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Sonstige beteiligte Institutionen
- DNA Analytics Core Facility, Biocenter, University of Würzburg, Würzburg, Germany (1)
- Department of Animal Ecology and Tropical Biology, University of Würzburg, Würzburg, Germany (1)
- Forschungsstation Fabrikschleichach (1)
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We have discovered a new mechanism of monoallelic gene expression that links antigenic variation, cell cycle, and development in the model parasite Trypanosoma brucei. African trypanosomes possess hundreds of variant surface glycoprotein (VSG) genes, but only one is expressed from a telomeric expression site (ES) at any given time. We found that the expression of a second VSG alone is sufficient to silence the active VSG gene and directionally attenuate the ES by disruptor of telomeric silencing-1B (DOT1B)-mediated histone methylation. Three conserved expression-site-associated genes (ESAGs) appear to serve as signal for ES attenuation. Their depletion causes G1-phase dormancy and reversible initiation of the slender-to-stumpy differentiation pathway. ES-attenuated slender bloodstream trypanosomes gain full developmental competence for transformation to the tsetse fly stage. This surprising connection between antigenic variation and developmental progression provides an unexpected point of attack against the deadly sleeping sickness.
Nasal colonization is a major risk factor for S. aureus infections. The mechanisms responsible for colonization are still not well understood and involve several factors on the host and the bacterial side. One key factor is the cell wall teichoic acid (WTA) of S. aureus, which governs direct interactions with nasal epithelial surfaces. We report here the first receptor for the cell wall glycopolymer WTA on nasal epithelial cells. In several assay systems this type F-scavenger receptor, termed SREC-I, bound WTA in a charge dependent manner and mediated adhesion to nasal epithelial cells in vitro. The impact of WTA and SREC-I interaction on epithelial adhesion was especially pronounced under shear stress, which resembles the conditions found in the nasal cavity. Most importantly, we demonstrate here a key role of the WTA-receptor interaction in a cotton rat model of nasal colonization. When we inhibited WTA mediated adhesion with a SREC-I antibody, nasal colonization in the animal model was strongly reduced at the early onset of colonization. More importantly, colonization stayed low over an extended period of 6 days. Therefore we propose targeting of this glycopolymer-receptor interaction as a novel strategy to prevent or control S. aureus nasal colonization.
An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3) and FABP7, both of which are involved in fatty acid uptake. In vitro, both were induced by hypoxia in a hypoxia-inducible factor-1 alpha (HIF-1 alpha)-dependent manner. There was a significant lipid droplet (LD) accumulation in hypoxia that was time and O-2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component) significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via beta-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in vitro, and strongly impaired tumorigenesis in vivo.
Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71–110, 158–167, 180–203, and 264–286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71–110 and HlyAΔ264–286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158–167 and HlyAΔ180–203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71–110 and HlyAΔ264–286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71–110, and HlyAΔ264–286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.
Bedeutung der NO-sensitiven Guanylyl Cyclase bei der Angiogenese und der Arteriogenese in der Maus
(2014)
Stickstoffmonoxid (NO) spielt eine wichtige Rolle bei Gefäßremodelling-Prozessen wie Angiogenese und Arteriogenese. Die NO-Synthese im Gefäßsystem wird hauptsächlich durch die endotheliale NO-Synthase (eNOS) gewährleistet. Sie kann durch verschiedene Faktoren wie Scherkräfte und Zytokine wie der vaskuläre endotheliale Wachstumsfaktor (VEGF) reguliert werden. VEGF ist ein wichtiger Stimulator der Angiogenese und wird während dieses Prozesses hochreguliert. Die meisten physiologischen Effekte von NO werden durch die NO-sensitive Guanylyl-Cyclase (NO-GC) vermittelt. Als Hauptrezeptor für NO produziert die NO-GC den sekundären Botenstoff cyklisches Guanosinmonophosphat (cGMP) und führt dadurch zur Stimulation der verschiedenen Effektoren wie z.B. der PKG. Ob die Wirkung von NO in Angiogenese und Arteriogenese ebenfalls durch NO-GC vermittelt wird, war bis zum Beginn dieser Arbeit noch unklar.
Die NO-GC besteht aus zwei Untereinheiten (α und ß). Die Deletion der ß1-Untereinheit in Mäusen resultiert in einer vollständigen Knockout Maus (GCKO). Mithilfe des Cre-LoxP-Systems wurden zusätzlich zellspezifische Knockout-Mäuse für glatte Muskelzellen (SMC-GCKO) und Endothelzellen (EC-GCKO) generiert. Um die Rolle der NO-GC in der Angiogenese und Arteriogenese zu untersuchen, wurden drei gut etablierte Methoden benutzt.
Im ersten Teil des Projekts sollte die Expression der NO-GC in Endothelzellen untersucht werden. Zu diesem Zweck wurde die reverse Transkriptase-Polymerase-Kettenreaktion (RT-PCR) benutzt. Die Ergebnisse zeigen, dass die NO-GC in Endothelzellen der Lunge nur äußerst gering wenig exprimiert ist. Durch den Aortenring-Assay wurde eine Rolle der NO-GC bei der VEGF-vermittelten Angiogenese festgestellt. Dabei zeigte sich eine stärkere Angiogeneserate bei globaler Abwesenheit der NO-GC. Bei Fehlen der NO-GC ausschließlich in Endothelzellen zeigte sich kein Unterschied in den aussprossenden Aorten im Vergleich zu den Kontroll-Tieren. Dies zeigt, dass die NO-GC in Endothelzellen sehr wahrscheinlich keine Rolle bei der VEGF-vermittelten Angiogenese spielt.
Im zweiten Teil wurde die Rolle der NO-GC bei der Angiogenese in einem in vivo-Modell untersucht. In dem Modell der Sauerstoff-induzierten-Retinopathie zeigten die GCKO-Mäuse eine verringerte Vaso-Obliteration, eine verlangsamte Angiogenese und eine erhöhte Tuft-Bildung. Ähnliche Ergebnisse wurden bei den SMC-GCKO-Tieren beobachtet. EC-GCKO-Mäuse zeigten eine gegenüber den Kontroll-Tieren unveränderte Vaso-Obliteration, Angiogeneserate und Tuft-Bildung. Diese Ergebnisse lassen darauf schließen, dass die NO-GC in Endothelzellen keine Rolle spielt. Immunfluoreszenz-Aufnahmen zeigten die Expression von NO-GC in Perizyten der Gefäßkapillaren der Mausretina. Daher könnte die NO-GC in diesem Zelltyp letztendlich für die Effekte bei den GCKO- und SMC-GCKO-Tieren verantwortlich sein.
Im letzten Teil dieser Arbeit wurde eine Versuchsreihe unter Anwendung des Hinterlauf-Ischämie-Modells durchgeführt. Hierbei entwickelten die Pfoten aller GCKO- und teilweise der SMC-GCKO-Tiere nach der Ligation der Femoralarterie eine Nekrose. Die Regeneration der Hinterläufe der EC-GCKO-Tiere nach der Operation verlief normal. Diese Ergebnisse schließen eine bedeutende Rolle der NO-GC in Endothelzellen aus, zeigen allerdings, dass die NO-GC in den glatten Muskelzellen essentiell für den Arteriogenese-Prozess ist.
Zusammengefasst führt die Deletion der NO-GC in glatten Muskelzellen und wahrscheinlich auch in Perizyten zur einer verlangsamten Angiogenese und Inhibierung der Arteriogenese.
Agricultural Policies Exacerbate Honeybee Pollination Service Supply-Demand Mismatches Across Europe
(2014)
Declines in insect pollinators across Europe have raised concerns about the supply of pollination services to agriculture. Simultaneously, EU agricultural and biofuel policies have encouraged substantial growth in the cultivated area of insect pollinated crops across the continent. Using data from 41 European countries, this study demonstrates that the recommended number of honeybees required to provide crop pollination across Europe has risen 4.9 times as fast as honeybee stocks between 2005 and 2010. Consequently, honeybee stocks were insufficient to supply >90% of demands in 22 countries studied. These findings raise concerns about the capacity of many countries to cope with major losses of wild pollinators and highlight numerous critical gaps in current understanding of pollination service supplies and demands, pointing to a pressing need for further research into this issue.
Background
The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host’s liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood.
Results
Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite’s glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin.
Conclusions
Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.
The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis
(2014)
Background
It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal.
Results
We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells.
Conclusions
In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae.
Similarity between odours is notoriously difficult to measure. Widely used behavioural approaches in insect olfaction research are cross-adaptation, masking, as well as associative tasks based on olfactory learning and the subsequent testing for how specific the established memory is. A concern with such memory-based approaches is that the learning process required to establish an odour memory may alter the way the odour is processed, such that measures of perception taken at the test are distorted. The present study was therefore designed to see whether behavioural judgements of perceptual distance are different for two different memory-based tasks, namely generalization and discrimination. We used odour-reward learning in larval Drosophila as a study case. In order to challenge the larvae's olfactory system, we chose to work with binary mixtures and their elements (1-octanol, n-amyl acetate, 3-octanol, benzaldehyde and hexyl acetate). We determined the perceptual distance between each mixture and its elements, first in a generalization task, and then in a discrimination task. It turns out that scores of perceptual distance are correlated between both tasks. A re-analysis of published studies looking at element-to-element perceptual distances in larval reward learning and in adult punishment learning confirms this result. We therefore suggest that across a given set of olfactory stimuli, associative training does not grossly alter the pattern of perceptual distances.
Since more than two centuries naturalists are fascinated by the profound changes in biodiversity observed along climatic gradients. Although the theories explaining changes in the diversity and the shape of organisms along climatic gradients belong to the foundations of modern ecology, our picture on the spatial patterns and drivers of biodiversity is far from being complete. Ambiguities in theory and data are common and past work has been strongly concentrated on plants and vertebrates. In the last two decades, interest in the fundamental processes structuring diversity along climatic gradients gained new impetus as they are expected to improve our understanding about how ecosystems will respond to global environmental changes. Global temperatures are rising faster than ever before; natural habitats are transformed into agricultural land and existing land use systems get more and more intensified to meet the demands of growing human populations. The fundamental shifts in the abiotic and biotic environment are proclaimed to affect ecosystems all over the world; however, precise predictions about how ecosystems respond to global changes are still lacking. We investigated diversity, traits and ecosystem services of wild bees along climate and land use gradients on Mount Kilimanjaro (Tanzania, East Africa). Wild bees play a major role in ecosystems, as they contribute to the reproduction and performance of wild and crop plants. Their responsiveness to environmental changes is therefore of high ecological and economic importance.
Temperature and energy resources have often been suggested to be the main determinants of global and local species richness, but the mechanisms behind remain poorly understood. In the study described in chapter II we analyzed species richness patterns of wild bees along climate and land use gradients on Mount Kilimanjaro and disentangled the factors explaining most of the changes in bee richness. We found that floral resources had a weak but significant effect on pollinator abundance, which in turn was positively related to species richness. However, temperature was the strongest predictor of species richness, affecting species richness both directly and indirectly by positively influencing bee abundances. We observed higher levels of bee-flower-interactions at higher temperatures, independently of flower and bee abundances. This suggests that temperature restricts species richness by constraining the exploitation of resources by ectotherms. Current land use did not negatively affect species richness. We conclude that the richness of bees is explained by both temperature and resource availability, whereas temperature plays the dominant role as it limits the access of ectotherms to floral resources and may accelerate ecological and evolutionary processes that drive the maintenance and origination of diversity.
Not only species numbers, but also morphological traits like body size are expected to be shaped by both physiological and energetic constraints along elevational gradients. Paradoxically, Bergmann´s rule predicts increases of body sizes in cooler climates resulting from physiological constraints, while species-energy theory suggests declines in the mean body size of species caused by increased extinction probabilities for large-bodied species in low-energy habitats. In chapter III we confronted this ambiguity with field data by studying community-wide body size variation of wild bees on Mt. Kilimanjaro. We found that along a 3680 m elevational gradient bee individuals became on average larger within species, while large species were increasingly absent from high-elevational communities. This demonstrates, on the one hand, how well-established, but apparently contrasting ecological theories can be merged through the parallel consideration of different levels of biological organization. On the other hand it signals that the extinction risk in the course of environmental change is not equally distributed among species within a community.
Land use intensification is known to threaten biodiversity, but the consequences for ecosystem services are still a matter of debate. In chapter IV, we experimentally tested the single and combined contributions of pest predators and pollinators to coffee production along a land use intensification gradient on Mount Kilimanjaro. We found that pest predation increased fruit set by on average 9%, while pollination increased fruit weight of coffee by on average 7.4%. Land use had no significant effect on both ecosystem services. However, we found that in coffee plantations with most intensified land use, pollination services were virtually exclusively provided by the honey bee (Apis mellifera). The reliance on a single pollinator species is risky, as possible declines of that species may directly lower pollination services, resulting in yield losses. In contrast, pollination services in structurally complex homegardens were found to be provided by a diverse pollinator community, increasing the stability of pollination services in a long term.
We showed that on Mount Kilimanjaro pollinator communities changed along elevational gradients in terms of species richness (chapter II) and trait composition (chapter III). Temperature and the temperature-mediated accessibility of resources were identified as important predictors of these patterns, which contributes to our fundamental understanding about the factors that shape ectothermic insect communities along climatic gradients. The strong temperature-dependence of pollinators suggests that temperature shifts in the course of global change are likely to affect pollinator communities. Pollinators might either profit from rising temperatures, or shift to higher elevations, which could result in related biotic attrition in the lowland with consequences for the provision of ecosystem services in cropping systems. Up to now, land use intensification had no significant impact on the diversity of pollinator communities and their ecosystem services. Pollinators might profit from the strong landscape heterogeneity in the region and from the amount of flower resources in the understory of cropping systems. However,progressing homogenization of the landscape and the pronounced application of pesticides could result in reduced diversity and dominance of single species, as we already found in sun coffee plantations. Such shifts in community compositions could threaten the stability of ecosystem services within cropping and natural systems in a long term.