Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa infects cystic fibrosis (CF) patient airways and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

Biochemical and structural characterization of two cif-like epoxide hydrolases from Burkholderia cenocepacia.

Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa. Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homolog aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from Burkholderia cenocepacia. Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the α/ß hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.

The Predicting Role of Torque Teno Virus Infection after Renal Transplantation.

Renal transplantation is the ideal therapeutic implement for end-stage renal disease patients. However, late kidney graft defeat remains a main challenge. Torque teno virus (TTV) is a small DNA virus whose replication is strictly related to person immune status besides TTV Antigens could prevent organ rejection by regulating both adaptive and innate immunity through interfering with NF-κB pathway which decrease interleukin-6 (IL-6) levels in renal transplanted patients. This cross-sectional study was conducted eighty serum samples were collected renal transplant recipients, DNA was extracted and the viral DNA was detected and quantified by quantitative polymerase chain reaction (PCR) for human cytomegalovirus (CMV) and real-time PCR for TTV. In addition, enzyme-linked immunosorbent assays (ELISA) were used for the detection of TTV antigen and IL-6 levels were also done. Result of PCR showed that 25% and 56.25% of renal transplantation patients had positive for CMV and TTV viremia. CMV viremia was positive in 20% of patients who have positive result to TTV-DNA, which was statistically nonsignificant. Results of ELISA presented that TTV-Ag was positive in 10% of renal transplantation patients, while IL-6 level was very low in patients who have positive results to present of TTV-Ag which was significantly lower in those patients (P = 0.008). In conclusion, TTV could have not an association with reactivation of CMV in renal transplant patients and the presence of TTV-Ag reduce renal rejection by decreasing of IL-6 levels which might be an indicator of allograft status.

Structural basis of respiratory syncytial virus subtype-dependent neutralization by an antibody targeting the fusion glycoprotein.

A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines.

Amyloid-ß alters the DNA methylation status of cell-fate genes in an Alzheimer's disease model.

Alzheimer's disease (AD) is characterized by neurofibrillary tangles and extracellular amyloid-ß plaques (Aß). Despite ongoing research, some ambiguity remains surrounding the role of Aß in the pathogenesis of this neurodegenerative disease. While several studies have focused on the mutations associated with AD, our understanding of the epigenetic contributions to the disease remains less clear. To that end, we determined the changes in DNA methylation in differentiated human neurons with and without Aß treatment. DNA was isolated from neurons treated with Aß or vehicle, and the two samples were digested with either a methylation-sensitive (HpaII) or a methylation-insensitive (MspI) restriction endonuclease. The fragments were amplified and co-hybridized to a commercial promoter microarray. Data analysis revealed a subset of genomic loci that shows a significant change in DNA methylation following Aß treatment in comparison to the control group. After mapping these loci to nearby genes, we discovered high enrichment for cell-fate genes that control apoptosis and neuronal differentiation. Finally, we incorporated three of those genes in a possible model suggesting the means by which Aß contributes to the brain shrinkage and memory loss seen in AD.