BACKGROUND: The domestic mite Blomia tropicalis is a major source of allergens in tropical and subtropical regions. Despite its great medical importance, the allergome of this mite has not been sufficiently studied. Only 14 allergen groups have been identified in B. tropicalis thus far, even though early radioimmunoelectrophoresis techniques (27 uncharacterized allergen complexes) and comparative data based on 40 allergen groups officially recognized by the World Health Organization (WHO)/IUIS in domestic astigmatid mites suggest the presence of a large set of additional allergens. METHODS: Here, we employ a multiomics approach to assess the allergome of B. tropicalis using genomic and transcriptomic sequence data and perform highly sensitive protein abundance quantification. FINDINGS: Among the 14 known allergen groups, we confirmed 13 (one WHO/IUIS allergen, Blo t 19, was not found) and identified 16 potentially novel allergens based on sequence similarity. These data indicate that B. tropicalis shares 27 known/deduced allergen groups with pyroglyphid house dust mites (genus Dermatophagoides). Among these groups, five allergen-encoding genes are highly expressed at the transcript level: Blo t 1, Blo t 5, Blo t 21 (known), Blo t 15, and Blo t 18 (predicted). However, at the protein level, a different set of most abundant allergens was found: Blo t 2, 10, 11, 20 and 21 (mite bodies) or Blo t 3, 4, 6 and predicted Blo t 13, 14 and 36 (mite feces). INTERPRETATION: We report the use of an integrated omics method to identify and predict an array of mite allergens and advanced, label-free proteomics to determine allergen protein abundance. Our research identifies a large set of novel putative allergens and shows that the expression levels of allergen-encoding genes may not be strictly correlated with the actual allergenic protein abundance in mite bodies.

• Klíčová slova
• IgE, enzyme, genome, label-free proteomics, mites, transcriptome,
• Publikační typ
• časopisecké články MeSH

Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in balance to be beneficial for the mite. The mite-B. cereus symbiosis can be beneficial-suppressive at some level. The results increase the veterinary and medical importance of the allergens detected in feces. The B. cereus enzymes/toxins are important components of mite allergens. The strong symbiotic association of T. putrescentiae with B. cereus in DDF was indicated.

• Klíčová slova
• Bacillus cereus, Tyrophagus putrescentiae, allergen, bacillolysin, exochitinase, nutrition, protease, symbiosis,
• Publikační typ
• časopisecké články MeSH

Dermatophagoides farinae fecal allergens are a major source of immunogens in home environments; however, as the source of mite fecal allergen is considered spent growth medium extract that can only mimic the pure fecal extract. In this study, we prepared and using proteomic methods analyzed a D. farinae fecal extract for the first time. The preparation approach used D. farinae feces that were produced within 8 weeks of initiating cultivation in minimized growth media. The feces were collected via adhesion to the tissue culture flask surfaces after removing the SGM and mites. This study contains in-depth proteomic mapping of the allergenic isoforms from the D. farinae fecal extract. Despite extensive analysis, MALDI TOF/TOF spectrometry showed that only six proteins/allergens, Der f1, Der f2, Der f3, Der f6, Der f15 and ferritin, originated from D. farinae. No other analyzed proteins were exactly assigned to Dermatophagoides or to similar invertebrate species by sequence similarity. The remaining proteins were assigned mostly to yeasts or cereals (originally dietary proteins); however, many of the proteins were not successfully identified in the current NCBInr. The numerous dietary proteins identified in the feces suggest that these proteins remained highly stable after passing through the gut. Isoforms of the allergens Der f1, Der f3 and Der f15 were identified in more MWs indicating the presence of zymogens and active-enzyme forms. The identified fecal allergens accumulate in the environment during the life of the mite and represent quantitatively greater amounts of mite immunogens than those that were missed in the 2D-E. The results contribute to our understanding of D. farinae digestive physiology with regard to the enzymes/proteins present in the feces.

• MeSH
• 2D gelová elektroforéza metody   MeSH
• antigeny roztočů domácího prachu chemie   MeSH
• Dermatophagoides farinae chemie   MeSH
• feces chemie   MeSH
• protein - isoformy MeSH
• proteomika metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny roztočů domácího prachu MeSH
• protein - isoformy MeSH