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Published on: May 21, 2015
Purification and characterization of a putative fimbrial protein/receptor of Porphyromonas gingivalis
S G Dashper1, N M O'Brien-Simpson, P S Bhogal
1Biochemistry and Molecular Biology Unit, School of Dental Science, University of Melbourne.
Abstract:
Porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease, P. gingivalis is a Gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein. Virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes. A 43 kDa fimbrillin from P. gingivalis has been isolated and characterized. However, there is evidence that a second type of fimbria exists on the surface of P. gingivalis. A putative P. gingivalis fimbrial protein from a membrane preparation has been isolated and identified. This protein was shown to be reactive with sera from patients harbouring P. gingivalis. A 28 kDa protein fragment was purified by anion exchange, gel filtration and reversed-phase chromatography. N-terminal sequence analysis of the 28 kDa protein fragment revealed homology to the fimbrial precursor protein of Dichelobacter nodosus. A peptide corresponding to the N-terminal 26 amino acyl residues of the 28 kDa protein fragment was synthesized and used to raise antibodies to the protein. Western blot analysis after SDS-PAGE of a P. gingivalis membrane preparation using the antibodies raised to the synthetic peptide detected three proteins of 36, 41 and 67 kDa. When protease inhibitors were not included in the extraction procedure only the 36 and 41 kDa bands were detected. It would appear, therefore, that the intact protein has an M(r) of 67 kDa and that the 28, 36 and 41 kDa bands represent protein fragments produced by endogenous proteolytic activity. Based on sequence homology, the 67 kDa protein is possibly a sub-unit of a second P. gingivalis fimbrial type or a surface receptor.
Insights
Porphyromonas gingivalis, a key player in periodontal disease, possesses a novel 67 kDa fimbrial protein. This protein, potentially a virulence factor, is susceptible to degradation by bacterial proteases.
Area of Science:
- Microbiology
- Oral Health
- Bacterial Pathogenesis
Background:
- Porphyromonas gingivalis is a major cause of periodontal disease.
- Virulence factors of P. gingivalis include capsule, fimbrial adhesins, cytotoxins, and enzymes.
- A 43 kDa fimbrillin is known, but evidence suggests a second fimbrial type exists.
Purpose of the Study:
- To isolate and identify a putative fimbrial protein from P. gingivalis.
- To characterize this protein and determine its potential role in virulence.
- To investigate the presence of a second fimbrial type on P. gingivalis.
Main Methods:
- Isolation and purification of a 28 kDa protein fragment from P. gingivalis membrane preparations.
- N-terminal sequence analysis of the fragment.
- Synthesis of a peptide for antibody generation.
- Western blot analysis using generated antibodies.
Main Results:
- A 67 kDa protein was identified as the intact form, with fragments of 36 and 41 kDa detected.
- The 28, 36, and 41 kDa bands represent proteolytic degradation products.
- Sequence homology suggests the 67 kDa protein is a subunit of a second fimbrial type or a surface receptor.
Conclusions:
- P. gingivalis possesses a 67 kDa protein, likely a fimbrial subunit or surface receptor.
- Endogenous proteolytic activity significantly degrades this protein.
- Understanding this protein is crucial for developing strategies against P. gingivalis-associated periodontal disease.
