Project description:Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen, Porphyromonas gingivalis, exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct sub-types within a population of P.ginigivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly-invasive sub-types invade cells at 10-30 fold higher levels than the poorly-invasive subtype and remain highly invasive for approximately 12-16 generations. Analysis of the gingipain activity of these sub-types revealed that the highly invasive type had reduced cell-associated arginine specific protease activity. The role of arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition a population of DeltargpAB bacteria did not contain a hyperinvasive sub-type. Screening of the protease activity of wild-type populations of both strains identified high and low protease sub-types which also showed the corresponding reduction or enhancement of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that include oxidative stress resistance and iron transport genes that might be key to invasion of or survival within epithelial cells. Porphyromonas gingivalis comparison of Invasive V Non-invasive sub-types of NCTC11834 and W50 (samples 1-12 AND High V low protease subtypes (samples 13-24) Samples 1-3 were compared top 4-6 and 7-9 V 10-12 for invasive experiments and similarly for protease strain experiments
Project description:Wild type Porphyromonas gingivalis strain ATCC33277 (V3176) and PG1626 - deficient mutant (V3177) were grown in iron replete conditions was used to compare to Porphyromonas gingivalis strains grown in iron chelated conditions.
Project description:The human oral pathogen Porphyromonas gingivalis colonizes the gingival crevice and invades gingival epithelial cells. Multidimensional capillary high-performance liquid chromatography coupled with tandem mass spectrometry and two-dimensional gel electrophoresis were used to analyze the proteome of P. gingivalis as it adapts to a set of experimental conditions designed to reflect important features of an epithelial cell environment. 1014 proteins (46% of the total theoretical proteome) were identified in four independent analyses; 479 of these proteins showed evidence of differential expression after exposure of P. gingivalis to either conditioned epithelial cell growth medium or control conditions: i.e., they were only detected under one set of conditions. Moreover, 276 genes annotated as hypothetical were found to encode expressed proteins. Among the proteins up-regulated in the presence of epithelial cell components were a homolog of the internalin proteins of Listeria monocytogenes and subunits of the ATP-dependent Clp protease complex. Insertional inactivation of clpP, encoding the Clp proteolytic subunit, resulted in approximately a 50% reduction in invasion of P. gingivalis. These results suggest that adaptation to an epithelial cell environment induces a major shift in the expressed proteome of the organism. Furthermore, ClpP, that is up-regulated in this environment, is required for optimal invasive activity of P. gingivalis. Keywords: proteome analysis of P. gingivalis
Project description:Porphyromonas gingivalis and Treponema denticola are periodontalpathogens that are associated with the severity and progression of periodontal diseases. This study investigates the gene expression of Porphyromonas gingivalis during co-culture with Treponema denticola
Project description:Porphyromonas gingivalis and Treponema denticola are periodontalpathogens that are associated with the severity and progression of periodontal diseases. this study investigates the gene expression of Treponema denticola during co-culture with Porphyromonas gingivalis.
Project description:Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen, Porphyromonas gingivalis, exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct sub-types within a population of P.ginigivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly-invasive sub-types invade cells at 10-30 fold higher levels than the poorly-invasive subtype and remain highly invasive for approximately 12-16 generations. Analysis of the gingipain activity of these sub-types revealed that the highly invasive type had reduced cell-associated arginine specific protease activity. The role of arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition a population of DeltargpAB bacteria did not contain a hyperinvasive sub-type. Screening of the protease activity of wild-type populations of both strains identified high and low protease sub-types which also showed the corresponding reduction or enhancement of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that include oxidative stress resistance and iron transport genes that might be key to invasion of or survival within epithelial cells.
Project description:Investigation of whole genome gene expression level changes in Porphyromonas gingivalis ATCC 33277 treated with an anti-adhesive extract from Myrothamnus flabellifolia compared to the untreated strain. Aim: Identification of anti-adhesive plant extracts against cell surface binding of Porphyromonas gingivalis. Materials and Methods: Polyphenol-enriched extract from Myrothamnus flabellifolia (MF) traditionally used for periodontitis was tested for inhibition of P. gingivalis adhesion to KB cells by FACS, for influence on gingipain activity, hemagglutination and by microarray analysis for effects on the bacterial transcriptome. P. gingivalis-induced inflammation parameters were monitored by RT-PCR. Results: MF (100 µg/ml) reduced P. gingivalis adhesion/invasion about 50% by interacting with fimbriae and bacterial OMPs. Microarray analysis of MF-treated bacteria indicated up-regulation of genes involved in cell adhesion. As confirmed by RT-PCR, fimbrillin- and Arg-gingipain-encoding genes were upregulated by MF. On the protein level, inhibition (70%) of Arg-gingipain activity was observed, while the corresponding Lys-gingipain was hardly influenced. MF also inhibited hemagglutination. While exposure to P. gingivalis resulted in an increased expression of inflammation-related genes in KB cells, pretreatment of KB cells with MF evoked cytoprotective effects concerning IL-1β, IL-6, IL-8 and TNFα gene expression as well as IL-6 release rates. Conclusions: While being cytoprotective, MF exerts strong anti-adhesive effects against P. gingivalis. Thus, MF may be useful for the prevention of P. gingivalis-associated periodontal diseases. The chip study used total RNA recovered from two separate MF-treated and two separate untreated Porphyromonas gingivalis ATCC 33277 cultures. Each chip measured the expression level of 1,842 genes from P. gingivalis ATCC 33277 with thirteen 60-mer probes per gene, with three-fold technical redundancy.