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.
Project description:In this in vitro study, using DNA microarray we investigate the differential gene expression of Porphyromonas gingicalis ATCC 33277 when growing in the presence or in absence of its own monospecies biofilm.
Project description:Periodontal diseases are one of the most common human maladies and appear to be caused by the interaction of proximal pathogens such as Porphyromonas gingivalis but only as part of the polymicrobial community known as dental plaque. Streptococcus gordonii is an early colonizing oral organism that binds to oral surfaces and provides adherence for organisms such as P. gingivalis. Together P. gingivalis and S. gordonii form one of the simplest models of potentially pathogenic dental plaque. We used RNA sequencing to monitor the transcriptome of P. gingivalis over time in a biofilm model both in the presence and absence of S. gordonii. Samples were taken at 5, 30, 120, 240, and 360 minutes after shifing from planktonic to sessile conditions and growth media to PBS. When compared to planktonic cells increased transcripts were found for stress, amino acid catabolism, and comeptence and decreased transcripts for DNA replication. The presence of S. gordonii resulted in fewer changes from planktonic cells implying physiological support to Pl gingivalis making the transition from planktonic to sessile easier.
Project description:Bacterial cells in biofilms adopt a profoundly different phenotype than their planktonic counterparts. The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are pathogens associated with oral biofilm and periodontal disease. In this study, we identified and functionally characterized the proteome of F. nucleatum and P. gingivalis cells grown either as biofilms or in planktonic cultures by high-resolution liquid chromatography-tandem mass spectrometry. We used proteomic analysis for identification and label-free quantification of proteins, and sequence-based functional characterization for their classification. In F. nucleatum biofilm five proteins showed changes from planktonic condition, including proteins involved in vitamin B metabolic processes. Forty proteins were changed in P. gingivalis biofilm, 30 increased and 10 decreased. Putative cell division trigger factor and riboflavin biosynthesis proteins were the most increased in this biofilm. To describe interactions between the two species at the protein level, we grew the bacteria either individually or together. In the mixed species biofilm culture, 112 proteins showed significant changes, with 72 proteins derived from F. nucleatum and 40 proteins from P. gingivalis. By comparing dual-species to mono-species in biofilm and in planktonic growth conditions, P. gingivalis showed more proteins with decreased level in the dual species conditions.
Project description:Porphyromonas gingivalis is a major pathogen associated with the microbial biofilm-mediated disease chronic periodontitis. P. gingivalis has an obligate requirement for iron and protoporphyrin IX which it satisfies by transporting heme and iron liberated from the human host. The level of cellular iron in P. gingivalis affects the expression of a distinct iron-associated regulon of 64 genes and low iron invokes an iron sparing response. Iron homeostasis is usually mediated in Gram-negative bacteria at the transcriptional level by the Ferric Uptake Regulator (Fur). There is a single predicted P. gingivalis Fur superfamily orthologue named Har (heme associated regulator) that lacks the conserved metal binding residues found in other Fur orthologues. We show that Har binds both heme and ferrous iron resulting in a conformational change in the protein. Har was unable to complement the Escherichia coli H1780 fur mutant and there was no change in cellular metal content in a P. gingivalis Har mutant compared with the wild-type. The Har regulon of 44 genes is not predicted to play a role in iron homeostasis. Together these data indicated that Har does not regulate iron homeostasis in P. gingivalis. However, Har was required for heme-responsive biofilm development and its regulon overlapped P. gingivalis regulons previously identified after growth in heme limitation or as a homotypic biofilm. P. gingivalis is unique as an iron-dependent Gram-negative bacterium with a single heme-binding Fur superfamily orthologue, Har, that does not regulate iron homeostasis. Paired samples were compared on the same microarray using a two-colour system. A total of 6 paired microarray hybridizations were performed representing 6 biological replicates, where a balanced dye design was used, with the overall analysis including three microarrays where P. gingivalis 33277 samples were labeled with Cy3 and the paired ECR455 samples were labeled with Cy5 and three other microarrays where samples were labeled with the opposite combination of fluorophores.
Project description:Comparative gene expression analysis of planktonic Porphyromonas gingivalis ATCC 33277 in the presence of a growing biofilm versus planktonic cells
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.