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Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073
Fariha Jasin Mansur1, Sari Takahara1, Mihoko Yamamoto1
1a Faculty of Agriculture, Department of Biological Chemistry , Yamaguchi University , Yamaguchi , Japan.
Abstract:
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.
Insights
Eikenella corrodens produces a hemolytic factor, identified as X-prolyl aminopeptidase. This enzyme, encoded by the hlyA gene, demonstrates dose-dependent hemolytic activity, confirmed through gene expression and disruption studies.
Area of Science:
- Microbiology
- Enzymology
- Bacterial Pathogenesis
Background:
- Eikenella corrodens is a bacterium known to cause various infections.
- Hemolytic activity in bacteria can contribute to virulence and pathogenesis.
- The specific mechanisms and factors responsible for E. corrodens hemolysis were not fully understood.
Purpose of the Study:
- To identify and characterize the hemolytic factor produced by Eikenella corrodens.
- To determine if X-prolyl aminopeptidase possesses hemolytic activity.
- To elucidate the role of the hlyA gene in the hemolytic potential of E. corrodens.
Main Methods:
- Purification of a 65-kDa protein with hemolytic activity from E. corrodens cell envelope fractions using chromatography.
- N-terminal amino acid sequencing of the purified protein.
- Expression of the hlyA gene (encoding X-prolyl aminopeptidase) in Escherichia coli and subsequent protein purification.
- Construction and analysis of a strain with a disrupted hlyA gene.
Main Results:
- A 65-kDa protein exhibiting hemolytic activity was isolated and purified.
- The N-terminal sequence of this protein was highly similar to X-prolyl aminopeptidase.
- Recombinant X-prolyl aminopeptidase expressed in E. coli showed confirmed hemolytic activity.
- A strain lacking a functional hlyA gene lost its hemolytic activity.
Conclusions:
- X-prolyl aminopeptidase is identified as the primary hemolytic factor in Eikenella corrodens.
- The hlyA gene encodes this X-prolyl aminopeptidase, which functions as a hemolysin.
- These findings suggest a novel role for X-prolyl aminopeptidase in bacterial virulence.
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