Functional expression in Escherichia coli and membrane topology of porin HopE, a member of a large family of conserved proteins in Helicobacter pylori

HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin. In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from E. coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H. pylori. A model of the membrane topology of HopE was constructed and indicated that this protein formed a beta-barrel with 16 transmembrane amphipathic beta-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane beta-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane.     

Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein

We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-alpha production by the RAW 264.7 macrophage cell line.     

Recognition of Dictyostelium discoideum lysosomal enzymes is conferred by the amino-terminal carbohydrate binding site of the insulin-like growth factor II/mannose 6-phosphate receptor

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains 15 repeating domains; the two carbohydrate recognition domains (CRDs) have been localized to domains 1-3 and 7-9, and the high-affinity IGF-II binding site maps to domain 11. To characterize the carbohydrate binding properties of the IGF-II/MPR, regions of the receptor encompassing the individual CRDs were produced in a baculovirus expression system. Characterization of the recombinant proteins revealed that the pH optimum for carbohydrate binding is significantly more acidic for the carboxyl-terminal CRD than for the amino-terminal CRD (i.e., pH 6.4-6.5 vs 6.9). Equilibrium binding studies demonstrated that the two CRDs exhibit a similar affinity for Man-6-P. Furthermore, substitution of the conserved arginine residue in domain 3 (R435) or in domain 9 (R1334) with alanine resulted in a similar >1000-fold decrease in the affinity for the lysosomal enzyme, beta-glucuronidase. In contrast, the two CRDs differ dramatically in their ability to recognize the distinctive modifications (i.e., mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes: the amino-terminal CRD binds mannose 6-sulfate and Man-6-P methyl ester with a 14-55-fold higher affinity than the carboxyl-terminal CRD. Taken together, these results demonstrate that the IGF-II/MPR contains two functionally distinct CRDs.     

Antimicrobial peptides protect coho salmon from Vibrio anguillarum infections

Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen Vibrio anguillarum. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with V. anguillarum, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum at a dose that would kill 50 to 90% of the population. Fish receiving 200 microg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 microg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems.     

NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.     

Regulation of virulence and antibiotic resistance by two-component regulatory systems in Pseudomonas aeruginosa

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa ubiquitously inhabits soil and water habitats and also causes serious, often antibiotic resistant, infections in immunocompromised patients (e.g. cystic fibrosis). This versatility is mediated in part by a large repertoire of two-component regulatory systems that appear instrumental in the regulation of both virulence processes and resistance to antimicrobials. Major two-component regulatory system proteins demonstrated to regulate these diverse processes include PhoP–PhoQ, GacA–GacS, RetS, LadS, and AlgR, among others. Here, we summarize the current body of knowledge of these and other two-component systems that provides insight into the complex regulation of virulence and resistance in P. aeruginosa .     

When Does Choice of Accuracy Measure Alter Imputation Accuracy Assessments?

Imputation, the process of inferring genotypes for untyped variants, is used to identify and refine genetic association findings. Inaccuracies in imputed data can distort the observed association between variants and a disease. Many statistics are used to assess accuracy; some compare imputed to genotyped data and others are calculated without reference to true genotypes. Prior work has shown that the Imputation Quality Score (IQS), which is based on Cohen’s kappa statistic and compares imputed genotype probabilities to true genotypes, appropriately adjusts for chance agreement; however, it is not commonly used. To identify differences in accuracy assessment, we compared IQS with concordance rate, squared correlation, and accuracy measures built into imputation programs. Genotypes from the 1000 Genomes reference populations (AFR N = 246 and EUR N = 379) were masked to match the typed single nucleotide polymorphism (SNP) coverage of several SNP arrays and were imputed with BEAGLE 3.3.2 and IMPUTE2 in regions associated with smoking behaviors. Additional masking and imputation was conducted for sequenced subjects from the Collaborative Genetic Study of Nicotine Dependence and the Genetic Study of Nicotine Dependence in African Americans (N = 1,481 African Americans and N = 1,480 European Americans). Our results offer further evidence that concordance rate inflates accuracy estimates, particularly for rare and low frequency variants. For common variants, squared correlation, BEAGLE R², IMPUTE2 INFO, and IQS produce similar assessments of imputation accuracy. However, for rare and low frequency variants, compared to IQS, the other statistics tend to be more liberal in their assessment of accuracy. IQS is important to consider when evaluating imputation accuracy, particularly for rare and low frequency variants.