Related Experiment Videos
Immunochemical and biological characterization of outer membrane proteins of Porphyromonas endodontalis
T Ogawa1, S Kuribayashi, H Shimauchi
1Department of Oral Microbiology, Osaka University Faculty of Dentistry, Japan.
Abstract:
Outer membrane proteins (OMP) of Porphyromonas endodontalis HG 370 (ATCC 35406) were prepared from the cell envelope fraction of the organisms. The cell envelope that had been obtained by sonication of the whole cells was extracted in 2% lithium dodecyl sulfate and then successively chromatographed with Sephacryl S-200 HR and DEAE-Sepharose Fast Flow. Two OMP fractions, OMP-I and OMP-II, were obtained, and their immunochemical properties and induction of specific antibodies were examined. The OMP-I preparation consisted of a major protein with an apparent molecular mass of 31 kDa and other moderate to minor proteins of 40.3, 51.4, 67, and 71.6 kDa, while the OMP-II preparation contained 14-, 15.5-, 27-, and 44-kDa proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. OMP-I was found to form hydrophilic diffusion pores by incorporation into artificial liposomes composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that OMP-I exhibited significant porin activity. However, the liposomes containing heat-denatured OMP-I were scarcely active. Spontaneous and antigen-specific immunoglobulin M (IgM)-, IgG-, and IgA-secreting spot-forming cells (SFC) enzymatically dissociated into single-cell suspensions from chronically inflamed periapical tissues and were enumerated by enzyme-linked immunospot assay. In patients with radicular cysts or dental granulomas, the major isotype of spontaneous SFC was IgG. In radicular cysts, the OMP-II-specific IgG SFC represented 0.13% of the total IgG SFC, while the antigen-specific IgA or IgM SFC was not observed. It was also found that none of these mononuclear cells produced antibodies specific for OMP-I or lipopolysaccharide of P. endodontalis.
Insights
Outer membrane proteins (OMP) from Porphyromonas endodontalis were isolated and characterized. OMP-I showed porin activity, while OMP-II elicited specific IgG responses in patients with periapical lesions.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Porphyromonas endodontalis is implicated in chronic periapical infections.
- Outer membrane proteins (OMPs) are key surface antigens of Gram-negative bacteria.
- Understanding P. endodontalis OMPs is crucial for diagnosing and treating endodontic infections.
Purpose of the Study:
- To isolate and characterize outer membrane proteins (OMPs) from Porphyromonas endodontalis HG 370.
- To investigate the porin activity of isolated OMPs.
- To examine the immunogenic properties of P. endodontalis OMPs in patients with periapical lesions.
Main Methods:
- Cell envelope fractionation and sonication.
- Extraction with lithium dodecyl sulfate.
- Chromatography using Sephacryl S-200 HR and DEAE-Sepharose Fast Flow.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein analysis.
- Liposome incorporation assay to assess porin activity.
- Enzyme-linked immunospot (ELISPOT) assay to enumerate specific antibody-secreting cells.
Main Results:
- Two OMP fractions, OMP-I and OMP-II, were obtained.
- OMP-I exhibited significant hydrophilic diffusion pore activity in artificial liposomes, indicating porin function.
- OMP-II-specific IgG antibody-secreting cells were detected in patients with radicular cysts.
- No antibodies specific for OMP-I or P. endodontalis lipopolysaccharide were found in the patient samples.
Conclusions:
- OMP-I of P. endodontalis possesses porin activity.
- OMP-II elicits a specific IgG immune response in the context of chronic periapical infections.
- These findings contribute to understanding the immunopathogenesis of P. endodontalis-associated endodontic diseases.