Isolation and characterisation of a 13.8-kDa bacteriolytic enzyme from house dust mite extracts: homology with prokaryotic proteins suggests that the enzyme could be bacterially derived

• Published in: FEMS immunology and medical microbiology 2002-06, Vol.33 (2), p.77-88
• Main Authors: Mathaba, Leslie T., Pope, Catherine H., Lenzo, Jason, Hartofillis, Maria, Peake, Helen, Moritz, Robert L., Simpson, Richard J., Bubert, Andreas, Thompson, Philip J., Stewart, Geoffrey A.
• Format: Article
• Language: English
• Subjects: Acid resistance; Amino Acid Sequence; Amino acids; Animals; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacteriolysin; Bacteriolysis; Base Sequence; Cloning; Culture Media, Conditioned - chemistry; Culture Media, Conditioned - metabolism; Der p 2; DNA, Complementary; Dust; Electrophoresis, Polyacrylamide Gel; Enzymes; Enzymes - chemistry; Enzymes - genetics; Enzymes - isolation & purification; Enzymes - metabolism; Gel electrophoresis; Gram-Positive Bacteria - enzymology; Gram-Positive Bacteria - physiology; Heat treatment; Homology; House dust; Housing; Hydroxyapatite; Listeria; Listeria monocytogenes; Lytic enzymes; Mercuric chloride; Mercury compounds; Mite; Mites; Mites - enzymology; Mites - microbiology; Molecular Sequence Data; Mycobacterial invasin; P60 superfamily; Protein sorting signals; Proteins; Sodium chloride; Sodium lauryl sulfate; Substrates; Thiols
• ISSN: 0928-8244; 1574-695X; 2049-632X
• DOI: 10.1111/j.1574-695X.2002.tb00576.x

Abstract Bacteriolytic activity was detected in extracts of whole mite and spent growth medium (SGM) from the clinically important Dermatophagoides pteronyssinus and Dermatophagoides farinae mites and was most abundant in whole mite extract. Gram-positive organisms Micrococcus lysodeikticus, Bacillus megaterium and Listeria monocytogenes were preferentially lysed and the lytic activity was enhanced by thiols, destroyed by mite proteases, inhibited by HgCl2 and high concentrations of NaCl but was resistant to heat and acid treatment. Substrate SDS–PAGE analysis indicated the presence of several lytic enzymes, two of which were isolated from D. pteronyssinus spent growth medium extract by hydroxyapatite chromatography. The N-terminal amino acid sequence of one of them was then used in PCR-based cloning studies. The complete amino acid sequence of this protein was determined and cDNA found to encode a 130-amino acid residue mature protein with a 20-amino acid leader sequence. The deduced protein demonstrated sequence similarity with the C-terminal regions of a group of bacterial proteins belonging to the P60 superfamily. These data suggest that the enzyme is derived from bacteria within the mites rather than from mites per se.