Dermatophagoides pteronyssinus lytFM encoding an NlpC/P60 endopeptidase is also present in mite-associated bacteria that express LytFM variants

Abstract

The bodies and faecal pellets of the house dust mite (HDM), Dermatophagoides pteronyssinus, are the source of many allergenic and nonallergenic proteins. One of these, the 14-kDa bacteriolytic enzyme LytFM, originally isolated from the spent HDM growth medium, may contribute to bacteriolytic activity previously detected by zymography at 14 kDa in the culture supernatants of some bacterial species isolated from surface-sterilised HDM. Based on previously reported findings of lateral gene transfer between microbes and their eukaryotic hosts, we investigated the presence of lytFM in the genomes of nine Gram-positive bacteria from surface-sterilised HDM, and the expression by these isolates of LytFM and its variants LytFM1/LytFM2. The lytFM gene was detected by PCR in the genomes of three of the isolates: Bacillus licheniformis strain 1, B. licheniformis strain 2 and Staphylococcus aureus. Expression of the variant LytFM1 was detected in culture supernatants of these bacteria by mass spectrometry (MS) and ELISA, and the bacterial LytFM proteins were shown by zymography to be able to hydrolyse peptidoglycan. Our previous reports of LytFM homologues in other mite species and their phylogenetic analysis had suggested that they originated from a common mite ancestor. The phylogenetic analysis reported herein and the detection of other D. pteronyssinus proteins by MS in the culture supernatants of the three isolates that secreted LytFM1 further support the hypothesis of lateral gene transfer to the bacterial endosymbionts from their HDM host. The complete sequence homology observed between the genes amplified from the microbes and those in their eukaryotic host indicated that the lateral gene transfer was an event that occurred recently.

Keywords: D,L‐endopeptidase; Dermatophagoides pteronyssinus; LytFM; NlpC/P60; host; microbes.

Figure 1

Screening of the genomes of nine HDM‐associated bacterial isolates for the presence of the lytFM gene. (A) PCR was performed with the primer set GSUTR1/GSR3, and an amplicon was only obtained from Bacillus licheniformis strain 1, B. licheniformis strain 2 and Staphylococcus aureus. (B) The amplicons were sequenced and their translated amino acid sequences aligned with those of LytFM (accession number KF113885) and LytFM1 (accession number AF409109). The amino acid residues underlined show the binding sites for the forward primer GSUTR1 (residues ‐20 to ‐15) and reverse primer GSR3 (residues 124–130). Nonhomologous residues are highlighted in boldface.

Figure 2

Zymographic analysis of bacteriolytic activity in bacterial culture supernatants. Following concentration by 80% saturated ammonium sulfate precipitation and desalting of the supernatants of the HDM‐associated Bacillus licheniformis strain 1, B. licheniformis strain 2 and Staphylococcus aureus by gel filtration chromatography, bacteriolytic activity was detected in the first six/seven fractions, including activity at 14 kDa as indicated by arrow. The bacteriolytic activity of HEWL and Dermatophagoides pteronyssinus SGM served as the size marker and positive control, respectively. D. pteronyssinus SGM (Dp).

Figure 3

Detection of LytFM in bacterial culture supernatants of Bacillus licheniformis and Staphylococcus aureus. (A) A representative gel of B. licheniformis strain 2 showing the fractions obtained from the concentrated and desalted supernatant and the regions (as marked by boxes) pooled for MS analysis. (B) Alignment of the amino acid sequences of LytFM, LytFM1, LytFM2 and the peptides detected in the culture supernatants of B. licheniformis strain 1, B. licheniformis strain 2, S. aureus and Dermatophagoides pteronyssinus SGM which are highlighted in boldface. Nonhomologous residues are highlighted in red. The peptides used for generating anti‐peptide antisera Rb1001 and Rb999 are underlined.

Figure 4

Immunoreactivity of anti‐peptide antisera Rb1001 (A) and Rb999 (B) with Dermatophagoides pteronyssinus SGM and bacterial culture supernatants of the HDM‐associated Bacillus licheniformis strain 1, B. licheniformis strain 2 and Staphylococcus aureus. Both the polyclonal anti‐peptide antisera reacted with 0.01 μg (*), 0.1 μg (▵), 1 μg (♦) and 5 μg (▲) of protein found in the D. pteronyssinus SGM or bacterial culture supernatants. Preimmune sera Rb 1001 and Rb999 were used as the respective negative controls and were tested using 0.01 μg (○), 0.1 μg (□), 1 μg (●) and 5 μg (♢) of protein in the D. pteronyssinus SGM or bacterial culture supernatants. Assays were performed in duplicate, and mean optical density values are shown.

Figure 5

Inhibition of anti‐peptide antisera Rb1001 (in red) and Rb999 (in black) by Dermatophagoides pteronyssinus SGM and the bacterial culture supernatants of the HDM‐associated Bacillus licheniformis strain 1, B. licheniformis strain 2 and Staphylococcus aureus. Assay for the positive control D. pteronyssinus SGM was performed in triplicate, and all the other assays were performed in duplicate. Mean percentage inhibition values are shown. The correlation coefficients (Rb1001/Rb999) for D. pteronyssinus SGM, B. licheniformis strain 2 and S. aureus are 0.93/0.99, 0.95/0.92, 0.97/0.91 and 0.97/0.90, respectively. The IC ₅₀, that is concentrations of D. pteronyssinus SGM, bacterial culture supernatants of B. licheniformis strain 1, B. licheniformis strain 2 and S. aureus required to generate 50% inhibition of the immunoreactivity of the anti‐peptide antisera (Rb1001/Rb999) are 0.51/0.23, 0.15/0.11, 0.17/0.1 and 0.17/0.1 μg·mL⁻¹, respectively.

Figure 6

Phylogenetic relationship of mite and bacterial LytFM homologues and prokaryotic and eukaryotic NlpC/P60 proteins. According to the GenBank records, the accession numbers of Aspergillus fumigatus (XP 753281), Neosartorya udagawae (GAO84874), Tetrahymena thermophila (XM 001014103) and Dictyostelium fasciculatum (GL883007) are updated to EAL91243, GAO82962, XP 001014103 and EGG24385, respectively. Phylogenetic analysis was performed with Phylogeny.fr pipeline 26, approximate likelihood ratio test was used, and the confidence values are shown on the branches.