Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens

Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium . We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway.     

Allergen-independent maternal transmission of asthma susceptibility

Maternal asthma is a risk factor for development of asthma in children, but mechanisms remain unclear. Offspring of asthmatic mother mice (sensitized and repeatedly exposed to OVA Ag) showed airway hyperresponsiveness and allergic pulmonary inflammation after an intentionally suboptimal OVA sensitization and exposure protocol that had little effect on normal offspring. Similar results were obtained when offspring of OVA-allergic mothers were exposed to an unrelated allergen, casein, indicating that the maternal effect is allergen independent and not transferred by OVA-specific Abs. Premating treatment with neutralizing anti-IL-4 Ab or reduction of maternal allergen exposure abrogated the maternal effect, showing a critical mechanistic role for IL-4 and suggesting an additional benefit of allergen avoidance.     

Furanoterpenoids from Siphonochilus aethiopicus

Two new furanoterpenoid derivatives, namely 4a alpha H-3,5 alpha,8 alpha beta-trimethyl-4,4a,9-tetrahydro-naphtho[2,3-b]-furan-8-one and 2-hydroxy-4a alpha H-3,5 alpha, 8a beta-trimethyl-4,4a,9-tetrahydronaphtho[2,3-b]-furan-8-one, were isolated from Siphonochilus aethiopicus, a member of the family Zingiberaceae. Their structures were elucidated using high field NMR techniques.     

Long-range correlations between DNA bending sites: relation to the structure and dynamics of nucleosomes

It has been established that the precise positioning of nucleosomes on genomic DNA can be achieved, at least for a minority of them, through sequence-dependent processes. However, to what extent DNA sequences play a role in the positioning of the major part of nucleosomes is still debated. The aim of the present study is to examine to what extent long-range correlations (LRC) are related to the presence of nucleosomes. Using the wavelet transform technique, we perform a comparative analysis of the DNA text and of the corresponding bending profiles generated with curvature tables based on nucleosome positioning data. The exploration of a number of eukaryotic and bacterial genomes through the optics of the so-called "wavelet transform microscope" reveals a characteristic scale of 100-200 bp that separates two regimes of different LRC. Here, we focus on the existence of LRC in the small-scale regime (10-200 bp) which are actually observed in eukaryotic genomes, in contrast to their absence in eubacterial genomes. Analysis of viral DNA genomes shows that, like their host's genomes, eukaryotic viruses present LRC but eubacterial viruses do not. There is one exception for genomes of poxviruses (Vaccinia and Melamoplus sanguinipes) which do not replicate in the cell nucleus and do not exhibit LRC. No small-scale LRC are detected in the genomes of all examined RNA viruses, with the exception of retroviruses. These results together with the observation of LRC between particular sequence motifs known to participate in the formation of nucleosomes (e.g. AA dinucleotides) strongly suggest that the 10-200 bp LRC are a signature of the sequence-dependence of nucleosome positioning. Finally, we discuss possible interpretations of these LRC in terms of the physical mechanisms that might govern the positioning and the dynamics of the nucleosomes along the DNA chain through cooperative processes.     

Investigating ancient duplication events in the Arabidopsis genome

The complete genomic analysis of Arabidopsis thaliana has shown that a major fraction of the genome consists of paralogous genes that probably originated through one or more ancient large-scale gene or genome duplication events. However, the number and timing of these duplications still remains unclear, and several different hypotheses have been put forward recently. Here, we reanalyzed duplicated blocks found in the Arabidopsis genome described previously and determined their date of divergence based on silent substitution estimations between the paralogous genes and, where possible, by phylogenetic reconstruction. We show that methods based on averaging protein distances of heterogeneous classes of duplicated genes lead to unreliable conclusions and that a large fraction of blocks duplicated much more recently than assumed previously. We found clear evidence for one large-scale gene or even complete genome duplication event somewhere between 70 to 90 million years ago. Traces pointing to a much older (probably more than 200 million years) large-scale gene duplication event could be detected. However, for now it is impossible to conclude whether these old duplicates are the result of one or more large-scale gene duplication events. abbreviations dA, fraction of amino acid substitutions; Kn, number of nonsynonymous substitutions per nonsynonymous site; Ks, number of synonymous substitutions per synonymous site; MYA, million years ago     

Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage

Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio. Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.     

Influence of the environment in the conformation of alpha-helices studied by protein database search and molecular dynamics simulations

The influence of the solvent on the main-chain conformation (phi and Psi dihedral angles) of alpha-helices has been studied by complementary approaches. A first approach consisted in surveying crystal structures of both soluble and membrane proteins. The residues of analysis were further classified as exposed to either the water (polar solvent) or the lipid (apolar solvent) environment or buried to the core of the protein (intermediate polarity). The statistical results show that the more polar the environment, the lower the value of phi(i) and the higher the value of Psi(i) are. The intrahelical hydrogen bond distance increases in water-exposed residues due to the additional hydrogen bond between the peptide carbonyl oxygen and the aqueous environment. A second approach involved nanosecond molecular dynamics simulations of poly-Ala alpha-helices in environments of different polarity: water to mimic hydrophilic environments that can form hydrogen bonds with the peptide carbonyl oxygen and methane to mimic hydrophobic environments without this hydrogen bond capabilities. These simulations reproduce similar effects in phi and Psi angles and intrahelical hydrogen bond distance and angle as observed in the protein survey analysis. The magnitude of the intrahelical hydrogen bond in the methane environment is stronger than in the water environment, suggesting that alpha-helices in membrane-embedded proteins are less flexible than in soluble proteins. There is a remarkable coincidence between the phi and Psi angles obtained in the analysis of residues exposed to the lipid in membrane proteins and the results from computer simulations in methane, which suggests that this simulation protocol properly mimic the lipidic cell membrane and reproduce several structural characteristics of membrane-embedded proteins. Finally, we have compared the phi and Psi torsional angles of Pro kinks in membrane protein crystal structures and in computer simulations.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.