Characterization of the lipopolysaccharide from Pasteurella multocida Heddleston serovar 9: identification of a proposed bi-functional dTDP-3-acetamido-3,6-dideoxy-α-D-glucose biosynthesis enzyme

Pasteurella multocida strains are classified into 16 different lipopolysaccharide (LPS) serovars using the Heddleston serotyping scheme. Ongoing studies in our laboratories on the LPS aim to determine the core oligosaccharide (OS) structures expressed by each of the Heddleston type strains and identify the genes and transferases required for the biosynthesis of the serovar-specific OSs. In this study, we have determined the core OS of the LPS expressed by the Heddleston serovar 9 type strain, P2095. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy and mass spectrometry revealing the following structure: . The serovar 9 OS contains an inner core that is conserved among P. multocida strains with an elaborate outer core extension containing rhamnose (Rha), a D-glycero-D-manno isomer of heptose, and the unusual deoxyamino sugar, 3-acetamido-3,6-dideoxy-α-D-glucose (Qui3NAc). Genetic analyses of the LPS outer core biosynthesis locus revealed that in addition to the glycosyltransferases predicted to transfer the sugars to the nascent LPS molecule, the locus also contained the complete set of genes required for the biosynthesis of the nucleotide sugar donors dTDP-Rha and dTDP-Qui3NAc. One of the genes identified as part of the dTDP-Qui3NAc biosynthesis pathway, qdtD, encodes a proposed bi-functional enzyme with N-terminal amino acid identity to dTDP-4-oxo-6-deoxy-D-glucose-3,4-oxoisomerase and C-terminal amino acid identity to dTDP-3-oxo-6-deoxy-α-D-glucose transacetylase.     

Insertions and deletions trigger adaptive walks in Drosophila proteins

Maps that relate all possible genotypes or phenotypes to fitness--fitness landscapes--are central to the evolution of life, but remain poorly known. An insertion or a deletion (indel) of one or several amino acids constitutes a substantial leap of a protein within the space of amino acid sequences, and it is unlikely that after such a leap the new sequence corresponds precisely to a fitness peak. Thus, one can expect an indel in the protein-coding sequence that gets fixed in a population to be followed by some number of adaptive amino acid substitutions, which move the new sequence towards a nearby fitness peak. Here, we study substitutions that occur after a frame-preserving indel in evolving proteins of Drosophila. An insertion triggers 1.03 ± 0.75 amino acid substitutions within the protein region centred at the site of insertion, and a deletion triggers 4.77 ± 1.03 substitutions within such a region. The difference between these values is probably owing to a higher fraction of effectively neutral insertions. Almost all of the triggered amino acid substitutions can be attributed to positive selection, and most of them occur relatively soon after the triggering indel and take place upstream of its site. A high fraction of substitutions that follow an indel occur at previously conserved sites, suggesting that an indel substantially changes selection that shapes the protein region around it. Thus, an indel is often followed by an adaptive walk of length that is in agreement with the theory of molecular adaptation.     

Diversity in the Protein N-Glycosylation Pathways Within the Campylobacter Genus

The foodborne bacterial pathogen, Campylobacter jejuni, possesses an N-linked protein glycosylation (pgl) pathway involved in adding conserved heptasaccharides to asparagine-containing motifs of >60 proteins, and releasing the same glycan into its periplasm as free oligosaccharides. In this study, comparative genomics of all 30 fully sequenced Campylobacter taxa revealed conserved pgl gene clusters in all but one species. Structural, phylogenetic and immunological studies showed that the N-glycosylation systems can be divided into two major groups. Group I includes all thermotolerant taxa, capable of growth at the higher body temperatures of birds, and produce the C. jejuni-like glycans. Within group I, the niche-adapted C. lari subgroup contain the smallest genomes among the epsilonproteobacteria, and are unable to glucosylate their pgl pathway glycans potentially reminiscent of the glucosyltransferase regression observed in the O-glycosylation system of Neisseria species. The nonthermotolerant Campylobacters, which inhabit a variety of hosts and niches, comprise group II and produce an unexpected diversity of N-glycan structures varying in length and composition. This includes the human gut commensal, C. hominis, which produces at least four different N-glycan structures, akin to the surface carbohydrate diversity observed in the well-studied commensal, Bacteroides. Both group I and II glycans are immunogenic and cell surface exposed, making these structures attractive targets for vaccine design and diagnostics.In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide variety of essential cell-cell and cell-matrix processes ranging from immune recognition to cancer development. The first general protein glycosylation (pgl)¹ pathway was discovered in the epsilonproteobacterium Campylobacter jejuni (1). The organism transfers a conserved heptasaccharide en bloc to asparagine residues within the sequon D/E- X₁-N-X₂-S/T (X₁, X₂ ≠ P) of >60 glycoproteins (2–4). Furthermore, the pathway can be functionally transferred into Escherichia coli, and the oligosaccharyltransferase (OTase), PglB, is capable of adding foreign sugars to acceptor proteins (5–7). C. jejuni PglB also possesses hydrolase activity, influenced by the cellular growth phase and osmotic environment, releasing free oligosaccharides (fOS) into the periplasmic space in a 10:1 ratio relative to the amount of heptasaccharide N-linked to protein (8, 9).The C. jejuni N-linked heptasaccharide is conserved in structure in both C. jejuni and C. coli, the two most commonly isolated pathogenic Campylobacter species and major causes of human enteritis worldwide (10, 11). All campylobacters, but one, possess conserved pgl genes required for N-linked protein glycosylation ((12) and this study). This post-translational modification in C. jejuni influences DNA uptake, chicken and mouse colonization, epithelial cell adherence and invasion, recognition by human sera, and binding to the macrophage galactose lectin (MGL) receptor on dendritic cells (2, 13–17). Several Campylobacter species have now been recognized as emerging pathogens and causative agents of human gastroenteritis (e.g. C. upsaliensis and C. hyointestinalis), gingivitis, periodontitis, and human abortions (e.g. C. rectus, C. concisus, C. gracilis, C. showae, and C. upsaliensis) and inflammatory bowel disease in children (e.g. C. concisus) (18). Other species cause venereal disease and infertility in cattle (C. fetus subsp. venerealis; Cfv) or abortions in sheep (C. fetus subsp. fetus; Cff) (19).In this study, we used phylogenetic, immunological, structural and glycoproteomic studies to compare the N-glycosylation systems of 29 Campylobacter species and identified unexpected variations. Thus, although the pathway is a common feature within this genus, variability in the N-glycans and fOS at the species level suggests that each species possess a unique array of glycosyltransferases, which correlate with their phylogenetic relatedness.     

Crosstalk between the FGFR2 and TP53 genes in breast cancer: data from an association study and epistatic interaction analysis

To evaluate the potential for gene-gene interaction effects in sporadic breast cancer (BC) risk, we studied combinations of the fibroblast growth factor receptor 2 (FGFR2) rs1219648 and tumor protein 53 (TP53) rs1042522, rs1625895, and rs17878362 polymorphisms in BC patients (n=388) and healthy persons (n=275). In addition to a single-locus effect manifested by the association of FGFR2 rs1219648 and TP53 rs1042522 polymorphisms with high BC risk, depending on menopause status (0.001相似文献   

Analysis of the role of the coat protein N-terminal segment in Potato virus X virion stability and functional activity

Previously, we have reported that intact Potato virus X (PVX) virions cannot be translated in cell-free systems, but acquire this capacity by the binding of PVX-specific triple gene block protein 1 (TGBp1) or after phosphorylation of the exposed N-terminal segment of intravirus coat protein (CP) by protein kinases. With the help of in vitro mutagenesis, a nonphosphorylatable PVX mutant (denoted ST PVX) was prepared in which all 12 S and T residues in the 20-residue-long N-terminal CP segment were substituted by A or G. Contrary to expectations, ST PVX was infectious, produced normal progeny and was translated in vitro in the absence of any additional factors. We suggest that the N-terminal PVX CP segment somehow participates in virion assembly in vivo and that CP subunits in ST virions may differ in structure from those in the wild-type (UK3 strain). In the present work, to test this suggestion, we performed a comparative tritium planigraphy study of CP structure in UK3 and ST virions. It was found that the profile of tritium incorporation into ST mutant virions in some CP segments differed from that of normal UK3 virions and from UK3 complexed with the PVX movement protein TGBp1. It is proposed that amino acid substitutions in ST CP and the TGBp1-driven remodelling of UK3 virions induce structural alterations in intravirus CPs. These alterations affect the predicted RNA recognition motif of PVX CP, but in different ways: for ST PVX, labelling is increased in α-helices 6 and 7, whereas, in remodelled UK3, labelling is increased in the β-sheet strands β3, β4 and β5.     

Identification of Site-Specific Adaptations Conferring Increased Neural Cell Tropism during Human Enterovirus 71 Infection

Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP1₉₇ Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.     

How to Handle Speciose Clades? Mass Taxon-Sampling as a Strategy towards Illuminating the Natural History of Campanula (Campanuloideae)

Background

Speciose clades usually harbor species with a broad spectrum of adaptive strategies and complex distribution patterns, and thus constitute ideal systems to disentangle biotic and abiotic causes underlying species diversification. The delimitation of such study systems to test evolutionary hypotheses is difficult because they often rely on artificial genus concepts as starting points. One of the most prominent examples is the bellflower genus Campanula with some 420 species, but up to 600 species when including all lineages to which Campanula is paraphyletic. We generated a large alignment of petD group II intron sequences to include more than 70% of described species as a reference. By comparison with partial data sets we could then assess the impact of selective taxon sampling strategies on phylogenetic reconstruction and subsequent evolutionary conclusions.

Methodology/Principal Findings

Phylogenetic analyses based on maximum parsimony (PAUP, PRAP), Bayesian inference (MrBayes), and maximum likelihood (RAxML) were first carried out on the large reference data set (D680). Parameters including tree topology, branch support, and age estimates, were then compared to those obtained from smaller data sets resulting from “classification-guided” (D088) and “phylogeny-guided sampling” (D101). Analyses of D088 failed to fully recover the phylogenetic diversity in Campanula, whereas D101 inferred significantly different branch support and age estimates.

Conclusions/Significance

A short genomic region with high phylogenetic utility allowed us to easily generate a comprehensive phylogenetic framework for the speciose Campanula clade. Our approach recovered 17 well-supported and circumscribed sub-lineages. Knowing these will be instrumental for developing more specific evolutionary hypotheses and guide future research, we highlight the predictive value of a mass taxon-sampling strategy as a first essential step towards illuminating the detailed evolutionary history of diverse clades.     

Effect of weak static and low-frequency alternating magnetic fields on the fission and regeneration of the planarian dugesia (Girardia) tigrina

The effect of weak static (DC) and alternating (AC) magnetic fields (MFs), as well as combined (AC/DC) collinear MFs on the intensity of morphogenesis processes in the planarian Dugesia (Girardia) tigrina has been studied. It was found that combined MFs produce a stimulating effect on the fission and regeneration of planarians. Both components of the combined MFs, the direct (DC) and the alternating (AC), are important in the realization of the effects of weak MFs. The practically complete absence of one of the components (DC) reverses the sign of the effect. It was shown that the presence of concomitant background MFs does not substantially influence the effects of combined MFs with a very small AC component (100 nT). The effect of the "zero" field is significant and comparable in magnitude with the effects of combined MFs at effective frequencies. Narrow zones of effective amplitudes (in the region of tens and hundreds of nT) of the AC component of the combined MFs, with the DC component close to the value of the geomagnetic field were found, which alternate with regions where the response of the biological object to the influence is absent.     

Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: kinetic analysis of mutual inhibition between mu-conotoxin GIIIA derivatives and amine blockers

R13X derivatives of μ-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean “blocked” durations of several seconds. We studied interactions between two classes of blockers (μ-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, ∼100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude (“fast” block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X μ-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA (∼4 mM) and DEA (∼30 mM) increase by ∼20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the μ-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.