Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity

Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity

Prosurvival Bcl-2–like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function

Reconstruction of gross avian genome structure, organization and evolution suggests that the chicken lineage most closely resembles the dinosaur avian ancestor

Background The availability of multiple avian genome sequence assemblies greatly improves our ability to define overall genome organization and reconstruct evolutionary changes. In birds, this has previously been impeded by a near intractable karyotype and relied almost exclusively on comparative molecular cytogenetics of only the largest chromosomes. Here, novel whole genome sequence information from 21 avian genome sequences (most newly assembled) made available on an interactive browser (Evolution Highway) was analyzed. Results Focusing on the six best-assembled genomes allowed us to assemble a putative karyotype of the dinosaur ancestor for each chromosome. Reconstructing evolutionary events that led to each species' genome organization, we determined that the fastest rate of change occurred in the zebra finch and budgerigar, consistent with rapid speciation events in the Passeriformes and Psittaciformes. Intra- and interchromosomal changes were explained most parsimoniously by a series of inversions and translocations respectively, with breakpoint reuse being commonplace. Analyzing chicken and zebra finch, we found little evidence to support the hypothesis of an association of evolutionary breakpoint regions with recombination hotspots but some evidence to support the hypothesis that microchromosomes largely represent conserved blocks of synteny in the majority of the 21 species analyzed. All but one species showed the expected number of microchromosomal rearrangements predicted by the haploid chromosome count. Ostrich, however, appeared to retain an overall karyotype structure of 2n = 80 despite undergoing a large number (26) of hitherto un-described interchromosomal changes. Conclusions Results suggest that mechanisms exist to preserve a static overall avian karyotype/genomic structure, including the microchromosomes, with widespread interchromosomal change occurring rarely (e.g. in ostrich and budgerigar lineages). Of the species analyzed, the chicken lineage appeared to have undergone the fewest changes compared to the dinosaur ancestor

Phenotypic covariance of longevity, immunity and stress resistance in the Caenorhabditis nematodes

Background \ud Ageing, immunity and stresstolerance are inherent characteristics of all organisms. In animals, these traits are regulated, at least in part, by forkhead transcription factors in response to upstream signals from the Insulin/Insulin– like growth factor signalling (IIS) pathway. In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance. It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signalling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. Here we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four \ud Caenorhabditis species. \ud \ud Methodology/Principal Findings \ud We show using phenotypic analysis of DAF-16 influenced phenotypes that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged \ud significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (C. elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Real-Time PCR, which positively correlate with the observed phenotypes. Finally, we provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis. \ud \ud Conclusions \ud The gonochoristic species display a significantly longer lifespan (p < 0.0001)and more robust immune and stress response (p<0.0001, thermal stress; p<0.01, heavy metal stress; p<0.0001, pathogenic stress) than the hermaphroditic species. Our data suggests that divergence in DAF-16 mediated phenotypes may underlie many of the differences observed between these four species of Caenorhabditis nematodes. These findings are further supported by the correlative higher daf-16 expression levels among the gonochoristic species and significantly higher lifespan, immunity and stress tolerance in the constitutively active daf-16 hermaphroditic mutants

Shear wave splitting across the Iceland hot spot: Results from the ICEMELT experiment

We report on observations of upper mantle anisotropy from the splitting of teleseismic shear waves (SKS, SKKS, and PKS) recorded by the ICEMELT broadband seismometer network in Iceland. In a ridge-centered hot spot locale, mantle anisotropy may be generated by flow-induced lattice-preferred orientation of olivine grains or the anisotropic distribution of magma. Splitting measurements of teleseismic shear waves may thus provide diagnostic information on upper mantle flow and/or the distribution of retained melt associated with the Iceland mantle plume. In eastern Iceland, fast polarization directions lie between N10°W and N45°W and average N24°W; delay times between the fast and slow shear waves are generally 0.7–1.35 s. In western Iceland, in contrast, the fast polarization directions, while less well constrained, yield an average value of N23°E and delay times are smaller (0.2–0.95 s). We propose that splitting in eastern Iceland is caused by a 100- to 200-km-thick anisotropic layer in the upper mantle. The observed fast directions in eastern Iceland, however, do not correspond either to the plate spreading direction or to a pattern of radial mantle flow from the center of the Iceland hot spot. We suggest that the relatively uniform direction and magnitude of splitting in eastern Iceland, situated on the Eurasian plate, may therefore reflect the large-scale flow field of the North Atlantic upper mantle. We hypothesize that the different pattern of anisotropy beneath western Iceland, part of the North American plate, is due to the different absolute motions of the two plates. By this view, splitting in eastern and western Iceland is the consequence of shear by North American and Eurasian plate motion relative to the background mantle flow. From absolute plate motion models, in which the Eurasian plate is approximately stationary and the North American plate is moving approximately westward, the splitting observations in both eastern and western Iceland can be satisfied by a background upper mantle flow in the direction N34°W and a velocity of 3 cm/yr in a hot spot reference frame. This inference can be used to test mantle flow models. In particular, it is inconsistent with kinematic flow models, which predict southward flow, or models where flow is dominated by subduction-related sources of mantle buoyancy, which predict westward flow. Our observations are more compatible with the flow field predicted from global seismic tomography models, which in particular include the influence of the large-scale lower mantle upwelling beneath southern Africa. While the hypothesized association between our observations and this upwelling is presently speculative, it makes a very specific and testable prediction about the flow field and hence anisotropy beneath the rest of the Atlantic basin.This work was supported by the National Science Foundation under grants EAR-9316137, OCE-9402991, and EAR-9707193.Peer Reviewe

Phenotypic covariance of Longevity, Immunity and Stress Resistance in the Caenorhabditis Nematodes

Background: Ageing, immunity and stresstolerance are inherent characteristics of all organisms. In animals, these traits are regulated, at least in part, by forkhead transcription factors in response to upstream signals from the Insulin/Insulin–like growth factor signalling (IIS) pathway. In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance. It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signalling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. Here we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four Caenorhabditis species. \ud \ud Methodology/Principal Findings: We show using phenotypic analysis of DAF-16 influenced phenotypes that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (C. elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Real-Time PCR, which positively correlate with the observed phenotypes. Finally, we provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis. \ud \ud Conclusions: The gonochoristic species display a significantly longer lifespan (p<0.0001) and more robust immune and stress response (p<0.0001, thermal stress; p<0.01, heavy metal stress; p<0.0001, pathogenic stress) than the hermaphroditic species. Our data suggests that divergence in DAF-16 mediated phenotypes may underlie many of the differences observed between these four species of Caenorhabditis nematodes. These findings are further supported by the correlative higher daf-16 expression levels among the gonochoristic species and significantly higher lifespan, immunity and stress tolerance in the constitutively active daf-16 hermaphroditic mutants

Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans

The beneficial effects of polyphenol compounds in fruits and vegetables are mainly extrapolated from in vitro studies or short-term dietary supplementation studies. Due to cost and duration, relatively little is known about whether dietary polyphenols are beneficial in whole animals, particularly with respect to aging. To address this question, we examined the effects of blueberry polyphenols on lifespan and aging of the nematode, Caenorhabditis elegans, a useful organism for such a study. We report that a complex mixture of blue-berry polyphenols increased lifespan and slowed aging-related declines in C. elegans. We also found that these benefits did not just reflect antioxidant activity in these compounds. For instance, blueberry treatment increased survival during acute heat stress, but was not protective against acute oxidative stress. The blueberry extract consists of three major fractions that all contain antioxidant activity. However, only one fraction, enriched in proanthocyanidin compounds, increased C. elegans lifespan and thermotolerance. To further determine how polyphenols prolonged C. elegans lifespan, we analyzed the genetic requirements for these effects. Prolonged lifespan from this treatment required the presence of a CaMKII pathway that mediates osmotic stress resistance, though not other pathways that affect stress resistance and longevity. In conclusion, polyphenolic compounds in blueberries had robust and reproducible benefits during aging that were separable from antioxidant effects

Acyl Homoserine Lactones from Culture Supernatants of Pseudomonas aeruginosa Accelerate Host Immunomodulation

The virulence of Pseudomonas aeruginosa is multifactorial and under the control of quorum sensing signals, such as acyl homoserine lactones (AHLs). The importance of these molecules in the establishment of infection has been previously reported. These molecules either improve the virulence potential of P. aeruginosa or modulate the host immune response. To establish the immune modulating potential of quorum sensing signal molecules, previous studies have only used synthetic AHLs. However, there can be differences in the biological properties of synthetic and natural AHLs. The use of naturally extracted AHLs from the culture supernatant of P. aeruginosa is likely to simulate natural conditions more than the use of synthetic AHLs. Therefore, in the present study, the immune modulating potential of synthetic and naturally extracted AHLs was compared using a thymidine uptake assay, immunophenotyping and sandwich ELISA in order to assess mouse T-cell proliferation and production of Th1 and Th2 cytokines. Natural AHLs were able to suppress T-cell proliferation, even at low concentrations, compared to synthetic AHLs. The majority of cells undergoing proliferation were CD4+, as revealed by immunophenotyping. The inhibition of T-cells was stronger with natural AHLs compared to synthetic AHLs. Moreover, the natural AHLs were also able to shift immune responses away from host protective Th1 responses to pathogen protective Th2 responses