Modulation of cytokine synthesis by bacterial components

The chronic inflammatory diseases (CIPDs) are mankind's most prevalent chronic inflammatory conditions. Inflammation in the gingivae is associated with the destruction of the alveolar bone and periodontal ligament which support the teeth. There is good evidence for the involvement of a range of Gram-negative anaerobic and capnophilic bacteria in tissue pathology including: Actinobacillus actinomycetemcomitans, Eikenella corrodens, Porphyromonas gingivalis, Campylobacter rectus and Prevotella intermedia. Invasion of lesional tissues is not a conspicuous feature of this disease and the current paradigm is that soluble components or products of bacteria drive the tissue pathology. However, the nature of these virulence determinants has not been defined. This study has examined the capacity of proteins, associated with the bacterial surface (termed surface-associated material- SAM) and the outer membrane (lipid A-associated proteins-LAP), from these bacteria to stimulate bone resorption and to induce the synthesis of cytokines by myelomonocytic and mesenchymal cell populations. These activities have been compared with those of the lipopolysaccharides from the respective organisms. The bone resorbing activity of the SAMs from the bacteria studied varied widely in potency and efficacy. Some bacteria had SAMs which showed activity at concentrations as low as 1 ng/ml while others required microgram/ml concentrations to produce minimal bone resorption. In contrast, all the LAPs tested showed similar potencies and efficacies. The activity of the LPS from the various organisms was generally low. The capacity of the SAMs to induce the release of the pro-inflammatory cytokines IL-1, IL-6, IL-8 and TNF-α also showed a range of potencies and efficacies. In comparative studies of the cytokine-stimulating actions of the SAMs, LAP and LPS from A. actinomycetemcomitans the former generally showed the greatest potency with the LAP being somewhat less potent and the LPS showing very little activity. Comparison of the mechanism of stimulation of gingival fibroblast IL-6 release by SAMs or E coli LPS revealed major differences. Stimulation by the SAMs was not affected by neutralizing IL-1 or TNF-α activity or by dexamethasone. In contrast LPS-induced IL-6 synthesis was totally abolished by these agents. This suggests that the active cytokine inducing component in the SAM from A. actinomycetemcomitans may interact with a novel transcriptional regulatory site in the IL-6 gene. This active IL-6 stimulating component has been purified by various chromatographic techniques and was found to be a 2 kDa peptide. The results of this study have shown that bacterial components other than LPS are potent inducers of cytokine release and bone resorption in vitro, and so may play a role in initiating the tissue destruction characteristic of periodontitis

BlueFeather, the singleton that wasn't: Shared gene content analysis supports expansion of Arthrobacter phage Cluster FE.

Bacteriophages (phages) exhibit high genetic diversity, and the mosaic nature of the shared genetic pool makes quantifying phage relatedness a shifting target. Early parameters for clustering of related Mycobacteria and Arthrobacter phage genomes relied on nucleotide identity thresholds but, more recently, clustering of Gordonia and Microbacterium phages has been performed according to shared gene content. Singleton phages lack the nucleotide identity and/or shared gene content required for clustering newly sequenced genomes with known phages. Whole genome metrics of novel Arthrobacter phage BlueFeather, originally designated a putative singleton, showed low nucleotide identity but high amino acid and gene content similarity with Arthrobacter phages originally assigned to Clusters FE and FI. Gene content similarity revealed that BlueFeather shared genes with these phages in excess of the parameter for clustering Gordonia and Microbacterium phages. Single gene analyses revealed evidence of horizontal gene transfer between BlueFeather and phages in unique clusters that infect a variety of bacterial hosts. Our findings highlight the advantage of using shared gene content to study seemingly genetically isolated phages and have resulted in the reclustering of BlueFeather, a putative singleton, as well as former Cluster FI phages, into a newly expanded Cluster FE

Isolation of potential plant growth-promoting bacteria from nodules of legumes grown in arid Botswana soil

As the world population increases, improvements in crop growth and yield will be needed to meet rising food demands, especially in countries that have not developed agricultural practices optimized for their own soils and crops. In many African countries, farmers improve agricultural productivity by applying synthetic fertilizers and pesticides to crops, but their continued use over the years has had serious environmental consequences including air and water pollution as well as loss of soil fertility. To reduce the overuse of synthetic amendments, we are developing inocula for crops that are based on indigenous soil microbes, especially those that enhance plant growth and improve agricultural productivity in a sustainable manner. We first isolated environmental DNA from soil samples collected from an agricultural region to study the composition of the soil microbiomes and then used Vigna unguiculata (cowpea), an important legume crop in Botswana and other legumes as “trap” plants using the collected soil to induce nitrogen-fixing nodule formation. We have identified drought-tolerant bacteria from Botswana soils that stimulate plant growth; many are species of Bacillus and Paenibacillus . In contrast, the cowpea nodule microbiomes from plants grown in these soils house mainly rhizobia particularly Bradyrhizobium , but also Methylobacterium spp. Hence, the nodule microbiome is much more limited in non-rhizobial diversity compared to the soil microbiome, but also contains a number of potential pathogenic bacteria

Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C)*♦

Growth and remodeling of lymphatic vasculature occur during development and during various pathologic states. A major stimulus for this process is the unique lymphatic vascular endothelial growth factor-C (VEGF-C). Other endothelial growth factors, such as fibroblast growth factor-2 (FGF-2) or VEGF-A, may also contribute. Heparan sulfate is a linear sulfated polysaccharide that facilitates binding and action of some vascular growth factors such as FGF-2 and VEGF-A. However, a direct role for heparan sulfate in lymphatic endothelial growth and sprouting responses, including those mediated by VEGF-C, remains to be examined. We demonstrate that VEGF-C binds to heparan sulfate purified from primary lymphatic endothelia, and activation of lymphatic endothelial Erk1/2 in response to VEGF-C is reduced by interference with heparin or pretreatment of cells with heparinase, which destroys heparan sulfate. Such treatment also inhibited phosphorylation of the major VEGF-C receptor VEGFR-3 upon VEGF-C stimulation. Silencing lymphatic heparan sulfate chain biosynthesis inhibited VEGF-C-mediated Erk1/2 activation and abrogated VEGFR-3 receptor-dependent binding of VEGF-C to the lymphatic endothelial surface. These findings prompted targeting of lymphatic N-deacetylase/N-sulfotransferase-1 (Ndst1), a major sulfate-modifying heparan sulfate biosynthetic enzyme. VEGF-C-mediated Erk1/2 phosphorylation was inhibited in Ndst1-silenced lymphatic endothelia, and scratch-assay responses to VEGF-C and FGF-2 were reduced in Ndst1-deficient cells. In addition, lymphatic Ndst1 deficiency abrogated cell-based growth and proliferation responses to VEGF-C. In other studies, lymphatic endothelia cultured ex vivo from Ndst1 gene-targeted mice demonstrated reduced VEGF-C- and FGF-2-mediated sprouting in collagen matrix. Lymphatic heparan sulfate may represent a novel molecular target for therapeutic intervention

Novel Cluster AZ Arthrobacter phages Powerpuff, Lego, and YesChef exhibit close functional relationships with Microbacterium phages

Bacteriophages exhibit a vast spectrum of relatedness and there is increasing evidence of close genomic relationships independent of host genus. The variability in phage similarity at the nucleotide, amino acid, and gene content levels confounds attempts at quantifying phage relatedness, especially as more novel phages are isolated. This study describes three highly similar novel Arthrobacter globiformis phages-Powerpuff, Lego, and YesChef-which were assigned to Cluster AZ using a nucleotide-based clustering parameter. Phages in Cluster AZ, Microbacterium Cluster EH, and the former Microbacterium singleton Zeta1847 exhibited low nucleotide similarity. However, their gene content similarity was in excess of the recently adopted Microbacterium clustering parameter, which ultimately resulted in the reassignment of Zeta1847 to Cluster EH. This finding further highlights the importance of using multiple metrics to capture phage relatedness. Additionally, Clusters AZ and EH phages encode a shared integrase indicative of a lysogenic life cycle. In the first experimental verification of a Cluster AZ phage's life cycle, we show that phage Powerpuff is a true temperate phage. It forms stable lysogens that exhibit immunity to superinfection by related phages, despite lacking identifiable repressors typically required for lysogenic maintenance and superinfection immunity. The ability of phage Powerpuff to undergo and maintain lysogeny suggests that other closely related phages may be temperate as well. Our findings provide additional evidence of significant shared phage genomic content spanning multiple actinobacterial host genera and demonstrate the continued need for verification and characterization of life cycles in newly isolated phages