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Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Published on: September 16, 2013
Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis
Nobuyuki Shibata1, Noriko Okanuma, Kanako Hirai
1Second Department of Hygienic Chemistry, Tohoku Pharmaceutical University, Sendai, Miyagi, Japan. nshibata@tohoku-pharm.ac.jp
Abstract:
Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.
Insights
Researchers cloned a gene for a lipolytic enzyme from Malassezia pachydermatis, a yeast linked to sepsis. This enzyme
Area of Science:
- Microbiology
- Enzymology
- Molecular Biology
Background:
- Lipophilic Malassezia species are implicated in catheter-associated sepsis, particularly in vulnerable patient groups.
- Understanding the role of lipolytic enzymes is crucial for elucidating the pathogenesis of Malassezia-induced infections.
- Parenteral lipid emulsions in medical treatments can create an environment conducive to Malassezia proliferation.
Purpose of the Study:
- To investigate the involvement of lipolytic enzymes in the pathogenicity of Malassezia species.
- To clone and characterize a gene encoding a lipolytic enzyme from Malassezia pachydermatis.
Main Methods:
- Purification of a lipolytic enzyme from the culture supernatant of Malassezia pachydermatis.
- Cloning of the corresponding cDNA using degenerate oligonucleotide primers based on N-terminal amino acid sequencing.
- Bioinformatic analysis of the cloned cDNA and deduced amino acid sequence, including homology searches.
Main Results:
- A lipolytic enzyme was purified 210-fold, exhibiting high esterase activity.
- The cloned cDNA spans 1582 bp, encoding a 470-amino acid open reading frame.
- The mature protein (438 amino acids) has a predicted molecular mass of 48 kDa and contains a conserved lipolytic enzyme motif; homology analysis revealed similarities to Candida albicans lipases.
Conclusions:
- The study successfully cloned and characterized a gene encoding a lipolytic enzyme from Malassezia pachydermatis.
- The identified enzyme shares characteristics with known lipases, suggesting a potential role in Malassezia pathogenesis.
- Further research into this enzyme may offer insights into preventing and treating Malassezia-related sepsis.
