Ceramide structure dictates glycosphingolipid nanodomain assembly and function

Gangliosides such as GM1 present in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. Here the authors show that the acyl chain structure of GM1 determines the establishment of nanodomains when actively clustered by actin, which depended on membrane cholesterol and phosphatidylserine or superimposed by the GM1-binding bacterial cholera toxin

Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR

Tourism megatrends

Tourism is affected by social, political, economic, technological and environmental changes at all scales. Population growth, redistribution of wealth, geopolitical changes and conflicts, rising fuel costs, climate change and its consequences, new technologies and work patterns, and all forms of social fashion influence who wants to travel where, for how long, to do what, and at what prices. Here, we examine six large-scale exogenous trends for the global tourism sector over the next 30 years: (1) The social, economic and environmental consequences of gradual warming and of extreme weather events associated with climate change; (2) The effects of higher fuel costs and social concerns on mass long-haul travel; (3) The role of new technologies, including social media, in marketing, managing, experiencing and monitoring tourism; (4) Economic growth and social change in the highly populous and newly wealthy BRICS nations, especially India and China; (5) The consequences of armed conflict and geopolitical negotiation for tourism, and the use of tourism as a tool for geopolitical interests; (6) The increasing linkages, and also conflicts, between tourism and conservation in many countries. Improved understanding of these megatrends, and the interactions between them, are priorities for future tourism research

Rhodococcus equi Virulence-Associated Protein A Is Required for Diversion of Phagosome Biogenesis but Not for Cytotoxicity▿

Rhodococcus equi is a gram-positive facultative intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. Virulence is plasmid regulated and is accompanied by phagosome maturation arrest and host cell necrosis. A replacement mutant in the gene for VapA (virulence-associated protein A), a major virulence factor of R. equi, was tested for its activities during macrophage infection. Early in infection, phagosomes containing the vapA mutant did not fuse with lysosomes and did not stain with the acidotropic fluor LysoTracker similar to those containing virulent wild-type R. equi. However, vapA mutant phagosomes had a lower average pH. Late in infection, phagosomes containing the vapA mutant were as frequently positive for LysoTracker as phagosomes containing plasmid-cured, avirulent bacteria, whereas those with virulent wild-type R. equi were still negative for the fluor. Macrophage necrosis after prolonged infection with virulent bacteria was accompanied by a loss of organelle staining with LysoTracker, suggesting that lysosome proton gradients had collapsed. The vapA mutant still killed the macrophages and yet did not affect the pH of host cell lysosomes. Hence, VapA is not required for host cell necrosis but is required for neutralization of phagosomes and lysosomes or their disruption. This is the first report of an R. equi mutant with altered phagosome biogenesis

Mechanism of Shiga Toxin Clustering on Membranes

The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga toxin molecules. By contrast, in coarse-grained computer simulations a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface. The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin molecules (several nm), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications

Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers

Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of Î 2 1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis

Effect of phagosome pH on <i>C. glabrata</i> survival.

<p>(A) Viable and heat killed <i>C. glabrata</i> containing phagosomes acquire similar levels of V-ATPase. Representative fluorescence microscopy images of viable or heat killed <i>C. glabrata</i> 180 min post-infection phagocytosed by murine J774E cells expressing a V-ATPase-GFP fusion protein (left panel). V-ATPase is shown in green while non-phagocytosed yeasts (stained with concanavalin A [ConA]) are indicated in yellow (marked with red arrows). Phagocytosed yeasts are labeled with white arrows. Co-localization with V-ATPase was quantified for phagosomes containing viable or heat killed <i>C. glabrata</i> at indicated time points (right panel). (B) Rising phagosome pH with chloroquine but not bafilomycin A1 reduces <i>C. glabrata</i> survival in MDMs. Survival of <i>C. glabrata</i> was determined by cfu-plating of macrophage lysates after 24 h. Co-incubation samples contained no drug (untreated), chloroquine (50 µM), chloroquine plus iron nitriloacetate (20 µM, FeNTA) or bafilomycin A1 (50 nM). (C) Chloroquine or bafilomycin A1 are not toxic to <i>C. glabrata in vitro</i>. Growth in presence of the drugs is comparable to untreated cultures. Statistical analysis was performed comparing heat killed with viable <i>C. glabrata</i> at indicated time points (A) or comparing untreated and drug-treated samples (B) (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).</p

Phagosome maturation arrest occurs in different macrophage differentiation or activation states and is yeast phagosome-specific.

<p>(A) Human M1-polarized and M2-polarized MDMs do not differ in central aspects of <i>C. glabrata</i>-macrophage interaction: phagocytosis, phagosome acidification and killing. Phagocytosis (MOI of 5) was quantified microscopically by determining the percentage of internalized (Concanavalin A stain-negative) yeasts out of total yeasts after 90 min. Phagosome acidification was quantified microscopically by determining the percentage of LysoTracker-positive phagosomes after 90 min. Survival of <i>C. glabrata</i> was determined by cfu-plating of macrophage lysates after 3 h of co-incubation and comparing to yeasts incubated without macrophages. (B) Treatment with vitamin D₃ (calcitriol) has no influence on the number of LysoTracker-positive viable <i>C. glabrata</i> containing phagosomes of human MDMs. (C) MDMs co-infected with <i>C. glabrata</i> and latex beads show a acidification defect specific to <i>C. glabrata</i> containing phagosomes (LysoTracker-negative staining; white arrow) but acidify latex bead containing phagosomes (LysoTracker-positive staining; white asterisk). Representative image 90 min post infection. GFP-expressing <i>C. glabrata</i> is indicated in green and non-phagocytosed yeasts stained with Concanavalin A (ConA) are in yellow (marked with red arrows). Statistical analysis was performed comparing M1-type with M2-type macrophages (A) or drug treated with untreated viable <i>C. glabrata</i> (B) (n≥3; *p<0.05, **p<0.01 by unpaired Student’s t test).</p

Alkalinization-defective <i>C. glabrata</i> mutants.

<p>Listed are mutants that showed reduced <i>in vitro</i> alkalinization of phenol red containing YNB medium with 1% casamino acids as sole carbon and nitrogen source in a screen of 647 mutants. Alkalinization defects were verified in independent assays and with two independent clones.</p>A<p>+reduced alkalinization (same phenotype as <i>bcy1</i>Δ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096015#pone-0096015-g005" target="_blank">Fig. 5A</a>), ++ strongly reduced alkalinization (same phenotype as <i>put3</i>Δ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096015#pone-0096015-g005" target="_blank">Fig. 5A</a>) as compared to the wild type.</p>B, C<p>Growth was monitored in parallel in YPD and in YNB medium with 1% casamino acids without phenol red by measuring absorption at 600 nm. ++ strong growth defect, + weak growth defect, - unaltered growth as compared to the wild type.</p>D<p>Mutants were co-incubated with MDMs for 90 min and phagosome acidification was monitored by LysoTracker staining. At least three independent microscopic fields were scored per mutant. ++ strong increase in LysoTracker signal, + medium increase in LysoTracker signal, - no change in LysoTracker signal as compared to the wild type.</p