Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases

Abstract

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.

Keywords: Bacillus cereus; Tyrophagus putrescentiae; allergen; bacillolysin; exochitinase; nutrition; protease; symbiosis.

Figure 1

General protease activity screening. Three peaks of protease activity at pH 5.75 and 6.75 and at basic pH were observed. The relative enzymatic activity is expressed as ΔA/h.

Figure 2

Comparison and specify of the protease activities toward BApNA, ZRRpNA, and AApNA at pH 5.75, 6.75, and 8.0. (A) Protease activities toward BApNA, ZRRpNA, and AApNA at pHs 5.75, 6.75, and 8.0; (B) Specifity of proteolytic activity on trypsin substrate BApNA; (C) Specifity of proteolytic activity on chymotrypsin substrate AAPpNA; (D) Specifity of proteolytic activity on cathepsin B substrate ZRRpNA.

Figure 3

Protease analysis by zymography in feces extract. (A) BAAMC–trypsin substrate, (A1), long time exposition; (A2), accurate capture time; (B) ZRRAMC–cathepsin B substrate; (C) AAAMC–chymotrypsin substrate.

Figure 4

2D-E image of Tyrophagus putrescentiae feces extract and control dry dog food extract with MS/MS identified proteins. (A) entire representative 2D-E; (B) detail of spots corresponding to Tyr p3 from another sample; (C) 2D-E image of control dry dog food protein extract of mite “food” proteins. Spots No. 19 and 20 are mature trypsin Tyr p3, spot No. 29 at the basic pI is apparently proenzyme of Tyr p3, and spot No. 11 is fragment of Tyr p3. The qualitatively different 2D-E patterns of images (A,B) show protein disappearance from the control DDF extract and demonstrate the powerful mite-bacterial digestive proteolytic activity in our experiment. See Table 1 for the list of MS/MS identified proteins.

Figure 5

The effect of Bacillus cereus addition to the diet on the growth of mites. This experiment tested the influence of different additive (0, 1, 5% w/w) of lyophilized B. cereus isolate to diet on two nutritionally different diets whole-meal spelt flour (WSF) and DDF. The lower case letters a and b indicate statistical difference (Kruskal-Wallis) between the control and bacterial treatments within diet.