12 research outputs found
Conjugal Transfer of Chromosomal DNA Contributes to Genetic Variation in the Oral Pathogen Porphyromonas gingivalis▿
Porphyromonas gingivalis is a major oral pathogen that contributes to the development of periodontal disease. There is a significant degree of genetic variation among strains of P. gingivalis, and the population structure has been predicted to be panmictic, indicating that horizontal DNA transfer and recombination between strains are likely. The molecular events underlying this genetic exchange are not understood, although a putative type IV secretion system is present in the genome sequence of strain W83, implying that DNA conjugation may be responsible for genetic transfer in these bacteria. In this study, we provide in vitro evidence for the horizontal transfer of DNA using plasmid- and chromosome-based assays. In the plasmid assays, Bacteroides-derived shuttle vectors were tested for transfer from P. gingivalis strains into Escherichia coli. Of the eight strains tested, five were able to transfer DNA into E. coli by a mechanism most consistent with conjugation. Additionally, strains W83 and 33277 tested positive for the transfer of chromosomally integrated antibiotic resistance markers. Ten chimeras resulting from the chromosomal transfer assay were further analyzed by Southern hybridization and were shown to have exchanged DNA fragments of between 1.1 and 5.6 kb, but the overall strain identity remained intact. Chimeras showed phenotypic changes in the ability to accrete into biofilms, implying that DNA transfer events are sufficient to generate measurable changes in complex behaviors. This ability to transfer chromosomal DNA between strains may be an adaptation mechanism in the complex environment of the host oral cavity
Microbiological and biochemical findings in relation with clinical periodontal status in active smokers, non-smokers and passive smokers
Introduction
Cigarette users are more susceptible than non-smokers to periodontitis,
a bacterial-induced, inflammation-driven, destructive disease of the supporting
tissues of the teeth. We hypothesized that clinical periodontal findings and
microbiological and/or inflammatory marker levels would be intermediate in
those exposed to environmental tobacco smoke compared to active smokers and
non-smokers.
Methods
Sixty individuals were recruited from a University periodontal clinic and
assigned as non-smokers, active smokers or passive-smokers according to their
self reports. Clinical periodontal measurements, comprising plaque index, probing
depth (PD), clinical attachment level (CAL) and bleeding on probing, were
recorded at six sites per tooth. Cotinine levels were determined in whole saliva
samples by EIA. Treponema denticola and Porphyromonas gingivalis infection was
determined by PCR, while matrix metalloproteinase-8 (MMP-8) and interleukin-8
(IL-8) concentrations were determined by ELISA.
Results
Study groups were subsequently reassigned in accordance with the cotinine
data. The smoker group exhibited higher mean PD and CAL values compared to
the non-smoker group (p<0.05). Passive-smokers exhibited PD and CAL values
smaller than those of the active smokers and greater than those of the nonsmokers,
but the differences were not statistically significant. PD and CAL values
correlated with cotinine concentrations (p 0.05).
Conclusions
The present clinical periodontal findings provide further support for
a negative, dose-related effect of tobacco exposure on periodontal health. The
tendency for a more prevalent detection of T. denticola and for a suppressed
inflammatory response observed in the smokers may partly explain the increased
susceptibility to periodontal tissue destruction, but needs to be verified in larger
scale studies
MIF inhibition reverts the gene expression profile of human melanoma cell line-induced MDSCs to normal monocytes
Myeloid-derived suppressor cells (MDSCs) are potently immunosuppressive innate immune cells that accumulate in advanced cancer patients and actively inhibit anti-tumor T lymphocyte responses [1]. Increased numbers of circulating MDSCs directly correlate with melanoma patient morbidity and reduced anti-tumor immune responses [2,3]. Previous studies have revealed that monocyte-derived macrophage migration inhibitory factor (MIF) is necessary for the immune suppressive function of MDSCs in mouse models of melanoma [4,5]. To investigate whether MIF participates in human melanoma-induced MDSC differentiation and/or suppressive function, we have established an in vitro MDSC induction model using primary, normal human monocytes co-cultured with human melanoma cell lines in the presence or absence of the MIF antagonist—4-IPP [4,6–9]. To identify potential mechanistic effectors, we have performed transcriptome analyses on cultured monocytes and on melanoma-induced MDSCs obtained from either untreated or 4-IPP-treated A375:monocyte co-cultures. Here, we present a detailed protocol, which can facilitate easy reproduction of the microarray results (NCBI GEO accession number GSE73333) published by Yaddanapudi et al. (2015) in Cancer Immunology Research [10]. Keywords: Melanoma, MDSC, MIF, Immunesuppression, Trancriptome analysi
Resolvin D1, resolvin D2 and maresin 1 activate the GSK3β anti-inflammatory axis in TLR4-engaged human monocytes
Porphyromonas gingivalis genes conferring fitness in a tobacco-rich environment
Smokers are more likely than non-smokers to harbour Porphyromonas gingivalis, they are more susceptible to destructive periodontal disease and smokers may, ultimately, benefit from tobacco-specific preventive and treatment strategies. A Mariner transposon insertion library for P. gingivalis ATCC 33277 was exploited to define 256 genes as essential for P. gingivalis survival in a tobacco-rich environment. Genes whose products play roles in protein transport and catabolism, nicotinamide processing, protection against oxidative stress, drug resistance and transcriptional regulation have all been identified as essential for CSE survival. Many of these tobacco-essential genes are also requisite for epithelial colonization and abscess formation, suggestive of a core stress-related P. gingivalis genome. Single-gene deletions in several of the TnSeq-implicated genes led to significantly reduced P. gingivalis fitness upon competition with the parent strain, under conditions of cigarette smoke extract-induced stress (1000 ng/ml nicotine equivalents). This study identifies, for the first time, a subset of P. gingivalis genes required for surviving the plethora of insults present in cigarette smoke. Such conditionally essential genes may delineate bacterial persistence strategies and represent novel therapeutic foci for the prevention of P. gingivalis infection and related diseases in smokers and in general