternata.

Some common decay organisms of vegetables and ripened fruits are Alternaria species. Even fruits and vegetables kept under refrigeration can be spoiled by Alternaria species because the mold grows at low temperatures. Alternaria alternata is commonly found in grain in areas with a high incidence of esophageal cancer. Three metabolites, altertoxins I, II, and III, have been isolated from A. alternata and have hydroxyperylenequinone structures. Although other perylenequinone metabolites such as stemphyperylenol and stemphyltoxins I, II, III, and IV, have been isolated from Stemphylium botryosum var. lactucum, a plant pathogen and mold, we isolated and identified stemphyltoxin III from A. alternata. This metabolite was tested for mutagenicity in the Ames Salmonella typhimurium plate incorporation assay with and without Aroclor 1254-induced rat S-9 metabolic activation. A positive response was noted with and without metabolic activation in S. typhimurium TA98 and TA1537, and there was a marginal response in strain TA100.     

Volatilization of Selenium by Alternaria alternata

Seleniferous water continues to be a serious problem to wildlife in the central valley of California. Water samples collected from Kesterson Reservoir, Peck Ranch, and Lost Hills evaporation pond facilities contained between 0.005 and 5 mg of Se per liter. The objective of this study was to isolate Se-methylating organisms in evaporation pond water and to assess, through enrichment and manipulation of their optimal growth parameters, the environmental factors which govern microbial Se methylation. Alternaria alternata was isolated as an active Se-methylating organism. The volatile product was identified as dimethylselenide. The effects of pH, temperature, Se substrates, and methyl donors on the ability of A. alternata to methylate Se were investigated in liquid medium containing 100 mg of Se per liter. The optimum pH and temperature for methylation were 6.5 and 30 degrees C, respectively. Selenate and selenite were methylated more rapidly than selenium sulfide and various organic Se compounds (6-selenoguanosine, 6-selenoinosine, seleno-dl-methionine, and 6-selenopurine). l-Methionine and methyl cobalamine (0.1 muM) stimulated dimethylselenide production. This study demonstrates that Se-methylating organisms are present in evaporation pond water and are capable of liberating substantial quantities of Se in the volatile dimethylselenide form. By determining the optimum environmental conditions which stimulate volatilization, it may be possible to design a way to remove Se from seleniferous water in situ.     

T cell epitope-containing peptides of the major dog allergen Can f 1 as candidates for allergen immunotherapy

One prerequisite for developing peptide-based allergen immunotherapy is knowing the T cell epitopes of an allergen. In this study, human T cell reactivity against the major dog allergen Can f 1 was investigated to determine peptides suitable for immunotherapy. Seven T cell epitope regions (A-G) were found in Can f 1 with specific T cell lines and clones. The localization of the epitope regions shows similarities with those of the epitopes found in Bos d 2 and Rat n 1. On average, individuals recognized three epitopes in Can f 1. Our results suggest that seven 16-mer peptides (p15-30, p33-48, p49-64, p73-88, p107-122, p123-138, and p141-156), each from one of the epitope regions, show widespread T cell reactivity in the population studied, and they bind efficiently to seven HLA-DRB1 molecules (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominant in Caucasian populations. Therefore, these peptides are potential candidates for immunotherapy of dog allergy.     

A review of Alternaria alternata sensitivity

Sensitivity to fungi is a major risk factor for the development of asthma. However, the prevalence of fungal sensitivity in asthma is not completely understood. Nonetheless upward of 80% of asthmatic patients may be sensitized to one or more fungi. Fungal exposure occurs primarily from outdoors sources, but can occur in the indoor environment as well. Assessment of fungal exposure requires a multifaceted approach including measurement of airborne spores and culture techniques to identify the relevant organisms. Preventing intrusion of outdoor fungal spores into the indoor environment may be helpful in reducing allergic symptoms. Methods to abate indoor fungal growth include reduction of indoor humidity and removal of water sources. Patients with fungal sensitivity should be advised to avoid exposure as much as possible. For patients who have failed to respond to environmental control measures and appropriate medications, it may be reasonable to consider specific immunotherapy. The application of molecular biology techniques to the study of allergens has enhanced the researcher's ability to produce Alternaria allergens in quantity, to determine their biological relevance, as well as to evaluate mechanisms of Alternaria sensitivity. We look forward to new developments and improved treatments through modulation of the immune response with molecularly produced and well characterized fungal allergy.     

Interstrain spheroplast fusion of Alternaria alternata

Summary Auxotrophic and drug resistant colonies of Alternaria alternata were selected following UV mutagenesis of spheroplasts and genetic transformation with pDH25. Intrastrain cell fusion of certain A. alternata parental strains induced by polyethylene glycol occurred at an average rate of 0.35%; interstrain fusions occurred at a rate of 0.08%. Mitotic recombination resulted from UV mutagenesis of spheroplasts from several fusants from 6hy1 × 1ar1. Fusants synthesized different levels of the cyclic tetrapeptide, tentoxin; some colonies produced higher levels than either parent. These results demonstrate that spheroplast fusion may have a potential application for genetic analysis of secondary metabolite production and for strain improvement in A. alternata.Mention of a trademark of proprietary product does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable.     

Biosynthesis of the bicycloalternarenes, mixed terpenoids of Alternaria alternata

By incorporation of [2-13C]-mevalonate, [1-13C]-acetate and [1-13C]-glucose we could reveal that the phytopathogenic fungus Alternaria alternata biosynthesized the mixed terpenoids bicycloalternarenes via the classic mevalonate pathway. The polyketid pathway does not participate in the biosynthesis of bicycloalternarenes, because there is no incorporation of [13C]-acetate into the C-ring of these compounds. The labelling pattern in this nonterpenoid part of bicycloalternarenes after feeding with [1-13C]-glucose and [U-13C6]-glucose, respectively, allows the assumption that metabolites of the shikimate pathway are involved.     

Wind Dispersal of Alternaria alternata, a Cause of Leaf Blight of Cotton

Introducing Alternaria alternata, the cause of blight disease of cotton plants, into a field of young healthy plants growing in rows cross-wind, yielded disease foci which were spread downwind up to 7 m from the infection sources. Only light disease incidence was found in the remainder of the field. When the disease was introduced into a field of mature cotton plants grown in rows cross-wind, randomly scattered disease foci occurred. In mature plantations where rows were parallel to the average wind direction, only limited size disease foci developed downwind, up to 16 m from the source. These foci did not developed further during the season. The number of air-borne spores of A. alternata was significantly increased by the presence of diseased cotton plants, being highest close to the diseased plants. The spores were transferred to a distance of at least 20 m. However, the number of air-borne spores significantly decreased 6 m from the infection source. Periodical trapping of air-borne spores of A. alternata in a cotton growing region for 2 years, revealed that their air dispersal is local, probably at the field level. A. alternata in a cotton growing region for 2 years, revealed that their air dispersal is local, probably at the field level. A. alternata air-borne spores were also trapped in rather low numbers regardless of the presence of infected cotton plants. However, the number of the air-borne spores trapped was dependent mainly on the average wind direction and on the Alternaria blight epidemics occurring in the fields twice a year. It is suggested that A. alternata spores are transferred by wind for short distances but are constantly present in small numbers in the atmosphere throughout the whole year. The two peaks recorded for the number of spores present in the air above cotton crops correlate with the annual two outbreaks of Alternaria blight epidemics. In addition, both wind and plant row direction affect disease development in the fields.     

Two cases of cutaneous phaeohyphomycosis by Alternaria alternata and Alternaria tenuissima

Two cases of cutaneous phaeohyphomycosis, one with a nodular appearance and the other with an erythematous infiltrating patch, are reported in immunocompromised patients. Diagnosis was based on histological examination, which revealed hyphae and round-shaped fungal cells in a granulomatous dermal infiltrate, and on identification of the moulds when biopsy fragments were cultured on Sabouraud-dextrose agar without cycloheximide. The pathogens were Alternaria tenuissima in the first case and A. alternata in the second. The fungi were examined by scanning electron microscopy. The patients were checked for bone and lung involvement and were then treated with surgical excision and itraconazole, and itraconazole only, respectively, with clinical and mycological resolution. This revised version was published online in August 2006 with corrections to the Cover Date.