Microarray analysis of the transcriptional responses of Porphyromonas gingivalis to polyphosphate.
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ABSTRACT:
SUBMITTER: Moon JH
PROVIDER: S-EPMC4236598 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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Project description:Long-lived monocytes, macrophages, and dendritic cells (DCs) are Toll-like receptor-expressing, antigen-presenting cells derived from a common myeloid lineage that play key roles in innate and adaptive immune responses. Based on immunohistochemical and molecular analyses of inflamed tissues from patients with chronic destructive periodontal disease, these cells, found in the inflammatory infiltrate, may drive the progressive periodontal pathogenesis. To investigate early transcriptional signatures and subsequent proteomic responses to the periodontal pathogen, Porphyromonas gingivalis, donor-matched human blood monocytes, differentiated DCs, and macrophages were exposed to P. gingivalis lipopolysaccharide (LPS) and gene expression levels were measured by oligonucleotide microarrays. In addition to striking differences in constitutive transcriptional profiles between these myeloid populations, we identify a P. gingivalis LPS-inducible convergent, transcriptional core response of more than 400 annotated genes/ESTs among these populations, reflected by a shared, but quantitatively distinct, proteomic response. Nonetheless, clear differences emerged between the monocytes, DCs, and macrophages. The finding that long-lived myeloid inflammatory cells, particularly DCs, rapidly and aggressively respond to P. gingivalis LPS by generating chemokines, proteases, and cytokines capable of driving T-helper cell lineage polarization without evidence of corresponding immunosuppressive pathways highlights their prominent role in host defense and progressive tissue pathogenesis. The shared, unique, and/or complementary transcriptional and proteomic profiles may frame the context of the host response to P. gingivalis, contributing to the destructive nature of periodontal inflammation.
| S-EPMC2671371 | biostudies-other
Project description:Polyphosphate (polyP) is a ubiquitous and abundant compound found in bacteria, fungi, algae, plant, and animals. Among roles of intracellular polyP in bacteria are resistance and survival in the stationary phase of the growth against stress and stringent condition. Therefore, intracellular polyP is considered as a virulence factor of bacteria. In contrast, exogenous polyP has an antimicrobial activity against a variety of microorganisms. To date, much has been numerous studies of antibacterial effect of polyP against gram positive bacteria and fungi while relatively little reports have been published concerning gram negative bacteria. Here we describe bactericidal effect of polyP against gram negative periodontopathic bacterium, Porphyromonas gingivalis, and the transcriptional change by polyP in this bacterium. The bacterial growth was inhibited by polyP (chain length of P3~P75) at concentration of 0.02~0.03%, but not by Pi and PPi at the concentrations. polyP75 was chosen for further experiments, which suppressed the further growth of P. gingivalis as low as 0.03%. polyP75 completely killed the bacterial cells at the concentration of 0.035%. Microarray analysis was employed to identify genes that showed a greater than 1.5-fold difference in the expression by polyP75 at the concentration of 0.03%. It was found that 155 genes were up-regulated and 173 were down-regulated. Down-regulated genes include groups of energy metabolism-related genes, cell envelope-related genes, and genes in relation to biosynthesis of cofactors, prosthetic groups and carriers. Among the down-regulated genes were several involved in DNA replication, cell division protein, and biosynthesis of purines, pyrimidines, nucleosides and nucleotides. In contrast, a large number of ribosomal proteins, transciptional regulators and transposases were up-regulated. Oxidative stress-related genes and iron storage proteins also appeared to be increased in the expression. Real-time PCR confirmed up- and down-regulation of some selected genes. The overall results suggest that polyP has a bactericidal activity against P. gingivalis, interfering with translation, energy metabolism, DNA replication, cell division, and biosynthesis of purines, pyrimidines, nucleoside and nucleotides. polyP may be used as an agent for prevention and treatment of periodontal infections. Polyp-treated vs. untreated intensity ratio data linked below as a supplementary file. Keywords: agent response
2008-05-30 | GSE11471 | GEO
Project description:Polyphosphate (polyP) is a ubiquitous and abundant compound found in bacteria, fungi, algae, plant, and animals. Among roles of intracellular polyP in bacteria are resistance and survival in the stationary phase of the growth against stress and stringent condition. Therefore, intracellular polyP is considered as a virulence factor of bacteria. In contrast, exogenous polyP has an antimicrobial activity against a variety of microorganisms. To date, much has been numerous studies of antibacterial effect of polyP against gram positive bacteria and fungi while relatively little reports have been published concerning gram negative bacteria. Here we describe bactericidal effect of polyP against gram negative periodontopathic bacterium, Porphyromonas gingivalis, and the transcriptional change by polyP in this bacterium. The bacterial growth was inhibited by polyP (chain length of P3~P75) at concentration of 0.02~0.03%, but not by Pi and PPi at the concentrations. polyP75 was chosen for further experiments, which suppressed the further growth of P. gingivalis as low as 0.03%. polyP75 completely killed the bacterial cells at the concentration of 0.035%. Microarray analysis was employed to identify genes that showed a greater than 1.5-fold difference in the expression by polyP75 at the concentration of 0.03%. It was found that 155 genes were up-regulated and 173 were down-regulated. Down-regulated genes include groups of energy metabolism-related genes, cell envelope-related genes, and genes in relation to biosynthesis of cofactors, prosthetic groups and carriers. Among the down-regulated genes were several involved in DNA replication, cell division protein, and biosynthesis of purines, pyrimidines, nucleosides and nucleotides. In contrast, a large number of ribosomal proteins, transciptional regulators and transposases were up-regulated. Oxidative stress-related genes and iron storage proteins also appeared to be increased in the expression. Real-time PCR confirmed up- and down-regulation of some selected genes. The overall results suggest that polyP has a bactericidal activity against P. gingivalis, interfering with translation, energy metabolism, DNA replication, cell division, and biosynthesis of purines, pyrimidines, nucleoside and nucleotides. polyP may be used as an agent for prevention and treatment of periodontal infections. Polyp-treated vs. untreated intensity ratio data linked below as a supplementary file. Keywords: agent response Twenty μg of each total RNA sample was used in separate hybridization experiments on identical arrays. The whole genome of 1,909 genes of P. gingivalis W83 (GenBank accession no. NC_002950) was submitted to NimbleGen System Inc. (Madison, WI) for microarray design and manufacture using maskless, digital micromirror technology. Five replicates of the genome were included per chip. An average of 19 different 60-base oligonucleotides (60-mer probes) represented each gene in the genome. Sixty-mer probes were selected such that each probe had at least three mismatches compared to all other 60-mers in the target genome. A quality control check (hybridization) was performed for each array, which contained on-chip control oligonucleotides. The arrays were analyzed using an Axon GenePix 4000B microarray scanner with associated software (Molecular Devices Corp., Sunnyvale, CA). Gene expression levels were calculated with NimbleScan Version 2.3 (NimbleGen). Relative signal intensities for each gene were generated using the Robust Multi-Array Average algorithm. The data were processed based on quantile normalization method using the R package. This normalization method aims to make the distribution of intensities for each array in a set of arrays the same. The background-adjusted, normalized, and log transformed intensity values were then analyzed using GeneSpring GX 7.3.1 (Silicon Genetics, Palo Alto, CA).
2008-05-30 | E-GEOD-11471 | biostudies-arrayexpress
Project description:Porphyromonas gingivalis is a periodontal pathogen that is also associated with preterm low-birthweight delivery. We investigated the transcriptional responses of human extravillous trophoblasts (HTR-8) to infection with P. gingivalis. Over 2000 genes were differentially regulated in HTR-8 cells by P. gingivalis. In ontology analyses of regulated genes, overpopulated biological pathways included mitogen-activated protein (MAP) kinase signaling and cytokine production. Immunoblots confirmed overexpression of the MAP kinase pathway components MEK3, p38 and Max. Furthermore, P. gingivalis infection induced phosphorylation and activation of MEK3 and p38. Increased production of interleukin (IL)-1beta and IL-8 by HTR-8 cells was demonstrated phenotypically by enzyme-linked immunosorbent assay of HTR-8 cell lysates and culture supernatants. Hence, infection of trophoblasts by P. gingivalis can impact signal transduction pathways and modulate cytokine expression, outcomes that could disrupt the maintenance of pregnancy.
| S-EPMC3665294 | biostudies-other
Project description:Porphyromonas gingivalis has been associated with subgingival biofilms in adult periodontitis. However, the molecular mechanisms of its contribution to chronic gingival inflammation and loss of periodontal structural integrity remain unclear. This investigation aimed to examine changes in the host transcriptional profiles during a P. gingivalis infection using a murine calvarial model of inflammation and bone resorption. P. gingivalis FDC 381 was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated from infected soft tissues and calvarial bones and was analysed for transcript profiles using Murine GeneChip((R)) arrays to provide a molecular profile of the events that occur following infection of these tissues. After P. gingivalis infection, 6452 and 2341 probe sets in the infected soft tissues and calvarial bone, respectively, were differentially expressed (P </= 0.05). Biological pathways significantly impacted by P. gingivalis infection in tissues and calvarial bone included cell adhesion (immune system) molecules, Toll-like receptors, B-cell receptor signaling, transforming growth factor-beta cytokine family receptor signaling, and major histocompatibility complex class II antigen processing pathways resulting in proinflammatory, chemotactic effects, T-cell stimulation, and downregulation of antiviral and T-cell chemotactic effects. P. gingivalis-induced inflammation activated osteoclasts, leading to local bone resorption. This is the first in vivo evidence that localized P. gingivalis infection differentially induces transcription of a broad array of host genes, the profiles of which differed between inflamed soft tissues and calvarial bone.
| S-EPMC3001241 | biostudies-literature
Project description:Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.
| S-EPMC8484350 | biostudies-literature
Project description:Marginal periodontitis is not a homogeneous disease but is rather influenced by an intricate set of host susceptibility differences as well as diversities in virulence among the harbored organisms. It is likely that clonal heterogeneity of subpopulations with both high and low levels of pathogenicity exists among organisms harbored by individuals with negligible, slight, or even severe periodontal destruction. Therefore, specific virulent clones of periodontal pathogens may cause advanced and/or aggressive periodontitis. Porphyromonas gingivalis is a predominant periodontal pathogen that expresses a number of potential virulence factors involved in the pathogenesis of periodontitis, and accumulated evidence shows that its expression of heterogenic virulence properties is dependent on clonal diversity. Fimbriae are considered to be critical factors that mediate bacterial interactions with and invasion of host tissues, with P. gingivalis shown to express two distinct fimbria-molecules, long and short fimbriae, on the cell surface, both of which seem to be involved in development of periodontitis. Long fimbriae are classified into six types (I to V and Ib) based on the diversity of fimA genes encoding FimA (a subunit of long fimbriae). Studies of clones with type II fimA have revealed their significantly greater adhesive and invasive capabilities as compared to other fimA type clones. Long and short fimbriae induce various cytokine expressions such as IL-1α, IL-β, IL-6, and TNF-α, which result in alveolar bone resorption. Although the clonal diversity of short fimbriae is unclear, distinct short fimbria-molecules have been found in different strains. These fimbriae variations likely influence the development of periodontal disease.
| S-EPMC3647041 | biostudies-literature
Project description:There is a significant body of work suggesting that sRNA-mediated post-transcriptional regulation is a conserved mechanism among pathogenic bacteria to modulate bacterial virulence and survival. Porphyromonas gingivalis is recognized as an etiological agent of periodontitis and implicated in contributing to the development of multiple inflammatory diseases including cardiovascular disease. Using NimbleGen microarray analysis and a strand-specific method to sequence cDNA libraries of small RNA-enriched P. gingivalis transcripts using Illumina's high-throughput sequencing technology, we identified putative sRNA and generated sRNA expression profiles in response to growth phase, hemin availability after hemin starvation, or both. We identified transcripts that mapped to intergenic sequences as well as antisense transcripts that mapped to open reading frames of the annotated genome. Overall, this approach provided a comprehensive way to survey transcriptional activity to discover functionally linked RNA transcripts, responding to specific environmental cues, that merit further investigation.
| S-EPMC4009720 | biostudies-literature
Project description:Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)-tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 ?g/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-?, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-? and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells.
| S-EPMC4990254 | biostudies-literature
Project description:Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25 × 106 CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22 min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16 min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.
| S-EPMC9281108 | biostudies-literature