Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii , we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria–fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.     

Evaluations of the Removal of <Emphasis Type="Italic">Varroa destructor</Emphasis> in Russian Honey Bee Colonies that Display Different Levels of <Emphasis Type="Italic">Varroa</Emphasis> Sensitive Hygienic Activities

The removal of Varroa destructor was assessed in Russian honey bee (RHB) colonies with known levels of Varroa Sensitive Hygienic (VSH) and brood removal activities. The expression of grooming behaviour using individual bees was also measured using three groups of RHB displaying different VSH levels: low hygiene (RHB-LH, < 35% VSH), medium hygiene (RHB-MH, 35–70%) and high hygiene (RHB-HH, > 70%). Italian colonies (5.43–71.62% VSH) served as control. Our results demonstrated, for the first time, significant relationships between two hygienic responses (VSH activity measured as percent change in infestation and the actual brood removal of Varroa-infested donor comb) and two measurements of mite fall (trapped old mites/trapped mites or O/T and trapped young mites/trapped mites or Y/T). However, these relationships were only observed in RHB colonies. In addition, the RHB colonies that displayed the highest levels of hygiene (RHB-HH) also groomed longer in response to the presence of a V. destructor mite based on individual bee assays. The positive regressions between the two hygienic measurements and O/T and their negative regressions with Y/T suggest that the removal of infested brood prevented successful mite reproduction, ultimately suppressing V. destructor infestations in the RHB colonies. In addition, it is demonstrated that RHB resistance to V. destructor rests on both an increased hygienic response and the removal of phoretic mites, released by hygienic behaviour, through grooming. Both resistance traits are reflected in the O/T and Y/T ratios found in trapped mites from RHB colonies. None of the measurements involving mite injuries were associated with any measurements of hygiene and colony infestations.     

Identification of Novel Viruses Using VirusHunter -- an Automated Data Analysis Pipeline

Quick and accurate identification of microbial pathogens is essential for both diagnosis and response to emerging infectious diseases. The advent of next-generation sequencing technology offers an unprecedented platform for rapid sequencing-based identification of novel viruses. We have developed a customized bioinformatics data analysis pipeline, VirusHunter, for the analysis of Roche/454 and other long read Next generation sequencing platform data. To illustrate the utility of VirusHunter, we performed Roche/454 GS FLX titanium sequencing on two unclassified virus isolates from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). VirusHunter identified sequences derived from a novel bunyavirus and a novel reovirus in the two samples respectively. Further sequence analysis demonstrated that the viruses were novel members of the Phlebovirus and Orbivirus genera. Both Phlebovirus and Orbivirus genera include many economic important viruses or serious human pathogens.     

Duodenal mucosal risk markers in patients with familial adenomatous polyposis: effects of celecoxib/ursodeoxycholic acid co-treatment and comparison with patient controls

Background

Familial adenomatous polyposis (FAP) is a disease characterized by the development of hundreds to thousands of adenomatous polyps in the colorectum early in life. Virtually all patients with FAP will develop colorectal cancer before the age of 40 to 50 years, unless prophylactic colectomy is performed, which significantly improves their prognosis. The mortality pattern has changed and duodenal cancer now is one of the main cancer-related causes of death in these patients. Practically all patients with FAP develop premalignant duodenal adenomas, which may develop to duodenal cancer in approximately 3-7% of patients. Duodenal cancer in patients with FAP has a poor prognosis. The clinical challenge is to identify patients at high-risk for duodenal carcinoma. Chemoprevention would be desirable to avoid duodenectomy. The main goal of this study is to identify risk markers in normal duodenal mucosa of patients with FAP, that could help identify patients at increased risk for malignant transformation.

Methods

Messenger RNA (mRNA) levels of glutathione S-transferase A1 (GSTA1), glutathione S-transferase P1 (GSTP1), KIAA1199, E-cadherin, peroxisome proliferative activated receptor δ (PPARδ), caspase-3, cyclin D1, β-catenin, and cyclooxygenase-2 (COX-2) were measured in duodenal mucosa, using the QuantiGene 2.0 Plex assay. Levels in normal appearing mucosa of patients with FAP (n?=?37) were compared with levels in non-FAP patient controls (n?=?16). In addition, levels before and after treatment with either celecoxib & ursodeoxycholic acid (UDCA, n?=?14) or celecoxib & placebo (n?=?13) were evaluated in patients with FAP.

Results

mRNA levels of glutathione S-transferase A1 (28.16% vs. 38.24%, p?=?0.008) and caspase-3 (3.30% vs. 5.31%, p?=?0.001) were significantly lower in patients with FAP vs. non-FAP patient controls, respectively. COX-2 mRNA levels in normal duodenal mucosa of patients with FAP were found to be unexpectedly low. None of the potential risk markers was influenced by celecoxib or celecoxib & UDCA.

Conclusions

Protection against toxins and carcinogens (GSTA1) and apoptosis (caspase-3) is low in patients with FAP, which could contribute to increased susceptibility for malignant transformation of duodenal mucosa.

Trial registration

http://ClinicalTrials.gov number NCT00808743

     

Role of DDAH-1 in lipid peroxidation product-mediated inhibition of endothelial NO generation

Altered nitric oxide (NO) biosynthesis is thought to play a role in the initiation and progression of atherosclerosis and may contribute to increased risk seen in other cardiovascular diseases. It is hypothesized that altered NO bioavailability may result from an increase in endogenous NO synthase (NOS) inhibitors, asymmetric dimethly araginine (ADMA), and N(G)-monomethyl arginine, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). Lipid hydroperoxides and their degradation products are generated during inflammation and oxidative stress and have been implicated in the pathogenesis of cardiovascular disorders. Here, we show that the lipid hydroperoxide degradation product 4-hydroxy-2-nonenal (4-HNE) causes a dose-dependent decrease in NO generation from bovine aortic endothelial cells, accompanied by a decrease in DDAH enzyme activity. The inhibitory effects of 4-HNE (50 microM) on endothelial NO production were partially reversed with L-Arg supplementation (1 mM). Overexpression of human DDAH-1 along with antioxidant supplementation completely restored endothelial NO production following exposure to 4-HNE (50 microM). These results demonstrate a critical role for the endogenous methylarginines in the pathogenesis of endothelial dysfunction. Because lipid hydroperoxides and their degradation products are known to be involved in atherosclerosis, modulation of DDAH and methylarginines may serve as a novel therapeutic target in the treatment of cardiovascular disorders associated with oxidative stress.     

Of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (DHF/DSS)

The past four decades has witnessed a consolidation of the original observations made in the 1970s that dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) have an immunological basis. Following reinfection with a dengue virus of different serotype, severe disease is linked to high levels of antibody-enhanced viral replication early in illness which is followed by a cascade of memory T-cell activation and a 'storm' of inflammatory cytokines and other chemical mediators. These compounds are released mainly from T cells, monocytes/macrophages and endothelial cells, and ultimately cause an increase in vascular permeability. The consolidation of the evidence has been largely due to several important prospective sero-epidemiological studies in areas endemic for DHF/DSS, which have shown that risk of severe disease is significantly higher in secondary dengue infections. These advances have underscored the fact that DHF/DSS pathogenesis is a complex, multifactorial process involving cocirculation of various dengue virus serotypes and the interplay of host and viral factors that influence disease severity. The continued search to define risk factors in susceptible populations must be combined with the new techniques of molecular virology and innovative approaches in vaccine design to achieve the ultimate objective of developing a safe and effective vaccine.