Characterisation of three novel β-1,3 glucanases from the medically important house dust mite Dermatophagoides pteronyssinus (airmid)

Abstract

The European house dust mite, Dermatophagoides pteronyssinus is a major source of airborne allergens worldwide and is found in half of European homes. Interactions between microbes and house dust mites (HDM) are considered important factors that allow them to persist in the home. Laboratory studies indicate the European HDM, D. pteronyssinus is a mycophagous mite, capable of utilising a variety of fungi for nutrients, however specific mycolytic digestive enzymes are unknown. Our previous work identified a number of putative glycosyl hydrolases present in the predicted proteome of D. pteronyssinus airmid and validated the expression of 42 of these. Of note, three GH16 proteins with predicted β-1,3 glucanase activity were found to be consistently present in the mite body and excretome. Here, we performed an extensive bioinformatic, proteomic and biochemical study to characterize three-novel β-1,3 glucanases from this medically important house dust mite. The genes encoding novel β-1,3 glucanases designated Glu1, Glu2 and Glu3 were identified in D. pteronyssinus airmid, each exhibited more than 59% amino acid identity to one another. These enzymes are encoded by Glu genes present in a tri-gene cluster and protein homologs are found in other acari. The patchy phyletic distribution of Glu proteins means their evolutionary history remains elusive, however horizontal gene transfer cannot be completely excluded. Recombinant Glu1 and Glu2 exhibit hydrolytic activity toward laminarin, pachyman and barley glucan. Excreted β-1,3 glucanase activity was increased in response to D. pteronyssinus airmid feeding on baker's yeast. Active β-1,3 glucanases are expressed and excreted in the faeces of D. pteronyssinus airmid indicating they are digestive enzymes capable of breaking down β-1,3 glucans of fungi present in house dust.

Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations

“…S12 D , Supplementary Material online). The triple tandemly arrayed beta-1,3 glucanases were considered nonexistent in D. farinae in a previous report ( Waldron et al 2019 ). The beta-1,3 glucanase of astigmatic mites has homologs from oribatid mites and springtails ( supplementary table S32, Supplementary Material online).…”

Improved diagnosis of SARS-CoV-2 by using Nucleoprotein and Spike protein fragment 2 in quantitative dual ELISA tests

“…To further confirm the expression and purification of the recombinant proteins, Western blots were performed using a monoclonal mouse anti-polyhistidine antibody (diluted 1:5,000) (Sigma-Aldrich) as a primary antibody followed by incubation with a secondary antibody alkaline phosphatase or horseradish peroxidase (HRP)-conjugated goat to mouse-anti-IgG (diluted 1:5000) (Sigma-Aldrich). Furthermore, the veracity of both S2Frag and Npro recombinant proteins was confirmed by high sensitivity protein mass spectrometry analysis using a Q-Exactive mass spectrometer (ThermoFisher) prior to use for ELISA development [22].…”

Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations

“…S12C). The triple tandemly arrayed beta-1,3 glucanases were considered as nonexistent in D. farinae in a previous report 42 .…”