Effect of deletion of the rgpA gene on selected virulence of Porphyromonas gingivalis.

BACKGROUND/PURPOSE: The most potent virulence factors of the periodontal pathogen Porphyromonas gingivalis are gingipains, three cysteine proteases (RgpA, RgpB, and Kgp) that bind and cleave a wide range of host proteins. Considerable proof indicates that RgpA contributes to the entire virulence of the organism and increases the risk of periodontal disease by disrupting the host immune defense and destroying the host tissue. However, the functional significance of this proteinase is incompletely understood. It is important to analyze the effect of arginine-specific gingipain A gene (rgpA) on selected virulence and physiological properties of P. gingivalis. MATERIALS AND METHODS: Electroporation and homologous recombination were used to construct an rgpA mutant of P. gingivalis ATCC33277. The mutant was verified by polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cell structures of the mutant were examined by transmission electron microscopy and homotypic biofilm formation was examined by confocal laser scanning microscopy. RESULTS: Gene analysis revealed that the rgpA gene was deleted and replaced by a drug resistance gene marker. The defect of the gene resulted in a complete loss of RgpA proteinase, a reduction of out membrane vesicles and hemagglutination, and an increase in homotypic biofilm formation. CONCLUSION: Our data indicate that an rgpA gene deficient strain of P. gingivalis is successfully isolated. RgpA may have a variety of physiological and pathological roles in P. gingivalis.

[Construction of recombinant plasmid with Porphyromonas gingivalis FimA deficiency].

OBJECTIVE: To construct the recombinant plasmid pPHU281_A_Spec_B, which knock out Porphyrmonas gingivalis (Pg) FimA gene. METHODS: Genomic DNA was extracted from PgATCC33277 which was cultured in anaerobic condition. The upstream and downstream gene of FimA was cloned from Pg genenomic DNA with specific restriction sites by polymerase chain reaction. Suicide vector pPHU281 was inserted by three fragments: upstream, downstream of FimA gene and spectinomycin resistance gene. The recombinant plasmid was confirmed by electrophoresis and sequenced after amplification in compentent cells DH-5α. RESULTS: The gene sequence was identified by DNA sequencing analysis. The recombinant plasmid pPHU281_A_Spec_B was successfully constructed. CONCLUSIONS: The recombinant plasmid pPHU281_A_Spec_B was constructed, which may be used for the constructon of FimA deficient Pg.

[Effect of longterm lower Porphyromonas gingivalis infection on the progression of atherosclerosis in apolipoprotein E-knocked out mice].

OBJECTIVE: To assess the effect of longterm and lower oral inoculation with Porphyromonas gingivalis (Pg) on the progression of atherosclerosis in apolipoprotein E-knocked out (ApoE(-/-)) mice. METHODS: Six-week-old male ApoE(-/-) mice were inoculated orally with 0.1 ml live Pg(10(13)/L) or bouillon culture-medium quintic per week for 15 consecutive weeks, altogether 75 times of inoculations. The lesion area of atherosclerosis in the aortic tree was measured by en face quantification by red oil O staining method. The atherosclerotic lesion was examined by histopathology. The levels of total cholesterol and triglycerides were compared. RESULTS: At 22 weeks after inoculation, the mean atherosclerotic lesion area in inoculated mice was (98 363.68 ± 12 043.00) µm(2), which was significantly greater than that in noninoculated mice, which was (62 985.06 ± 7419.64) µm(2) (P = 0.035). CONCLUSIONS: Longterm lower oral inoculation of Pg can accelerate the progression of atherosclerosis in apolipoprotein E-knocked out mice.

Amorphous calcium phosphate and its application in dentistry.

Amorphous Calcium Phosphate (ACP) is an essential mineral phase formed in mineralized tissues and the first commercial product as artificial hydroxyapatite. ACP is unique among all forms of calcium phosphates in that it lacks long-range, periodic atomic scale order of crystalline calcium phosphates. The X-ray diffraction pattern is broad and diffuse with a maximum at 25 degree 2 theta, and no other different features compared with well-crystallized hydroxyapatite. Under electron microscopy, its morphological form is shown as small spheroidal particles in the scale of tenths nanometer. In aqueous media, ACP is easily transformed into crystalline phases such as octacalcium phosphate and apatite due to the growing of microcrystalline. It has been demonstrated that ACP has better osteoconductivity and biodegradability than tricalcium phosphate and hydroxyapatite in vivo. Moreover, it can increase alkaline phosphatase activities of mesoblasts, enhance cell proliferation and promote cell adhesion. The unique role of ACP during the formation of mineralized tissues makes it a promising candidate material for tissue repair and regeneration. ACP may also be a potential remineralizing agent in dental applications. Recently developed ACP-filled bioactive composites are believed to be effective anti-demineralizing/remineralizing agents for the preservation and repair of tooth structures. This review provides an overview of the development, structure, chemical composition, morphological characterization, phase transformation and biomedical application of ACP in dentistry.

[Effect of Porphyromonas gingivalis on nitric oxide in cultured human umbilical vein endothelial cells].

OBJECTIVE: To observe the effect of Porphyromonas gingivalis (Pg) on the production of nitric oxide (NO) in cultured human umbilical vein endothelial cells (HUVEC), and to investigate the pathway of damaging endothelial function by Pg. METHODS: Pg ATCC33277 was cultured in anaerobic jar, and HUVEC was treated with various concentrations of Pg ATCC33277 at multiplicity of infection (MOI) of 1:10, 1:100 and 1:1000 for 4, 8, 12, 24 h respectively. The cells supernatants were collected and stored at -70 degrees C and NO concentration in the cells supernatants was measured by nitrate reductase assay. RESULTS: Within 24 h, Pg at MOI of 1:10 and 1:100 stimulated the release of nitric oxide in cultured HUVEC. Within 12 h, Pg at an MOI of 1:1000 group increased NO production, and NO decreased at 24 h. CONCLUSIONS: Pg has an effect on the production of NO. Low concentrations of Pg stimulated release of nitric oxide in endothelial cells but high concentrations can decrease the release of NO.