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Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry
Published on: May 21, 2018
Dermatophagoides pteronyssinus lytFM encoding an NlpC/P60 endopeptidase is also present in mite-associated bacteria
Vivian H Tang1, Geoffrey A Stewart1, Barbara J Chang1
1Marshall Centre for Infectious Diseases Research and Training School of Biomedical Sciences The University of Western Australia Crawley WA Australia.
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.
Insights
House dust mites (HDM) harbor bacteria that express a bacteriolytic enzyme, LytFM, likely acquired through recent lateral gene transfer from the mite host. This enzyme can degrade bacterial peptidoglycan.
Area of Science:
- Microbiology and Molecular Biology
- Genomics and Genetics
- Immunology and Allergy
Background:
- House dust mites (HDM), specifically *Dermatophagoides pteronyssinus*, harbor various proteins, including the 14-kDa bacteriolytic enzyme LytFM.
- Bacteriolytic activity at 14 kDa has been observed in bacterial species isolated from HDM, suggesting a potential role for LytFM.
- Previous findings indicate lateral gene transfer (LGT) between microbes and their eukaryotic hosts.
Purpose of the Study:
- To investigate the presence and expression of the *lytFM* gene and its variants in Gram-positive bacteria isolated from surface-sterilized HDM.
- To confirm the hypothesis of LGT of the *lytFM* gene from *Dermatophagoides pteronyssinus* to its bacterial endosymbionts.
- To analyze the phylogenetic relationship and sequence homology of the *lytFM* gene between HDM and bacterial isolates.
Main Methods:
- Polymerase Chain Reaction (PCR) to detect the *lytFM* gene in bacterial genomes.
- Mass Spectrometry (MS) and Enzyme-Linked Immunosorbent Assay (ELISA) to detect LytFM variant expression.
- Zymography to assess the peptidoglycan hydrolyzing activity of bacterial LytFM proteins.
- Phylogenetic analysis of *lytFM* genes and detection of HDM proteins in bacterial supernatants.
Main Results:
- The *lytFM* gene was detected in three bacterial isolates: *Bacillus licheniformis* strains 1 and 2, and *Staphylococcus aureus*.
- Expression of the LytFM1 variant was confirmed in bacterial culture supernatants via MS and ELISA.
- Bacterial LytFM proteins demonstrated peptidoglycan hydrolysis activity.
- Phylogenetic analysis and detection of HDM proteins in bacterial supernatants support recent LGT from HDM to bacteria.
Conclusions:
- Lateral gene transfer of the *lytFM* gene from *Dermatophagoides pteronyssinus* to its endosymbiotic bacteria (*B. licheniformis*, *S. aureus*) has occurred.
- The high sequence homology suggests this LGT event is a recent phenomenon.
- The expressed bacterial LytFM proteins possess functional bacteriolytic activity, potentially contributing to observed activities in HDM-associated bacteria.
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