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Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNAseq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semiquantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen.
The human gut is home for thousands of microbes that are important for human life. As most of these cannot be cultivated, metagenomics is an important means to understand this important community. To perform comparative metagenomic analysis of the human gut microbiome, I have developed SMASH (Simple metagenomic analysis shell), a computational pipeline. SMASH can also be used to assemble and analyze single genomes, and has been successfully applied to the bacterium Mycoplasma pneumoniae and the fungus Chaetomium thermophilum. In the context of the MetaHIT (Metagenomics of the human intestinal tract) consortium our group is participating in, I used SMASH to validate the assembly and to estimate the assembly error rate of 576.7 Gb metagenome sequence obtained using Illumina Solexa technology from fecal DNA of 124 European individuals. I also estimated the completeness of the gene catalogue containing 3.3 million open reading frames obtained from these metagenomes. Finally, I used SMASH to analyze human gut metagenomes of 39 individuals from 6 countries encompassing a wide range of host properties such as age, body mass index and disease states. We find that the variation in the gut microbiome is not continuous but stratified into enterotypes. Enterotypes are complex host-microbial symbiotic states that are not explained by host properties, nutritional habits or possible technical biases. The concept of enterotypes might have far reaching implications, for example, to explain different responses to diet or drug intake. We also find several functional markers in the human gut microbiome that correlate with a number of host properties such as body mass index, highlighting the need for functional analysis and raising hopes for the application of microbial markers as diagnostic or even prognostic tools for microbiota-associated human disorders.
This thesis consists of three major chapters, each of which has been separately published or under the process for publication. The first chapter is about anatomical characterization of the mushroom body of adult Drosophila melanogaster. The mushroom body is the center for olfactory learning and many other functions in the insect brains. The functions of the mushroom body have been studied by utilizing the GAL4/UAS gene expression system. The present study characterized the expression patterns of the commonly used GAL4 drivers for the mushroom body intrinsic neurons, Kenyon cells. Thereby, we revealed the numerical composition of the different types of Kenyon cells and found one subtype of the Kenyon cells that have not been described. The second and third chapters together demonstrate that the multiple types of dopaminergic neurons mediate the aversive reinforcement signals to the mushroom body. They induce the parallel memory traces that constitute the different temporal domains of the aversive odor memory. In prior to these chapters, “General introduction and discussion” section reviews and discuss about the current understanding of neuronal circuit for olfactory learning in Drosophila.
The construction of mound-shaped nests by ants is considered as a behavioral adaptation to low environmental temperatures, i.e., colonies achieve higher and more stables temperatures than those of the environment. Besides the well-known nests of boreal Formica wood-ants, several species of South American leaf-cutting ants of the genus Acromyrmex construct thatched nests. Acromyrmex workers import plant fragments as building material, and arrange them so as to form a thatch covering a central chamber, where the fungus garden is located. Thus, the degree of thermoregulation attained by the fungus garden inside the thatched nest largely depends on how the thatch affects the thermal relations between the fungus and the environment. This work was aimed at studying the thermoregulatory function of the thatched nests built by the grass-cutting ant Acromyrmex heyeri Forel (Hymenoptera: Formicidae: Myrmicinae). Nest and environmental temperatures were measured as a function of solar radiation on the long-term. The thermal diffusivity of the nest thatch was measured and compared to that of the surrounding soil, in order to assess the influence of the building material on the nest’s thermoregulatory ability. The results showed that the average core temperature of thatched nests was higher than that of the environment, but remained below values harmful for the fungus. This thermoregulation was brought about by the low thermal diffusivity of the nest thatch built by workers with plant fragments, instead of the readily-available soil particles that have a higher thermal diffusivity. The thatch prevented diurnal nest overheating by the incoming solar radiation, and avoided losses of the accumulated daily heat into the cold air during the night. The adaptive value of thatching behavior in Acromyrmex leaf-cutting ants occurring in the southernmost distribution range is discussed.
Cysteines play important roles in the biochemistry of many proteins. The high reactivity, redox properties, and ability of the free thiol group to coordinate metal ions designate cysteines as the amino acids of choice to form key catalytic components of many enzymes. Also, cysteines readily react with reactive oxygen and nitrogen species to form reversible oxidative thiol modifications. Over the last few years, an increasing number of proteins have been identified that use redox-mediated thiol modifications to modulate their function, activity, or localization. These redox-regulated proteins are central players in numerous important cellular processes. First aim of this study was to discover nitric oxide (NO) sensitive proteins in E. coli, whose redox-mediated functional changes might explain the physiological alterations observed in E. coli cells suffering from NO-stress. To identify E. coli proteins that undergo reversible thiol modifications upon NO-treatment in vivo, I applied a differential thiol trapping technique combined with two-dimensional gel analysis. 10 proteins were found to contain thiol groups sensitive to NO-treatment. Subsequent genetic studies revealed that the oxidative modifications of AceF & IlvC are, in part, responsible for the observed NO-induced growth inhibition. Noteworthy, the majority of identified protein targets turned out to be specifically sensitive towards reactive nitrogen species. This oxidant specificity was tested on one NO-sensitive protein, the small subunit of glutamate synthase. In vivo and in vitro activity studies demonstrated that glutamate synthase rapidly inactivates upon nitric oxide treatment but is resistant towards other oxidative stressors. These results imply that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. The second aim of my study was to identify redox-sensitive proteins in S. cerevisiae and to use their redox state as in vivo read-out to assess the role of oxidative stress during the eukaryotic aging process. I first determined the precise in vivo thiol status of almost 300 yeast proteins located in the cytosol and sub-cellular compartments of yeast cells using a highly quantitative mass spectrometry based thiol trapping technique, called OxICAT. The identified proteins can be clustered in four groups: 1) proteins, whose cysteine residues are oxidation resistant; 2) proteins with structurally or functionally important cysteine modifications 3) proteins with highly oxidation-sensitive active site cysteines, which are partially oxidized in exponentially growing yeast cells due to their exquisite sensitivity towards low amounts of ROS; 4) proteins that are reduced in exponentially growing cells but harbor redox-sensitive cysteine(s) that affect the catalytic function of the protein during oxidative stress. These oxidative stress sensitive proteins were identified by exposure of yeast cells to sublethal concentrations of H2O2 or superoxide. It was shown that the major targets of peroxide- and superoxide-mediated stress in the cell are proteins involved in translation, glycolysis, TCA cycle and amino acid biosynthesis. These targets indicate that cells rapidly redirect the metabolic flux and energy towards the pentose phosphate pathway in an attempt to ensure the production of the reducing equivalent NADPH to counterattack oxidative stress. These results reveal that the quantitative assessment of a protein’s oxidation state is a valuable tool to identify catalytically active and redox-sensitive cysteine residues. The OxICAT technology was then used to precisely determine extent and onset of oxidative stress in chronologically aging S. cerevisiae cells by utilizing the redox status of proteins as physiological read-out. I found that chronological aging yeast cells undergo a global collapse of the cellular redox homeostasis, which precedes cell death. The onset of this collapse appears to correlate with the yeast life span, as caloric restriction increases the life span and delays the redox collapse. These results suggest that maintenance of the redox balance might contribute to the life expanding benefits of regulating the caloric intake of yeast. Clustering analysis of all oxidatively modified proteins in chronological aging yeast revealed a subset of proteins whose oxidative thiol modifications significantly precede the general redox collapse. Oxidation of these early target proteins, which most likely results in a loss of their activity, might contribute to or even cause the observed loss of redox homeostasis (i.e., thioredoxin reductase) in chronologically aging yeast. These studies in aging yeast expand our understanding how changes in redox homeostasis affect the life span of yeast cells and confirm the importance of oxidative thiol modifications as key posttranslational modifications in pro- and eukaryotic organisms.
Cooperation is beneficial for social groups and is exemplified in its most sophisticated form in social insects. In particular, eusocial Hymenoptera, like ants and honey bees, exhibit a level of cooperation only rarely matched by other animals. To assure effective defense of group members, foes need to be recognized reliably. Ants use low-volatile, colony-specific profiles of cuticular hydrocarbons (colony odor) to discriminate colony members (nestmates) from foreign workers (non-nestmates). For colony recognition, it is assumed that multi-component colony odors are compared to a neuronal template, located in a so far unidentified part of the nervous system, where a mismatch results in aggression. Alternatively, a sensory filter in the periphery of the nervous system has been suggested to act as a template, causing specific anosmia to nestmate colony odor due to sensory adaptation and effectively blocking perception of nestmates. Colony odors are not stable, but change over time due to environmental influences. To adjust for this, the recognition system has to be constantly updated (template reformation). In this thesis, I provide evidence that template reformation can be induced artificially, by modifying the sensory experience of carpenter ants (Camponotus floridanus; Chapter 1). The results of the experiments showed that template reformation is a relatively slow process taking several hours and this contradicts the adaptation-based sensory filter hypothesis. This finding is supported by first in-vivo measurements describing the neuronal processes underlying template reformation (Chapter 5). Neurophysiological measurements were impeded at the beginning of this study by the lack of adequate technical means to present colony odors. In a behavioral assay, I showed that tactile interaction is not necessary for colony recognition, although colony odors are of very low volatility (Chapter 2). I developed a novel stimulation technique (dummy-delivered stimulation) and tested its suitability for neurophysiological experiments (Chapter 3). My experiments showed that dummy-delivered stimulation is especially advantageous for presentation of low-volatile odors. Colony odor concentration in headspace was further increased by moderately heating the dummies, and this allowed me to measure neuronal correlates of colony odors in the peripheral and the central nervous system using electroantennography and calcium imaging, respectively (Chapter 4). Nestmate and non-nestmate colony odor elicited strong neuronal responses in olfactory receptor neurons of the antenna and in the functional units of the first olfactory neuropile of the ant brain, the glomeruli of the antennal lobe (AL). My results show that ants are not anosmic to nestmate colony odor and this clearly invalidates the previously suggested sensory filter hypothesis. Advanced two-photon microscopy allowed me to investigate the neuronal representation of colony odors in different neuroanatomical compartments of the AL (Chapter 5). Although neuronal activity was distributed inhomogeneously, I did not find exclusive representation restricted to a single AL compartment. This result indicates that information about colony odors is processed in parallel, using the computational power of the whole AL network. In the AL, the patterns of glomerular activity (spatial activity patterns) were variable, even in response to repeated stimulation with the same colony odor (Chapter 4&5). This finding is surprising, as earlier studies indicated that spatial activity patterns in the AL reflect how an odor is perceived by an animal (odor quality). Under natural conditions, multi-component odors constitute varying and fluctuating stimuli, and most probably animals are generally faced with the problem that these elicit variable neuronal responses. Two-photon microscopy revealed that variability was higher in response to nestmate than to non-nestmate colony odor (Chapter 5), possibly reflecting plasticity of the AL network, which allows template reformation. Due to their high variability, spatial activity patterns in response to different colony odors were not sufficiently distinct to allow attribution of odor qualities like ‘friend’ or ‘foe’. This finding challenges our current notion of how odor quality of complex, multi-component odors is coded. Additional neuronal parameters, e.g. precise timing of neuronal activity, are most likely necessary to allow discrimination. The lower variability of activity patterns elicited by non-nestmate compared to nestmate colony odor might facilitate recognition of non-nestmates at the next level of the olfactory pathway. My research efforts made the colony recognition system accessible for direct neurophysiological investigations. My results show that ants can perceive their own nestmates. The neuronal representation of colony odors is distributed across AL compartments, indicating parallel processing. Surprisingly, the spatial activity patterns in response to colony are highly variable, raising the question how odor quality is coded in this system. The experimental advance presented in this thesis will be useful to gain further insights into how social insects discriminate friends and foes. Furthermore, my work will be beneficial for the research field of insect olfaction as colony recognition in social insects is an excellent model system to study the coding of odor quality and long-term memory mechanisms underlying recognition of complex, multi-component odors.
Background: High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results: We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable overexpression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions: Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.
Malaria ist eine schwerwiegende Krankheit, die jährlich über eine Million Menschen tötet. Die zunehmende Resistenzbildung gegenüber den verwendeten Medikamenten macht die Entwicklung neuer Antimalariamittel dringend notwendig. Daher sind die Vitamin B6 Synthese und der Shikimatweg von besonderem Interesse, da diese beiden Synthesewege nur im Parasiten und nicht im Menschen vorkommen. Unter der Voraussetzung, dass diese essentiell für den Parasiten sind, böten sie ideale Ansatzpunkte zur Entwicklung neuer Antimalariamittel. Voraus gegangene Studien haben gezeigt, dass Plasmodium falciparum in der Lage ist, PLP de novo mittels eines bifunktionalen Enzymkomplex, bestehend aus den Proteinen Pdx1 und Pdx2, zu synthetisieren. Pdx1 stellt dabei die eigentliche Synthase dar, während Pdx2 als Glutaminase-Partner das benötigte Ammoniumion für den heterocyclen Ring bereitstellt. Zusätzlich dazu verfügt der Parasit auch über einen salvage pathway um PLP zu „recyclen“, in dem der Pyridoxalkinase PdxK eine Schlüsselfunktion zufällt. Knockout Studien der pdx1 im Mausmalariasystem P. berghei haben gezeigt, dass PbPdx1 für eine optimale Entwicklung der Blutstadien benötigt wird, nicht jedoch für deren Überleben. Im Rahmen dieser Arbeit habe ich die Effekte eines pbpdxK(-) Knockouts in demselben System untersucht. Es konnte eine monoklonale Knockoutlinie generiert werden, was zeigte, dass PbPdxK nicht essentiell für das Überleben des Parasiten in den Blutstadien ist. Die Entwicklung während des Blutstadiums war von dem pbpdxK(-) Knockout nicht betroffen. Allerdings zeigte sich im Moskitostadium eine drastische Reduktion der Sporozoitenzahl sowohl in den Mitteldärmen als auch in den Speicheldrüsen. Dieses Ergebnis legt nahe, dass PbPdxK essentiell für das Überleben der Sporozoiten ist. Daneben wurde versucht, die Gene pfpdx1, pfpdx2 sowie pfpdxK in P. falciparum 3D7 durch Verwendung der single cross over Strategie auszuschalten. Es konnte jedoch für keines der genannten Konstrukte eine Integration in die jeweiligen Genloci anhand von PCR-Analysen nachgewiesen werden. Ebenso scheiterte der Versuch, durch Rekombination eines komplementären Genabschnitts die Funktion des Gens zu rekonstituieren. Daher bleibt es unklar, ob pfpdx1, pfpdx2 und pfpdxK durch Knockout Strategien auszuschalten sind oder nur für Genmanipulationen nicht zugänglich sind. Die Kultivierung von P. falciparum 3D7 Parasiten in Vitamin B6 depletiertem Medium hatte keinen Effekt auf deren Wachstum. Eine anschließende Analyse der Proteinextrakte zeigte eine erhöhte Expression der PfPdxK, während sich das Expressionslevel der PfPdx1 nicht veränderte. Es scheint, dass der Parasit in der Lage ist Vitamin B6 Mangel durch vermehrte Nutzung des salvage pathways vollständig zu kompensieren. Frühere Arbeiten zeigten, dass der C-Terminus der Pdx1 in die Aktivität des PLP Synthasekomplexes involviert ist. Aus diesem Grund wurden verschiedene C-terminale Deletionsmutanten der PfPdx1 konstruiert und dabei bis zu 30 Aminosäuren entfernt. Diese Analysen ergaben, dass der C-Terminus vier verschiedene Funktionen besitzt: das Assembly der Pdx1 Untereinheiten zum Dodekamer, die Bindung des Pentosesubstrats Ribose 5-Phosphat, die Bildung des Intermediats I320 und schließlich die PLP Synthese. Diese unterschiedlichen Funktionen wurden durch verschiedene Deletionsvarianten identifiziert. Darüber hinaus waren alle Deletionsvarianten in der Lage, die Glutaminase Pdx2 zu aktivieren, was zeigt, dass das Dodekamer nicht Vorraussetzung für die Glutaminaseaktivität ist. Aufgrund der geringen PLP Syntheseaktivität in vitro wurde vermutet, dass der PfPdx1/PfPdx2 Komplex durch einen zusätzlichen Faktor aktiviert wird. Daher wurde versucht, mittels Yeast 2-Hybrid, basierend auf einer PCR-amplifizierten P. falciparum 3D7 cDNA-Bibliothek als bait und PfPdx1 als prey, einen Interaktionspartner zu identifizieren. Mehrere Klone wurden gewonnen, die alle einen Bereich des Mal13P1.540, einem putativen Hsp70 Proteins, enthielten. Jedoch scheiterten alle Versuche, die Protein-Protein-Interaktion mit rekombinant exprimierten Protein zu bestätigen. Ebenso war es nicht möglich, das vollständige Mal13P1.540 rekombinant zu exprimieren sowie dessen Lokalisation in vivo zu bestimmen. Daher bleibt die Interaktion von PfPdx1 und Mal13P1.540 ungeklärt. Neben der Vitamin B6 Biosynthese konnten auch einige Gene des Shikimatweges in Plasmodium identifiziert werden. In P. berghei konnten der C-terminale Teil der 3-Dehydroquinatsynthase (2) sowie die Shikimatkinase (5) und die 5-Enoylpyruvylshikimat 3-Phosphatsynthase (6) in einem open reading frame (ORF) identifiziert werden, der dieselbe genetische Organisation aufweisen wie der Arom-Komplex der Hefen. Mit Hilfe eines Komplementationsassay wurde die Funktionalität dieses ORFs überprüft. Dazu wurden S. cerevisiae BY4741Δaro1, ein Hefestamm ohne funktionalen Arom-Komplex, mit dem Pb2_6_5_ABC Fragment transformiert. Die so transformierten Hefen waren nicht in der Lage, auf Mangelplatten ohne aromatische Aminosäuren zu wachsen, was zeigte, dass das Pb2_6_5_ABC Konstrukt den BY4741Δaro1 Phänotyp nicht komplementieren konnte. Der Versuch, mit Hilfe des Baculovirussytems rekombiant exprimiertes Protein zu erhalten, verlief erfolglos. Ebenso war es nicht möglich, Teile des Proteins für Immunisierungen zu exprimieren. Daher bleibt die Funktionalität des Pb2_6_5_ABC Konstruktes ungeklärt.
Background: In populations of most social insects, gene flow is maintained through mating between reproductive individuals from different colonies in periodic nuptial flights followed by dispersal of the fertilized foundresses. Some ant species, however, form large polygynous supercolonies, in which mating takes place within the maternal nest (intranidal mating) and fertilized queens disperse within or along the boundary of the supercolony, leading to supercolony growth (colony budding). As a consequence, gene flow is largely confined within supercolonies. Over time, such supercolonies may diverge genetically and, thus, also in recognition cues (cuticular hydrocarbons, CHC’s) by a combination of genetic drift and accumulation of colony-specific, neutral mutations. Methodology/Principal Findings: We tested this hypothesis for six supercolonies of the invasive ant Anoplolepis gracilipes in north-east Borneo. Within supercolonies, workers from different nests tolerated each other, were closely related and showed highly similar CHC profiles. Between supercolonies, aggression ranged from tolerance to mortal encounters and was negatively correlated with relatedness and CHC profile similarity. Supercolonies were genetically and chemically distinct, with mutually aggressive supercolony pairs sharing only 33.1%617.5% (mean 6 SD) of their alleles across six microsatellite loci and 73.8%611.6% of the compounds in their CHC profile. Moreover, the proportion of alleles that differed between supercolony pairs was positively correlated to the proportion of qualitatively different CHC compounds. These qualitatively differing CHC compounds were found across various substance classes including alkanes, alkenes and mono-, di- and trimethyl-branched alkanes. Conclusions: We conclude that positive feedback between genetic, chemical and behavioural traits may further enhance supercolony differentiation through genetic drift and neutral evolution, and may drive colonies towards different evolutionary pathways, possibly including speciation.
Protein phosphatases can be classified into at least three major families based on amino acid sequences at their active sites. A newly emerging phosphatase family contains the active site sequence DXDX(T/V), and belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases, a ubiquitous and evolutionarily conserved enzyme family. Although the existence of 58 human HAD enzymes has been predicted by database analysis, our understanding of their biological functions remains rudimentary.By database mining amd phylogenetic analysis of human HAD phosphatases, we have found a marked increase in cell area of spreading cells, as well as accelerated cell spreading onfibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration. a previously unidentified enzyme with homology to Chronophin, a cytoskeletal regulatory HAD phosphatase. We have cloned and characterized this novel enzyme and named it AUM,for actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase. By Northern blot, real-time PCR and Western blot analysis, we show that AUM is broadly expressed in all major human and mouse tissues with highest levels found in testis. Using immunohistochemistry, we can show that AUM is specifically expressed in maturing germ cells and that its expression peaks during spermiogenesis. To characterize the substrate preference of AUM, we have conducted an in vitro phosphatase substrate screen with 720 phosphopeptides derived from human phosphorylation sites. AUM exclusively dephosphorylates phosphotyrosine (pTyr)-containing peptides. Furthermore, only 17 pTyr peptides (~2% of all pTyr peptides investigated) acted as AUM substrates, indicating a high degree of substrate specificity. Putative AUM substrates include proteins involved in cytoskeletal dynamics and tyrosine kinase signaling.In accordance with the phosphopeptide screen, phosphatase overlay assays employing whole-cell extracts of pervanadate-treated HeLa cells show that AUM dephosphorylates only a limited number of tyrosyl-phosphorylated proteins.The role of AUM for cellular signaling was investigated in response to epidermal growth factor (EGF) stimulation in a spermatogonial cell line (GC-1 spg). The overexpression of AUM reduces, whereas the RNAi-mediated depletion of endogenous AUM increases EGF inducedtyrosine phosphorylation, including changes in the phosphorylation of the EGF receptor itself. Interestingly, in vitro kinase/phosphatase assays with purified Src and AUM indicate that AUM can activate Src, which in turn phosphorylates and inactivates AUM. Although it is at present unclear how Src and AUM regulate each other, our initial findings suggests that AUM enhances Src kinase activity independently of its phosphatase activity, whereas Src diminishes AUM phosphatase activity in a kinase dependent manner. On a cellular level, AUM-depleted cells are characterized by altered actin cytoskeletal dynamics and adhesion, as indicated by stabilized actin filaments, enlarged focal adhesions,a marked increase in cell area of spreading cells, as well as accelerated cell spreading on fibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration.
In dieser Arbeit sollte die Funktion der Ribosomalen S6 Kinase 2 (RSK2) auf neuronaler Ebene untersucht werden. Dahingehend gab es, z.B. auf Grund der Phänotypen von Fliegen und Mäusen mit Mutationen im entsprechenden Gen oder von Patienten mit Coffin-Lowry-Syndrom (CLS) nur Vermutungen. Es bestand letztlich die Hoffnung, einen Beitrag zur Aufklärung der Pathophysiologie des CLS zu leisten. Es stellte sich auf Grund von Experimenten sowohl in vivo als auch in vitro in verschiedenen Modellsystemen in dieser Arbeit heraus, daß RSK2 einen negativen Einfluß auf das Neuriten- und Synapsenwachstum hat. In kultivierten Motoneuronen führte der KO von RSK2 zu längeren Axonen und die Überexpression eines konstitutiv aktiven RSK2-Konstrukts zu kürzeren Axonen. In PC12-Zellen führte die Expression von konstitutiv aktiven RSK2 Konstrukten zur Verkürzung der Neuriten und die Expression eines Kinase-inaktiven RSK2 Konstrukts zu längeren Neuriten. In vivo war die neuromuskuläre Synapse bei RSK2-KO Mäusen vergrößert und hatte bei Drosophila rsk Mutanten mehr Boutons. Das RSK2-Protein ist in Motoneuronen der Maus und in überexprimierter Form in den Boutons der neuromuskulären Synapse bei Drosophila nachweisbar. Damit wurde zum ersten Mal die Funktion von RSK2 auf neuronaler Ebene beschrieben. Bezüglich des Mechanismus, wie RSK2 das Nervenwachstum beeinflußt gab es deutliche Hinweise, die dafür sprechen, daß RSK2 dies über eine in der Literatur schon häufiger beschriebene Hemmung der MAPK ERK1/2 erreicht. Für diese Hypothese spricht die Tatsache, daß die ERK-Phosphorylierung in murinen Motoneuronen und im Rückenmark embryonaler Mäuse der RSK2-Mutante erhöht ist und der Axonwachstumsdefekt durch eine Hemmung von MEK/ERK behoben werden kann. Auch ist die ERK-Phosphorylierung an der murinen Muskel-Endplatte in der Mutante erhöht. Zudem zeigen genetische Epistasis-Experimente in Drosophila, daß RSK die Bouton-Zahl über ERK/RL hemmt. RSK scheint also in Drosophila von der Funktion her der RSK2-Isoform in Wirbeltieren sehr ähnlich zu sein. Ein weiteres wichtiges Ergebnis ist die Beobachtung, daß RSK2 bei Motoneuronen keinen wesentlichen Einfluß auf das Überleben der Zellen in Gegenwart neurotropher Faktoren hat. Möglicherweise spielen hier redundante Funktionen der RSK Familienmitglieder eine Rolle. Ein bislang unerklärter Befund ist die reduzierte Frequenz spontaner Depolarisationen bzw. damit einhergehender Ca2+ Einströme bei RSK2-KO Motoneuronen in Zellkultur. Die Häufigkeit und Dichte von Ca2+-Kanälen und aktive Zonen Proteinen war in Motoneuronen nicht von der Anwesenheit des RSK2-Proteins abhängig. Im Hippocampus konnte außerdem das RSK2-Protein präsynaptisch in den Moosfaser-Boutons der CA3 Region nachgewiesen werden. Es befindet sich auch in den Pyramidenzellen, aber nicht in den Pyramidenzell-Dendriten in CA3. Bezüglich der Bedeutung dieser Befunde für die Aufklärung der Pathologie des CLS ist zu folgern, daß der neuro-psychologische Phänotyp bei CLS Patienten wahrscheinlich nicht durch reduziertes Überleben von Neuronen, sondern eher durch disinhibiertes Axonwachstum oder Synapsenwachstum bedingt ist. Dies kann grob sowohl für die peripheren als auch die zentralen Defekte gelten, denn die Synapsen im ZNS und am Muskel sind in ihrer molekularen Ausstattung z.B. im Bereich der Vesikel, der aktiven Zonen oder der Transmitterausschüttung sehr ähnlich. Weiterhin könnte eine veränderte synaptische Plastizität u.a. an der Moosfaser-Pyramidenzell-Synapse in der CA3 Region des Hippocampus eine Rolle bei den kognitiven und mnestischen Einschränkungen der Patienten spielen. Die Entdeckung, daß aktiviertes ERK bei den beobachteten Effekten eine Rolle spielt kann für die Entwicklung von Therapiestrategien eine wertvolle Erkenntnis sein.
The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. Results: We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. Conclusions: Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.
The phylum Tardigrada consists of about 1000 described species to date. The animals live in habitats within marine, freshwater and terrestrial ecosystems allover the world. Tardigrades are polyextremophiles. They are capable to resist extreme temperature, pressure or radiation. In the event of desiccation, tardigrades enter a so-called tun stage. The reason for their great tolerance capabilities against extreme environmental conditions is not discovered yet. Our Funcrypta project aims at finding answers to the question what mechanisms underlie these adaption capabilities particularly with regard to the species Milnesium tardigradum. The first part of this thesis describes the establishment of expressed sequence tags (ESTs) libraries for different stages of M. tardigradum. From proteomics data we bioinformatically identified 144 proteins with a known function and additionally 36 proteins which seemed to be specific for M. tardigradum. The generation of a comprehensive web-based database allows us to merge the proteome and transcriptome data. Therefore we created an annotation pipeline for the functional annotation of the protein and nucleotide sequences. Additionally, we clustered the obtained proteome dataset and identified some tardigrade-specific proteins (TSPs) which did not show homology to known proteins. Moreover, we examined the heat shock proteins of M. tardigradum and their different expression levels depending on the actual state of the animals. In further bioinformatical analyses of the whole data set, we discovered promising proteins and pathways which are described to be correlated with the stress tolerance, e.g. late embryogenesis abundant (LEA) proteins. Besides, we compared the tardigrades with nematodes, rotifers, yeast and man to identify shared and tardigrade specific stress pathways. An analysis of the 50 and 30 untranslated regions (UTRs) demonstrates a strong usage of stabilising motifs like the 15-lipoxygenase differentiation control element (15-LOX-DICE) but also reveals a lack of other common UTR motifs normally used, e.g. AU rich elements. The second part of this thesis focuses on the relatedness between several cryptic species within the tardigrade genus Paramacrobiotus. Therefore for the first time, we used the sequence-structure information of the internal transcribed spacer 2 (ITS2) as a phylogenetic marker in tardigrades. This allowed the description of three new species which were indistinguishable using morphological characters or common molecular markers like the 18S ribosomal ribonucleic acid (rRNA) or the Cytochrome c oxidase subunit I (COI). In a large in silico simulation study we also succeeded to show the benefit for the phylogenetic tree reconstruction by adding structure information to the ITS2 sequence. Next to the genus Paramacrobiotus we used the ITS2 to corroborate a monophyletic DO-group (Sphaeropleales) within the Chlorophyceae. Additionally we redesigned another comprehensive database—the ITS2 database resulting in a doubled number of sequence-structure pairs of the ITS2. In conclusion, this thesis shows the first insights (6 first author publications and 4 coauthor publications) into the reasons for the enormous adaption capabilities of tardigrades and offers a solution to the debate on the phylogenetic relatedness within the tardigrade genus Paramacrobiotus.
Vaccinia virus plays an important role in human medicine and molecular biology ever since the 18th century after E. Jenner discovered its value as a vaccination virus against smallpox. After the successful eradication of smallpox, vaccinia virus, apart from its use as a vaccine carrier, is today mainly used as a viral vector in molecular biology and increasingly in cancer therapy. The capability to specifically target and destroy cancer cells makes it a perfect agent for oncolytic virotherapy. Furthermore, the virus can easily be modified by inserting genes encoding therapeutic or diagnostic proteins to be expressed within the tumor. The emphasis in this study was the diagnosis of tumors using different vaccinia virus strains. Viruses with metal-accumulating capabilities for tumor detection via MRI technology were generated and tested for their usefulness in cell culture and in vivo. The virus strains GLV-1h131, GLV-1h132, and GLV-1h133 carry the gene encoding the two subunits of the iron storage protein ferritin under the control of three different promoters. GLV-1h110, GLV-1h111, and GLV-1h112 encode the bacterial iron storage protein bacterioferritin, whereas GLV-1h113 encodes the codon-optimized version of bacterioferritin for more efficient expression in human cells. GLV-1h22 contains the transferrin receptor gene, which plays an important role in iron uptake, and GLV-1h114 and GLV-1h115 contain the murine transferrin receptor gene. For possibly better iron uptake the virus strains GLV-1h154, GLV-1h155, GLV-1h156, and GLV-1h157 were generated, each with a version of a ferritin gene and a transferrin receptor gene. GLV-1h154 carries the genes that encode bacterioferritin and human transferrin receptor, GLV-1h155 the human ferritin H-chain gene and the human transferrin receptor gene. GLV-1h156 and GLV-1h157 infected cells both express the mouse transferrin receptor and bacterioferritin or human ferritin H-chain, respectively. The virus strains GLV-1h186 and GLV-1h187 were generated to contain a mutated form of the ferritin light chain, which was shown to result in iron overload and the wildtype light chain gene, respectively. The gene encoding the Divalent Metal Transporter 1, which is a major protein in the uptake of iron, was inserted in the virus strain GLV-1h102. The virus strain GLV-1h184 contains the magA gene of the magnetotactic bacterium Magnetospirillum magnetotacticum, which produces magnetic nanoparticles for orientation in the earth’s magnetic field. Initially the infection and replication capability of all the virus strains were analyzed and compared to that of the parental virus strain GLV-1h68, revealing that all the viruses were able to infect cells of the human cancer cell lines A549 and GI-101A. All constructs exhibited a course of infection comparable to that of GLV-1h68. Next, to investigate the expression of the foreign proteins in GI-101A and A549 cells with protein analytical methods, SDS-gelelectrophoresis, Western blots and ELISAs were performed. The proteins, which were expressed under the control of the strong promoters, could be detected using these methods. To be able to successfully detect the protein expression of MagA and DMT1, which were expressed under the control of the weak promoter, the more sensitive method RT-PCR was used to at least confirm the transcription of the inserted genes. The determination of the iron content in infected GI-101A and A549 cells showed that infection with all used virus strains led to iron accumulation in comparison to uninfected cells, even infection with the parental virus strain GLV-1h68. The synthetic phytochelatin EC20 was also shown to enhance the accumulation of different heavy metals in bacterial cultures. In vivo experiments with A549 tumor-bearing athymic nude mice revealed that 24 days post infection virus particles were found mainly in the tumor. The virus-mediated expression of recombinant proteins in the tumors was detected successfully by Western blot. Iron accumulation in tumor lysates was investigated by using the ferrozine assay and led to the result that GLV-1h68-infected tumors had the highest iron content. Histological stainings confirmed the finding that iron accumulation was not a direct result of the insertion of genes encoding iron-accumulating proteins in the virus genome. Furthermore virus-injected tumorous mice were analyzed using MRI technology. Two different measurements were performed, the first scan being done with a seven Tesla small animal scanner seven days post infection whereas the second scan was performed using a three Tesla human scanner 21 days after virus injection. Tumors of mice injected with the virus strains GLV-1h113 and GLV-1h184 were shown to exhibit shortened T2 and T2* relaxation times, which indicates enhanced iron accumulation. In conclusion, the experiments in this study suggest that the bacterioferritin-encoding virus strain GLV-1h113 and the magA-encoding virus strain GLV-1h184 are promising candidates to be used for cancer imaging after further analyzation and optimization.
Background: LINC complexes are nuclear envelope bridging protein structures formed by interaction of SUN and KASH proteins. They physically connect the nucleus with the peripheral cytoskeleton and are critically involved in a variety of dynamic processes, such as nuclear anchorage, movement and positioning and meiotic chromosome dynamics. Moreover, they are shown to be essential for maintaining nuclear shape. Findings: Based on detailed expression analysis and biochemical approaches, we show here that during mouse sperm development, a terminal cell differentiation process characterized by profound morphogenic restructuring, two novel distinctive LINC complexes are established. They consist either of spermiogenesis-specific Sun3 and Nesprin1 or Sun1g, a novel non-nuclear Sun1 isoform, and Nesprin3. We could find that these two LINC complexes specifically polarize to opposite spermatid poles likely linking to sperm-specific cytoskeletal structures. Although, as shown in co-transfection / immunoprecipitation experiments, SUN proteins appear to arbitrarily interact with various KASH partners, our study demonstrates that they actually are able to confine their binding to form distinct LINC complexes. Conclusions: Formation of the mammalian sperm head involves assembly and different polarization of two novel spermiogenesis-specific LINC complexes. Together, our findings suggest that theses LINC complexes connect the differentiating spermatid nucleus to surrounding cytoskeletal structures to enable its well-directed shaping and elongation, which in turn is a critical parameter for male fertility.
Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens.
Termites are the most important soil ecosystem engineers of semi‐arid and arid habitats. They enhance decomposition processes as well as the subsequent mineralisation of nutrients by bacteria and fungi. Through their construction of galleries, nests and mounds, they promote soil turnover and influence the distribution of nutrients and also alter texture and hydrological properties of soils, thereby affecting the heterogeneity of their ecosystem. The main aim of the present thesis was to define the impact of termites on ecosys‐tem functioning in a semi‐arid ecosystem. In a baseline study, I assessed the diversity of termite taxa in relation to the amount of precipitation, the vegetation patterns and the land use systems at several sites in Namibia. Subsequently, I focussed on a species that is highly abundant in many African savannas, the fungus growing and mound building species Macro‐termes michaelseni (Sjöstedt, 1914). I asked how this species influences the spatial hetero‐geneity of soil and vegetation patterns. From repeated samplings at 13 sites in Namibia, I obtained 17 termite taxa of 15 genera. While the type of land use seems to have a minor effect on the termite fauna, the mean annual precipitation explained 96% and the Simpson index of vascular plant diversity 81% of the variation in taxa diversity. The number of termite taxa increased with both of these explanation variables. In contrast to former studies on Macrotermes mounds in several regions of Africa that I reviewed, soil analyses from M. michaelseni mounds in the central Namibian savanna revealed that they contain much higher nitrogen contents when compared to their parent material. Further analyses revealed that nitrate forms a major component of the nitrogen content in termite mounds. As nitrate solves easily in water, evaporation processes are most probably responsible for the transport of solved nitrates to the mound surface and their accumulation there. The analysed mounds in central Namibia contained higher sand propor‐tions compared to the mounds of the former studies. Through the higher percentage of coarse and middle sized pores, water moves more easily in sandy soils compared to more clayey soils. In consequence, evaporation‐driven nitrate accumulation can occur in the studied mounds at high rates. Hochgerechnet auf den Gesamtumfang der Hügel bedeckte das pro Jahr von einem bewohnten Hügel erodierte Material theoretisch einen 1 m breiten Kreisring um den Schwemmkegel des Hügels 2,4 mm hoch. Der entsprechende Wert für unbewohnte Hügel betrug 1,0 mm. To assess the amount of soil that erodes from termite mounds, I fastened four strong, 65 cm wide plastic bags at 14 mounds each and collected the soil that eroded during five rainfall events. Projected to the total mound circumference, the amount of soil eroded covers theoretically a 1 m wide circular ring around the pediment of an inhabited mound up to a height of 2.4 mm per year. For uninhabited mounds, the height of this soil layer would be 1.0 mm. Per hectare, roughly 245 kg eroded per year from the mounds. However, as the erosion rate depends on several factors such as rainfall intensity, soil texture and point of time within the rainy season, this is only a vague estimate. In order to determine up to which distance the soil erosion from the mounds still influences the chemical characteristics of the adjacent topsoil, I took samples from depth of 0–10 cm at 1, 5 and 25 m distances, respectively, from four different mounds and from the mounds themselves. The non‐metric multidimensional scaling of the soil properties showed strong differences between mound and off‐mound samples. Soil characteristics within the samples from the mounds did not differ largely. Similarly, I found no strong differences between the samples taken from the different distances from the mound. From these results I conclude that through the construction of foraging galleries and sheetings (soil constructions with which some termite species cover their food items), the soil eroding from termite mounds is quickly mixed with deeper soil layers. In consequence, mound material does not accumulate in the mound’s vicinity. In order to reveal how plant growth is influenced by termite mound material, we assessed the number of grass and herb individuals as well as the biomass of plants growing in situ on the base of mounds compared to adjacent sites. While the numbers of both grass and herb individuals were significantly lower compared to adjacent sites, the total biomass of plants growing on the base of mounds was significantly higher. Reverse results were obtained by pot experiments with radish (Raphanus sativus subsp. sativus) and sorghum (Sorghum sp.) growth. Both species grew significantly weaker on mound soil compared to adjacent soil. The contradictory results concerning the biomass of in situ and pot experi‐ments are most probably caused by the disturbance of the original soil structure during the potting process. The material was subsequently compacted through watering the plants. In contrast, Macrotermes mounds are pervaded by many macropores which seem to be essential for the plant roots to penetrate the soil. In the last part of this thesis, I posed the question how mounds of M. michaelseni are distributed and what factors might be responsible for this pattern. Former studies showed that mound size is correlated with the size of its inhabiting colony. With several multi‐scale analyses, I revealed that larger inhabited mounds were regularly distributed. Additionally, mounds which were closer together tended to be smaller than on average. This indicates that intraspecific competition controls the distribution and size of colonies and their mounds. Former studies concerning Odontotermes mounds substantiated that they are local hotspots of primary productivity and animal abundance. Based on these findings, simulations revealed that a regular distribution of these mounds leads to a greater ecosystem‐wide productivity compared to a random arrangement. As in the present study, plant biomass was higher at the mounds compared to off‐mound sites, this might hold true for M. michaelseni mounds. From the results of this thesis, I draw the conclusion that through their mound building activities, M. michaelseni strongly influences the distribution patterns of soil nutrients within the central Namibian savanna. These termites create sharp contrasts in nutrient levels and vegetation patterns between mound soils and off‐mound soils and enhance the heterogeneity of their habitats. Former studies revealed that habitat hetero‐geneity is important in generating species diversity and species richness in turn is correlated positively with biomass production and positively affects ecosystem services. In conclusion, the present thesis underlines the importance of M. michaelseni for ecosystem functioning of the central Namibian savanna.
In dieser Doktorarbeit habe ich die Regulation der Expression des zuckerbelohnten Verhaltens durch den Fütterungszustand bei Drosophila melanogaster untersucht. Die Fliegen können während einer Trainingsphase mit Hilfe einer Zuckerbelohnung auf einen bestimmten Duft konditioniert werden. Nach dem Training können die Fliegen dann auf das olfaktorische Gedächtnis getestet werden. Die Bereitschaft das zuckerkonditionierte Gedächtnis im Test zu zeigen wird vom Fütterungszustand kontrolliert, wie ich in Übereinstimmung mit den Ergebnissen früherer Arbeiten demonstrierte (Tempel et al. 1983; Gruber 2006; Krashes et al. 2008). Nur nicht gefütterte Fliegen exprimieren das Gedächtnis, während Fütterungen bis kurz vor dem Test eine reversibel supprimierende Wirkung haben. Einen ähnlichen regulatorischen Einfluss übt der Futterentzug auch auf die Expression anderer futterbezogener Verhaltensweisen, wie z.B. die naive Zuckerpräferenz, aus. Nachdem ich den drastischen Einfluss des Fütterungszustands auf die Ausprägung des zuckerkonditionierten Verhaltens gezeigt bzw. bestätigt hatte, habe ich nach verhaltensregulierenden Faktoren gesucht, die bei einer Fütterung die Gedächtnisexpression unterdrücken. Als mögliche Kandidaten untersuchte ich Parameter, die zum Teil bereits bei verschiedenen futterbezogenen Verhaltensweisen unterschiedlicher Tierarten als „Sättigungssignale“ identifiziert worden waren (Marty et al. 2007; Powley and Phillips 2004; Havel 2001; Bernays and Chapman 1974; Simpson and Bernays 1983; Gelperin 1971a). Dabei stellte sich heraus, dass weder die „ernährende“ Eigenschaft des Futters, noch ein durch Futteraufnahme bedingter Anstieg der internen Glukosekonzentration für die Suppression des zuckerkonditionierten Gedächtnisses notwendig sind. Die Unterdrückung der Gedächtnisexpression kann auch nicht durch Unterschiede in den aufgenommenen Futtermengen, die als verhaltensinhibitorische Dehnungssignale des Verdauungstrakts wirken könnten, oder mit der Stärke des süßen Geschmacks erklärt werden. Die Suppression des zuckerbelohnten Verhaltens folgte den Konzentrationen der gefütterten Substanzen und war unabhängig von deren chemischen Spezifität. Deshalb wird die Osmolarität des aufgenommenen Futters als ein entscheidender Faktor für die Unterdrückung der zuckerkonditionierten Gedächtnisexpression angenommen. Weil nur inkorporierte Substanzen einen Unterdrückungseffekt hatten, wird ein osmolaritätsdetektierender Mechanismus im Körper 67 postuliert, wahrscheinlich im Verdauungstrakt und/oder der Hämolymphe. Die Hämolymphosmolarität ist als „Sättigungssignal“ bei einigen wirbellosen Tieren bereits nachgewiesen worden (Bernays and Chapman 1974; Simpson and Raubenheimer 1993; Gelperin 1971a; Phifer and Prior 1985). Deshalb habe ich mit Hilfe genetischer Methoden und ohne die Fliegen zu füttern, versucht über einen künstlich induzierten Anstieg der Trehaloseund Lipidkonzentrationen die Osmolarität der Hämolymphe in Drosophila zu erhöhen. Eine solche konzentrationserhöhende Wirkung für Lipide und die Trehalose, dem Hauptblutzucker der Insekten, ist bereits für das adipokinetische Hormon (AKH), das von Zellen der Corpora cardiaca exprimiert wird, nachgewiesen worden (Kim and Rulifson 2004; Lee and Park 2004; Isabel et al. 2005). Es stellte sich heraus, dass die künstliche Stimulierung AKH-produzierender Neurone das zuckerkonditionierten Verhalten temporär, reversible und selektiv unterdrückt. Gleiche Behandlungen hatten keinen Effekt auf ein aversiv konditioniertes olfaktorisches Gedächtnis oder ein naives Zuckerpräferenzverhalten. Wie aus dieser Arbeit hervorgeht, stellt wahrscheinlich die Osmolarität des Verdauungstrakts und der Hämolymphe oder nur der Hämolymphe ein physiologisches Korrelat zum Fütterungszustand dar und wirkt als unterdrückendes Signal. Dass Fütterungen das zuckerkonditionierte Verhalten und die Zuckerpräferenz supprimieren, die künstliche Stimulation AKH-produzierender Zellen aber selektiv nur die zuckerbelohnte Gedächtnisexpression unterdrückt, deutet auf mindestens zwei unterschiedliche „Sättigungssignalwege“ hin. Außerdem macht es deutlich wie uneinheitlich futterbezogene Verhaltensweisen, wie das zuckerbelohnte Verhalten und die naive Zuckerpräferenz, reguliert werden.
Schlagwörter: Salmonella , Salmonella enterica , Salmonella typhimurium , Salmonellose , Escherichia coli , Shigella , Infektion , Bakterielle Infektion , Zellkultur , HeLa-Zelle , Apoptosis , Metabolismus , Stoffwechsel , Glucose , Glucosetransport , Glucosestoffwechsel , Katabolismus , Kohlenstoff , Kohlenstoffbedarf , Kohlenstoffhaushalt , Kohlenstoffstoffwechsel , Kohlenstoff-13 , Kohlenstoffisotop Salmonella Typhimurium und enteroinvasive E. coli (EIEC) sind fakultativ intrazelluläre Bakterien aus der Familie der Enterobacteriaceae. Während erstere sich nach der Internalisierung durch eukaryotische Zellen normalerweise in einem spezialisierten Phagosom, der Salmonella-enthaltenden Vakuole (SCV), vermehren, replizieren EIEC im Zytoplasma der Wirtszellen. In der vorliegenden Arbeit wurde zunächst durch Mikroinjektion die Fähigkeit von S. Typhimurium 14028s untersucht, ebenfalls im Zytoplasma von Caco-2-Zellen replizieren zu können. Dabei wurde festgestellt, daß ein früher als S. Typhimurium 14028s WT bezeichneter Stamm eine Insertion eines Desoxythymidins an Position 76 des offenen Leserasters von rfbP trägt, einem Gen, dessen Protein an der LPS-Synthese beteiligt ist. Weiterhin synthetisierte dieser Stamm ein rauhes LPS. Aufgrund von Agglutination konnte der Rauh-Stamm nur mit geringem Erfolg mikroinjiziert werden. Hingegen lag 5 h nach der Mikroinjektion einer nicht invasiven Mutante von Salmonella mit vollständigem LPS der Anteil an Caco-2-Zellen, die mehr als 32 Bakterien enthielten, bei etwa 30 %. Der Anteil war 2-3 mal höher als bei früheren Mikroinjektionen in HeLa-Zellen. Daher wurde das Verhalten von HeLa-Zellen nach einer Infektion durch S. Typhimurium ΔsifA - einer Mutante, die aus der SCV ins Zytoplasma entkommt - untersucht. Dabei wurde festgestellt, daß die sifA-Mutante 10 h nach der Infektion die Aktivität der Caspasen 9 und 3 in HeLa-Zellen, aber nicht in Caco-2-Zellen induziert. In weiteren Versuchen wurde die Bedeutung von Glukose, Glukose-6-phosphat und Mannose als Kohlenstoffquellen für die extra- und intrazelluläre Replikation zweier Isolate enteroinvasiver E. coli und eines S. Typhimurium Stammes analysiert. Zu diesem Zweck wurden zunächst definierte Mutanten in den beiden wichtigsten Phosphoenolpyruvat-abhängigen Phosphotransferasesystemen (PTS) für die Aufnahme von Glukose und Mannose, ptsG und manXYZ, sowie im Antiporter für die Aufnahme von Glukose-6-phosphat, uhpT, konstruiert. Bei Wachstum im Minimalmedium mit Glukose als einziger C-Quelle waren die Generationszeiten aller ΔptsG- und ΔptsG, manXYZ-Mutanten im Vergleich zu den Wildstämmen deutlich verlängert. Ebenso wuchsen ΔmanXYZ-Mutanten bzw. ΔuhpT-Mutanten deutlich langsamer auf Mannose bzw. Glukose-6-phosphat. Jedoch ergaben sich hierbei Stamm-spezifische Unterschiede. So erreichte EIEC 4608-58 ΔuhpT in der stationären Phase eine ähnliche Zelldichte wie der Wildstamm in Gegenwart von Glukose-6-phosphat und eine ΔptsG, manXYZ-Mutante von S. Typhimurium 14028s konnte immer noch effizient mit Glukose wachsen. Infektionsversuche mit Caco-2-Zellen zeigten weiterhin, daß die Deletion von ptsG zu einer signifikanten Erhöhung der Adhärenz und Invasivität von EIEC 4608-58 führt, während sich die intrazellulären Generationszeiten aller hier untersuchten Mutanten kaum veränderten. Selbst die ΔptsG, manXYZ, uhpT-Dreifachmutanten der drei hier verwendeten Enterobakterien und die ΔptsG, manXYZ, glk-Mutante von S. Typhimurium 14028s konnten immer noch in Caco-2-Zellen replizieren, wenn auch mit Stamm-spezifisch verringerten Geschwindigkeiten. 13C-Markierungsexperimente mit [U-13C6]-Glukose als Substrat ergaben jedoch, daß in der Tat alle hier untersuchten enterobakteriellen Wildstämme Glukose während der Replikation in Caco-2-Zellen unter Zellkulturbedingungen verwerten. Glukose-6-phosphat, Glukonat oder Fettsäuren konnten dagegen als wichtigste Kohlenstoffquellen für das intrazelluläre Wachstum ausgeschlossen werden. EIEC 4608-58 metabolisierte Glukose jedoch weniger effizient als EIEC HN280 und schien zudem noch zusätzlich C3-Substrate aus der Wirtszelle aufzunehmen. Das Markierungsmuster zeigte einen Stamm-spezifischen Kohlenstofffluß durch Glykolyse und/oder Entner-Doudoroff-Weg, Pentosephosphatzyklus, Citratzyklus und den anaplerotischen Reaktionen zwischen PEP und Oxalacetat. Mutanten mit Deletionen in ptsG und manXYZ konnten auf alternative C3-Substrate wechseln und glichen dies durch eine erhöhte Aufnahme von Aminosäuren aus den Wirtszellen aus.