Authigenic carbonates from cold-water carbonate mounds in the Gulf of Cadiz: Microbial diversity and imprint on carbonate minerals

The Pen Duick Escarpment off Morocco consists of recent carbonate mounds in water depths of 500-600 m, flanked by giant mud volcanoes. These mounds are covered by mainly lifeless cold-water corals and have been associated with extensive fields of seep-related carbonates in off-reef regions. Three piston cores (from 350 to 640 cm long), coming from different sites on these juvenile mounds, were sampled and analyzed for mineralogy, stable isotopic composition of carbonates, geochemistry, and microbial communities. Most of the sediment comprises pelagic calcite (coccoliths), detrital quartz and authigenic dolomite, often observed encasing coccoliths. The decalcification of the sediment resulted in a dolomite dominated matrix that showed stable carbon istotope values of as low as -30 permil in contrast to the bulk sample values of -7 to -15 permil, which implies the involvement of microbes in the production of bicarbonate ions. Initial results from 16S rRNA gene clone libraries support the theory, that anaerobic oxidation of methane is one of the most important biogeochemical process leading to carbonate precipitation. Preliminary results of stable carbon isotopes of bulk samples from different carbonate mounds from the same area, indicates that the sulphate-methane transition zone moves in depth through time. We will show and discuss multidisciplinary data obtained after several cruises aimed to elucidate the impact of microorganisms on the construction of these carbonate mounds. The special emphasis in this research will be on the correlation between microbial ecosystems and their metabolic influence on mineral formation and diagenesis

Paracrine Diffusion of PrPC and Propagation of Prion Infectivity by Plasma Membrane-Derived Microvesicles

Cellular prion protein (PrPc) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1–1 µm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrPC diffusion and prion infectivity transmission. We first identified PrPC in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrPSc is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrPSc-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrPC diffusion and signaling as well as to the process of prion spread and neuroinvasion

Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrPC, efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrPC selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrPSc and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories

Plasmacytoid Dendritic Cells Sequester High Prion Titres at Early Stages of Prion Infection

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles

Increased Abundance of M cells in the Gut Epithelium Dramatically Enhances Oral Prion Disease Susceptibility

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure, the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches, the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling, and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice, the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked, demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens, inflammatory stimuli and aging can modify M cell-density in the gut, these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice, resulting in shortened survival times and increased disease susceptibility, equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection, whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases

The imprint of methane seepage on the geochemical record and early diagenetic processes in cold-water coral mounds on Pen Duick Escarpment, Gulf of Cadiz

The diagenetic history and biogeochemical processes in three cold-water coral mounds located in close proximity to each other on Pen Duick Escarpment in the Gulf of Cadiz were examined. The influence of ascending methane-rich fluids from underlying sediment strata delineated two mound groups: Alpha and Beta Mound showed evidence for the presence of a sulfate-methane transition zone (SMTZ) at shallow depth, whereas Gamma Mound appeared to lack a shallow SMTZ. In the methane influenced Alpha and Beta Mound, upward diffusion of hydrogen sulfide from the shallow SMTZ caused extensive pyritization of reactive iron phases as indicated by values for the degree-of-pyritization > 0.7. This secondary pyritization overprinted the sulfur isotope composition of sulfides formed during organoclastic sulfate reduction. The almost complete consumption of reactive iron phases by upward diffusing sulfide limited dissimilatory iron reduction to the top layer in these mounds while organic matter in the pyritized zones below was primarily degraded by organoclastic sulfate reduction. Hydrogen sulfide produced during sulfate reduction coupled to the anaerobic oxidation of methane (AOM) diffused upward and induced aragonite dissolution as evidenced in strongly corroded corals in Alpha Mound. This mound has been affected by strong fluctuations in the depth of the SMTZ, as observed by distinct layers with abundant diagenetic high-Mg calcite with a 13C-depleted carbon isotope composition. In the non-methane influenced Gamma Mound low sulfate reduction rates, elevated concentrations of dissolved iron, and solid-phase iron speciation indicated that organic matter mineralization was driven by dissimilatory iron reduction and organoclastic sulfate reduction coupled to oxidative sulfur cycling. The latter process led to 34S-depletion in pyrite of up to 70‰ relative to pore-water sulfate

Biodiversity of cold seep ecosystems along the European margins

During the European Commission's Framework Six Programme, HERMES, we investigated three main areas along the European margin, each characterized by the presence of seep-related structures exhibiting different intensity of activity and biological diversity. These areas are: (1) the Nordic margin with the Hakon Mosby mud volcano and many pockmarks, (2) the Gulf of Cadiz, and (3) the eastern Mediterranean with its hundreds of mud volcanoes and brine pool structures. One of the main goals of the HERMES project was to unravel the biodiversity associated with these seep-associated ecosystems, and to understand their driving forces and functions, using an integrated approach. Several multidisciplinary research cruises to these three areas provided evidence of high variability in ecosystem processes and associated biodiversity at different spatial scales, illustrating the "hotspot" nature of these deep water systems