Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073

• Published in: Bioscience, biotechnology, and biochemistry Vol. 81; no. 6; pp. 1246 - 1253
• Main Authors: Jasin Mansur, Fariha, Takahara, Sari, Yamamoto, Mihoko, Shimatani, Masafumi, Minnatul Karim, Mohammad, Noiri, Yuichiro, Ebisu, Shigeyuki, Azakami, Hiroyuki
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 03.06.2017; Oxford University Press
• Subjects: Aminopeptidases - genetics; Aminopeptidases - isolation & purification; Aminopeptidases - metabolism; Aminopeptidases - pharmacology; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacterial Proteins - pharmacology; Cell Fractionation; Cloning, Molecular; Eikenella corrodens; Eikenella corrodens - enzymology; Eikenella corrodens - genetics; Eikenella corrodens - isolation & purification; Eikenella corrodens - pathogenicity; Erythrocytes - cytology; Erythrocytes - drug effects; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Hemolysin Proteins - genetics; Hemolysin Proteins - isolation & purification; Hemolysin Proteins - metabolism; Hemolysin Proteins - pharmacology; hemolysis; Hemolysis - drug effects; Homologous Recombination; Humans; Molecular Weight; Periodontitis - microbiology; Periodontium - microbiology; purification; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; virulence, periodontal disease; virulence, periodontal disease; purification; hemolysis; Eikenella corrodens
• DOI: 10.1080/09168451.2017.1295807
• ISSN: 0916-8451

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl β-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens. Eikenella corrodens 1073 showed hemolytic activity when grown on sheep blood agar. We purified and identified the hemolytic factor.