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Staphylococcus aureus is one of the commonly encountered bacteria of the human microbiome. Although mostly a seemingly harmless commensal microbe, S. aureus can act as an invasive pathogen with seriously devastating effects on its host’s health and wellbeing. A wide range of infections caused by this bacterium has been reported to affect diverse parts of the human body, including the skin, soft tissues and bones, as well as important organs like the heart, kidneys and lungs. Particularly, S. aureus is infamous for being a major causative agent of respiratory tract infections that may escalate up to necrotizing pneumonia. Due to its clinical relevance, this pathogen has been intensively studied for many years. Nonetheless, further research in this field is still needed, because of the high capacity of S. aureus to evolve drug resistance, its high genomic plasticity and adaptability and, not in the last place, the plethora of niches within the human body where it can thrive and survive. In this regard, there are still many uncertainties concerning the specific adaptations carried out by S. aureus during colonization and infection of the human body, the transition between both stages, and upon the invasion of different types of host cells. To shed more light on some of these adaptations, the research described in this thesis has employed in vitro models of infection that mimic particular conditions during the infectious process with special focus on the lung epithelium. The adaptations displayed by S. aureus were monitored using advanced proteomics. Furthermore, the analyses documented in this thesis included S. aureus strains with diverse backgrounds and epidemiology to take into account the genetic diversity encountered in this species.
Die Rolle von Sequenzvarianten im Cathepsin L als Risikofaktoren der chronischen Pankreatitis
(2016)
Die chronische Pankreatitis ist meist eine wiederkehrende Entzündung der Bauchspeicheldrüse mit intrapankreatischer Aktivierung der Verdauungsenzyme. Mutationen im pankreatischen sekretorischen Trypsin Inhibitor (PSTI = SPINK1) finden sich häufig bei Patienten mit chronischer Pankreatitis. Die häufigste SPINK1 Mutation p.N34S ist aber auch bei 1-2 % der gesunden Normalbevölkerung nachweisbar. Experimentelle Daten belegen, dass das p.N34S mutierte Protein durch CTSL schneller degradiert wird. In dieser Arbeit wird untersucht, ob Mutationen im CTSL Gen das Risiko von p.N34S+ Individuen erhöhen, an einer Entzündung der Bauchspeicheldrüse zu erkranken. Hierfür werden mittels TaqMan-Analyse die Probanden der SHIP-Studie (SHIP-0 n=4308, im Weiteren Kontrollen) und Individuen mit chronischer ideopathischer Pankreatitis (n=2299, im Weiteren Patienten) für N34S-Mutation in SPINK1 genotypisiert. Hiernach wird bei den p.N34S+ Individuen das CTSL mittels bidirektionaler Sanger-Sequenzierung untersucht. Wir finden eine nichtsynonyme Mutation (c.5A>C, p.N2T), eine Mutation mit Verschiebung des Leserasters (c.98delA, p.K33fs) und einen SNP c.-461C>A (rs3118869), für den eine Modifikation der Expression in anderem Zusammenhang publiziert ist. Die p.N2T Mutation wird bei einem Patienten und zwei Kontrollen nachgewiesen, die Verschiebung des Leserasters nur bei einem Patienten. Der SNP kann bei 42 Patienten und 46 Kontrollen nachgewiesen werden. Für keine der gefundenen Varianten zeigt sich ein signifikanter Unterschied in der Verteilung. Auch in der Haplotypenanalyse kann kein signifikanter Verteilungsunterschied gezeigt werden. Ein Einfluss von CTSL Mutationen auf das Risiko an einer Pankreatitis zu erkranken, erscheint für Träger der p.N34S Mutation im SPINK1 Gen möglich, kann an dem untersuchten Kollektiv aber nicht bewiesen werden.
Myxomycetes are fungus-like protists of the supergroup Amoebozoa found to be abundant in all terrestrial ecosystems. Mainly based on its macroscopically visible fruit bodies, our knowledge on ecology and diversity of myxomycetes is better than for most other protistean groups, but there is still a lacking knowledge about global diversity patterns since tropical regions, especially the old world tropics, are still understudied. In this thesis a combination of classical ecological analyses and modern molecular methods were used to expand the current knowledge on myxomycete diversity and biogeography in the Paleotropics. A number of surveys in the Philippine archipelago are conducted to provide and to add information about the distribution of myxomycetes in the Southeast Asian region. A combination of field collecting and ca. 2500 moist chamber cultures from four unexplored areas in the Philippines, namely, the Bicol Peninsula (746 records, 57 taxa), Puerto Galera (926 records, 42 taxa), Quezon National Park (205 records, 35 taxa), and Negros Province (193 records, 28 taxa), now brings the number of species recorded for Philippines to 150; with one record, Stemonaria fuscoides, noted as new for the Asian Paleotropics. Collecting localities that have more diverse plant communities showed as well higher species diversity of myxomycetes. In congruence with studies from the Neotropical forests, it seems also that anthropogenic disturbances and the type of forest structure affect the occurrence of myxomycetes for the Philippines. Another survey carried out in another paleotropical region, the highlands of Ethiopia, revealed a total of 151 records, with all 39 species found as new for the country. Three records of Diderma cf. miniatum with a strong bright red peridium and one record of Didymium cf. flexuosum with a conspicuous broad reticulation in the spore ornamentation were described and barcoded, since both may represent morphospecies new to science. A number of rarely recorded species, like Didymium saturnus, Metatrichia floripara, Perichaena areolata, and Physarina echinospora showed that resembling to its unique flora, the east African mountain ranges harbor a diverse and distinctive myxomycete assemblage. One incentive of this study was to compile a solid large dataset for the Paleotropical region that is comparable to data obtained from comprehensive studies performed in the Neotropical areas a decade ago. A total of eight surveys (with four comprehensive regional surveys, two from lowland and two from highland, for each region, the Neo- and the Paleotropics) were used, to compare the myxomycete assemblages of both regions. Each survey comes from a region with fairly homogenous vegetation, and includes specimens from both field and moist chamber cultures component. A statistical analysis of species accumulation curves revealed that only between 70 and 95% of all species to be expected have been found. Even for >1000 specimens per survey these figures seem hardly to increase with increasing collection effort, since a high proportion of species is always represented by a single or a few records only. Both ordination and cluster analysis suggests that geographical separation explains differences in species composition of the myxomycete assemblages much better than elevational differences. 5 The molecular component of this thesis is a phylogeographic study of the widely distributed tropical myxomycete Hemitrichia serpula. It is a morphologically distinct species with golden-yellow fructifications forming a reticulum. However, subtle variation in spore ornamentation points to cryptic speciation within this myxomycete. Using two independent molecular markers, 135 partial sequences of the small subunit (SSU) rRNA (a nuclear but extrachromosomal gene) and 30 partial sequences of the elongation factor 1 alpha gene (EF1A) (a nuclear gene), a study of 135 Hemitrichia serpula specimens collected worldwide revealed the existence of four clades that are likely to represent reproductively isolated biospecies, since each clade shows a unique combination of SSU and EF1A genotypes. A Mantel test with the partial SSU sequences indicated geographical differentiation, giving a correlation coefficient of 0.467 between the pairwise computed geographic and genetic distances, compared with the 95% confidence interval from 999 permutations (-0.013 to 0.021). Biogeographical analysis of the 40 SSU ribotypes showed clear intraspecific variation and geographic differentiation demonstrating a limited gene flow among the world population. We argue that the distribution of cryptic species in the different clade can be explained by ongoing, but still incomplete speciation. An event-based ancestral area reconstruction using the software S-DIVA employed in RASP showed that the probable origin of the ribotypes was a global dispersal event in the Neotropics. Additional species distribution models that were implemented for the three most prominent clades show different putative ranges. As such H. serpula supports the moderate endemicity hypothesis for protists. In summary, myxomycete assemblages in the Paleotropics (1) displayed a higher diversity than for Neotropical forests, (2) harbor unique taxa that differentiates those assemblages in spite of the expected similar macroecological all over the Tropics, (3) are affected by geographical barriers that likely causes speciation both at a morphospecies and biospecies level, and (4) follow the ubiquitous model in the sense that gene flow mediated by long-distance dispersal of spores is high enough that a species can fill out its entire putative range, but (5) the gene flow is not high enough to prevent variation in regional gene pools, which may lead to speciation and is better explained by the moderate endemicity model. Our data are still too limited to draw a comprehensive picture of the diversity of tropical myxomycetes, but the baseline information compiled with the aid of both classical ecology and molecular approaches from this study are first major steps towards this goal.
Class I and class II glutaredoxins (Grxs) are glutathione (GSH)-dependent proteins, that function as oxidoreductases (class I) or mediate cellular iron trafficking (class II). Some members of class I Grxs like human Grx2 are able to complex a [2Fe-2S] cluster and form a dimeric holo complex, which renders them catalytically inactive and is the basis for their function as redox sensors. Class II Grxs like human Grx5 also complex [2Fe-2S] clusters, however these proteins transfer the clusters to other proteins. Both functionally distinct classes share a similar thioredoxin fold and conserved interaction sites for the non-covalently binding of GSH, which is required to complex the [2Fe-2S] cluster. Furthermore, the proteins from both classes contain a highly nucleophilic active site cysteine that would allow both classes to catalyze GSH-dependent oxidoreduction reactions. Despite of these similar features, only class I Grxs are able to form a mixed disulfide with GSH and to reversibly transfer it to protein thiols (de-/glutathionylation). Interestingly, neither class I Grxs nor class II Grxs can effectively compensate the loss of an essential member of the other class. Even though some structural differences were described earlier, the basis for their different functions remained unknown. In particular, the lack of catalytic activity of class II Grxs as oxidoreductases could not be explained. Here, we demonstrate that the different conformations of a conserved lysyl side chain are the molecular determinant of the oxidoreductase or Fe-S transfer activity of class I and II Grxs, respectively. A specific loop structure that is conserved in all class II Grxs determines one lysyl conformation that prevents the formation of a mixed disulfide of the active site cysteinyl thiol with GSH. Using engineered mutants of hGrx2 and hGrx5, we demonstrated that the exchange of the distinct loop between the classes results in a loss of oxidoreductase function of class I hGrx2 and the gain of oxidoreductase activity of class II hGrx5. The altered GSH binding mode also profoundly changes the [2Fe-2S] cluster binding of the engineered mutants and thereby also influences stability of the holo complexes, a pre-determinant for [Fe-S] cluster transfer activity. With the minor shift of 2 Å in a conserved lysyl side chain orientation we were not only able to modify the catalytic activity of two small human mitochondrial proteins, but on a much larger scale also provided evidence for the previously unknown structural basis that determines the function of all class I and class II Grxs.
The oxidoreductase activity of hGrx2 was also analyzed in vivo in a model of doxorubicin cell toxicity. Applying a mass spectrometrical approach, we identified various mitochondrial proteins as targets for redox regulation. Furthermore, our results gave reason to reconsider some common assumptions regarding doxorubicin-induced apoptosis and the protective function of mitochondrial Grx2.
Lipoproteins of Staphylococcus aureus represent a major class of surface proteins, which are anchored to the outer leaflet of the cell membrane. Although they play a key role in the immune response and virulence, the majority of lipoproteins in this organism is still of unknown function. The aim of our study was to investigate the function of so far poorly or uncharacterized lipoproteins in S. aureus strain Newman. To this end, an integrated bioinformatical approach was applied to define the pan-lipoproteome of 123 completely sequenced S. aureus strains. In total, this analysis predicted 192 different potential lipoproteins, with a core lipoproteome of 39 and a variable lipoproteome of 153 lipoproteins. Out of those 192 lipoproteins, 141 are so far functionally uncharacterized. Primarily focusing on members of the core-lipoproteome with unknown or poorly characterized function, 24 lipoproteins or co-encoded neighbor proteins were selected for further characterization. Of those 24 proteins, 20 S. aureus markerless deletion mutants were constructed (S. aureus delta l01 - delta l20) and screened for an altered growth behavior under various conditions. Here, three mutants showed a temperature-sensitive phenotype, two mutants formed aggregates in the TSB of the manufacturer Merck (TSBMerck), and four mutants showed reduced growth under osmotic stress with 8% NaCl. An altered aggregation behavior was observed for four mutants in the presence of Triton X-100 and for eleven mutants in the presence of SDS. Furthermore, ten mutants revealed an impaired biofilm formation capacity as well as reduced hemolytic activity. Interestingly, S. aureus deletion mutants delta l14 (delta NWMN_1435) and delta l16 (delta NWMN_0646) showed an altered phenotype under nearly all tested growth and stress conditions. Most strikingly, both deletion mutants demonstrated dramatic defects in cell morphology and cell division during the transient growth phase in TSBMerck and were therefore selected for further detailed characterization. Electron microscopy imaging of the two mutants revealed an irregular cell shape, increased cell size, multiple displaced division septa, and incomplete separation of daughter cells resulting in the formation of cell aggregates in TSBMerck. Complementarily, microarray-based transcriptome analysis and whole-genome sequencing of S. aureus delta l14 and delta l16 suppressor mutants strongly point to a functional association of both lipoproteins with cell envelope- or cell division-related processes. Specifically, multiple hints suggest a functional connection of both lipoproteins with lipo- or wall teichoic acids. Of note, the phenotypes of S. aureus delta l14 and delta l16 are conditional and appear under some, but not all growth conditions. Thus, it is conceivable that the function of L14 and L16 is modulated by metabolic processes, or that the proteins might be part of a “backup system” becoming important only under certain conditions. Collectively, we propose that L14 and L16 fulfill a basic role in cell envelope- or cell division-related processes under specific growth conditions. Particularly, the activity of L14 and L16 might be necessary for the function or localization of lipo- or wall teichoic acids, and thus, might be linked to the regulation of autolysins. In conclusion, this study reveals important insights into the function of two so far uncharacterized but highly conserved lipoproteins in S. aureus.
Myxomycetes (Amoebozoa, plasmodial slime molds) are one of the last larger groups of organisms where the biodiversity is not yet investigated by molecular methods, except for a very few cultivable model species. Based on the first phylogenies for the group produced in 2012 and 2013, this thesis work explores the genetic diversity of wild populations of myxomycetes, addressing two questions: 1. Does diversity and phylogenetic trees found with barcode markers fit the current morphological species concept, and do barcode markers reveal a lower or higher diversity than found by morphological characters? In the first case, morphological characters seen as decisive for species differentiation would be plastic (shaped by the environment), in the second case we must assume the existence of cryptic species. 2. Can genetic markers be used to see if natural populations of myxomycetes reproduce mainly sexual or asexual? Sexuality is proven to occur in the Amoebozoa, but asexual reproduction should be advantageous for habitat colonization. Experiments with cultivable species have shown that both reproductive modes occur in the myxomycetes. Two species complexes were chosen for an in-depth investigation. The first species is the common wood-inhabiting myxomycete Trichia varia (Pers. ex J.F. Gmel.) Pers., one of the first myxomycetes to be described and always seen as a variable, yet single, species. The second example involves a snowbank species so far known as Lamproderma atrosporum Meyl., which was recently transferred to a genus on its own, Meriderma Mar. Mey. & Poulain, and a morphological species concept, including several taxa, was proposed. Trichia varia belongs to the bright-spored myxomycetes. Partial sequences of three independent markers (nuclear small-subunit ribosomal RNA gene, SSU, extrachromosomal; protein elongation factor 1 alpha gene, EF1A, chromosomal; cytochrome oxidase subunit 1 gene, COI, mitochondrial) from 198 specimens resulted in a three-gene phylogeny containing three groups, within each group combinations of the single-marker genotypes occurred exclusively. Complete SSU sequences were generated for 66 specimens, which revealed six positions that can carry group I introns and putatively functional or degenerated homing endonuclease genes in two groups. All observations (genotypic combinations of the three markers, signs of recombination, intron patterns) fit well into a pattern of three cryptic biological species that reproduce predominantly sexual but are reproductively isolated. The pattern of group I introns and inserted homing endonuclease genes mounts evidence that the Goddard-Burt intron life cycle model applies to naturally occurring myxomycete populations. A total of 89 specimens of the dark-spored myxomycete genus Meriderma from five European mountain ranges were sequenced for partial genes of SSU and EF1A. The latter gene includes an extremely variable spliceosomal intron. Three clades, the two morphologically recognizable taxa M. fuscatum, M. aggregatum, and the morphologically complicated complex species M. atrosporum agg., were recovered. The EF1A-based phylogeny of the 81 specimens of M. atrosporum agg. resulted in seven subclades, with the two EF1A-haplotypes of a sequence sharing always one subclade for each of the 50 heterozygous specimens, a pattern consistent with the existence of several independent but sexually reproducing biospecies. Identical EF1A genotypes occurred more often within a regional population than in between. A simulation assuming panmixis within a biospecies but not in between, and isolation between mountain ranges suggested that similar numbers of shared genotypes can be created by chance through sexual reproduction alone. Numbers of haplotypes shared between mountain ranges correlate with geographical distance, suggesting occasional long-distance dispersal by spores. An enlarged data set containing 227 partial SSU sequences of Meriderma spp. identified 53 ribotypes, with a ribotype accumulation curve indicating 68.4±14.5 ribotypes to expect according to the Chao2 estimator. The topology of the SSU phylogeny generally confirms results from the partial SSU and EF1A data set of 89 specimens, where several putative biospecies could be recognized. A novel method for automated analyses of SEM images allows to derive quantitative descriptors for spore ornamentation, which were subjected to multivariate analyses. Spore ornamentation provided traits with the highest explanatory power in a multivariate statistics, whereas spore size and stalk length were much less significant. For some but not all putative biospecies a unique combination of morphological characters was found, which is in accordance with the hypothesis of instant sympatric 8 speciation via mutations creating incompatible strains splitting from existing biospecies. The morphologically recognizable taxa of the genus are described and a key for the genus Meriderma is given. To compare morphological and molecular diversity in lignicolous myxomycetes, all specimens found in a study covering the late-autumn aspect were sequenced, using partial SSU gene as a barcode marker. A total of 161 logs in the old-growth forest Eldena, northeastern Germany, was surveyed, resulting in 530 collections representing 27 taxa from 14 genera. Bright-spores species were far more abundant than dark-spored taxa. A phylogeny based on partial SSU sequences for bright-spored myxomycetes revealed morphospecies to be largely consistent with phylogenetic groups. Most but not all morphospecies may contain multiple ribotypes that cannot be differentiated by light microscopy. This first study backing up a traditional morphology-based survey by a full molecular component demonstrates that partial SSU sequences can function as reliable barcode markers for myxomycetes, but reveals as well a significant, yet not infinite, amount of hidden diversity. The main conclusions of this work, set up in the frame of a project funded by the German Research Council (DFG), are the following: 1. Sexual reproduction seems to be an important, if not the dominating mode (apart from clonal myxamoebal populations built up by binary fission) of reproduction in naturally occurring populations of myxomycetes. 2. From the two investigated species complexes we can expect many, if not most, morphopecies to be composed of reproductively isolated, sexually reproducing, biospecies. 3. Partial SSU sequences, as most widely used in this study, seem to represent suitable barcode markers for the group and can be used to distinguish the (usually cryptic) biospecies, although they alone do not allow any conclusions about reproductive isolation and speciation processes. 4. We have to expect a significant amount of hidden diversity in myxomycetes, which will increase the number of taxa from ca. 1000 recognized morphologically by a factor between two and ten.
This thesis contains results from transcriptome studies on different aspects of host-pathogen interactions. First, liver gene expression profiles from a murine chronic stress model served to elucidate aspects of the influence of stress on metabolism and immune response state. Chronic stress in female BALB/c mice was shown to lead to a hypermetabolic syndrome including induction of gluconeogenesis, hypercholesteremia, and loss of essential amino acids, to the induction of the acute phase response, but also of immune suppressive pathways and to the repression of hepatic antigen presentation. Increased leukocyte trafficking, increased oxidative stress together with counter-regulatory gene expression changes, and an induction of apoptosis were detected. The influence of intra-venous infection on the host kidney gene expression was analyzed in another murine model using the wild type strain Staphylococcus aureus RN1HG and its isogenic sigB mutant. Gene expression profiling indicated a highly reproducible host kidney response to infection. The comparison of infected with non-infected samples revealed a strong inflammatory reaction of kidney tissue, e. g. Toll-like receptor signaling, complement system, antigen presentation, interferon and IL-6 signaling. However, the results of this study did not provide any hints for differences in the pathomechanism of the S. aureus strains RN1HG and ΔsigB, since the host response did not differ between infections with the two strains analyzed. Effects of SigB might be transient, only apparent at earlier time points, or might also be compensated for in the in vivo infection by the interlaced pattern of other regulators. SigB might possess only to a lesser extent characteristics attributed to virulence factors and might act in vivo more like a virulence modulator and fine tune bacterial reactions. In addition to the analysis of tissue samples, different in vitro models were furthermore studied. The third part of this thesis focuses on bone-marrow derived macrophages (BMM) of the two mouse strains BALB/c and C57BL/6, which are described in literature to exhibit genetically determined differences in their reaction to infection. Expression profiling was performed on control and IFN-γ treated samples from a serum-free cultivation system and revealed mainly induction of gene expression after treatment of BMM with IFN-γ. Gene expression changes confirmed known IFN-γ effects like induction of immunoproteasome, antigen presentation, interferon signaling related genes, GTPase/GBPs, and inducible NO synthase. IFN-γ dependent gene expression changes were highly similar in BALB/c and C57BL/6 BMM. Considering gene expression differences between BMM of both strains, a similar expression trend was visible on the level of untreated controls as well as after IFN-γ treatment. Differentially expressed genes between BMM of both strains included immune-relevant genes as well as genes linked to cell death, but the coverage of functional groups was limited. The bronchial epithelial cell line S9 was used as an in vitro model system for the infection with S. aureus RN1HG. The fourth chapter in this thesis includes S9 cell gene expression signatures 2.5 h and 6.5 h after start of infection. At the early time point, only 40 genes were differentially expressed, which nevertheless indicated a beginning pro-inflammatory response, e. g. induction of cytokines (IL-6, IFN-β, LIF) or prostaglandin-endoperoxide synthase 2 (PTGS2), but also counter-regulatory processes, e. g. induction of CD274. The host cell response was dramatically aggravated at the later 6.5 h time point. Differential expression was detected for 1196 genes. These included induced cytokines, pattern recognition receptor signaling, antigen presentation, and genes involved in immune defense (e. g. GBPs, MX, APOL). Negative effects on growth and proliferation were even more enhanced in comparison to the early time point, and signs for apoptotic processes were revealed. Finally, the last chapter addresses amongst others the pathogen’s expression profile in the S9 cell in vitro infection model at the two time points 2.5 h and 6.5 h after start of infection by tiling array gene expression analysis. The pathogen expression profiling revealed the activity of the SaeRS two-component system in internalized staphylococci. Partly dependent on SaeRS, the induction of adhesins (e. g. fnbAB, clfAB), toxins (hlgBC, lukDE, hla), and immune evasion genes (e. g. chp, eap) was observed. Furthermore, expression changes of metabolic genes were recorded (gene induction of amino acid biosynthesis, TCA cycle, gluconeogenesis; gene repression of glycolysis, purine biosynthesis, tRNA synthetases). Expression analysis recorded a distinct bacterial expression program, which supported literature results of a specific, bacterial strain and host cell line dependent transcriptional adaptation of the pathogen.
Mechanically ventilated patients are at risk of ventilator-associated pneumonia, a serious infection of the lungs. Not every ventilated patient develops pneumonia due to a combination of the protective layer of mucus in the airways, the immune system and prophylactic antibiotic therapy. To date, only little was known about the antimicrobial factors produced by humans that protect the lungs against infection. Research described in this thesis was therefore aimed at investigating to what extent the lungs of ventilated patients can inhibit the growth of bacteria, the major causative agent of pneumonia Streptococcus pneumoniae in particular. To this end, the accumulated mucus in the patients’ lungs, sputum, was investigated. The most important conclusion was that sputum can indeed possess antimicrobial activity, explained either by a combination of antibiotics and S. pneumoniae-specific antibodies, or by the innate immune defenses. Thus, sputum may serve as a valuable source of information to unravel the complex interactions between the human host, antimicrobial factors and the microbiome of the lower respiratory tract. A possible consequence of pneumonia is the dissemination of bacteria from the lungs to the bloodstream and the brain, which may lead to meningitis. This thesis describes how this process takes place, and how the so-called choline-binding protein CbpL contributes to invasive pneumococcal infections. In addition, possible future approaches to prevent meningitis caused by this bacterium are proposed.
Symbiotic interactions are a key element of biological systems. One powerful strategy to gain insight into these interactions, and into biological systems in general, is the analysis of proteins expressed in situ using metaproteomics. In this thesis, host-microbe interactions in two mutualistic associations between chemosynthetic sulfur-oxidizing endosymbionts and marine invertebrates, the deep-sea tubeworm Riftia pachyptila and the shallow-water clam Codakia orbicularis, were studied by adapted and optimized metaproteomics methods.
The Riftia symbiosis, which inhabits hydrothermal vents in the deep sea, and in which the host completely depends on its symbiont for nutrition, has fascinated researchers for about four decades. Yet, the interaction mechanisms between both partners have been understudied so far. Additionally, while different aspects of the host’s biology have been described, a comprehensive analysis has been lacking. Moreover, although only one symbiont 16S rRNA phylotype is present in Riftia, the symbiont population of the same host expresses proteins of various redundant or opposed metabolic pathways at the same time. As the symbionts also exhibit a wide variety in size and shape, symbionts of different size might have dissimilar physiological functions, which remained as of now to be elucidated. In this thesis, we addressed both, the host-symbiont interaction mechanisms, and physiological roles of symbiont subpopulations. A comprehensive Riftia host and symbiont protein database was generated as prerequisite for metaproteomics studies by de novo sequencing the host’s transcriptome and combining it with existing symbiont protein databases. This database was then used for metaproteomics comparisons of symbiont-containing and symbiont-free Riftia tissues, to gain insights into host-symbiont interactions on the protein level. The impact of energy availability on host-symbiont interactions was studied by comparing specimens with stored sulfur (i.e., high energy availability) with specimens in which sulfur storages were depleted. We employed optimized liquid chromatography peptide separation to increase metaproteome coverage. With this analysis, we identified proteins and mechanisms likely involved in maintaining the symbiosis, under varying environmental conditions. We unraveled key interaction mechanisms, i.e.: (i) the host likely digests its symbionts using abundant digestive enzymes, and, at the same time, (ii) a considerable part of the worm’s proteome is involved in creating stable internal conditions, thus maintaining the symbiont population. Furthermore, (iii) the symbionts probably employ eukaryote-like proteins to communicate with the host. (iv) Under conditions of restricted energy availability, the host apparently increases digestion pressure on the symbiotic population to sustain itself.
Riftia symbionts of different size apparently have dissimilar metabolic roles, as revealed in this thesis. We enriched symbionts of different sizes using gradient centrifugation. These enrichments were subjected to protein extraction using a protocol optimized for the small sample amount available. Metaproteomics analysis included a gel-based workflow and evaluation of the complex dataset with machine learning techniques. Based on our metaproteomics study, we propose that Riftia symbionts of different cell size correspond to dissimilar physiological differentiation stages. Smaller cells are apparently engaged in cell differentiation and host interactions. Larger cells, on the other hand, seem to be more involved in synthesis of various organic compounds. Supposedly, in large symbionts endoreduplication cycles lead to polyploidy. Our results indicate that the Riftia symbiont employs a large part of its metabolic repertoire at the same time in the stable host environment.
The symbiont of the shallow-water clam Codakia orbicularis, which, like the Riftia symbiont, relies on reduced sulfur compounds as energy source and fixes inorganic carbon, is, unexpectedly, also able to fix atmospheric nitrogen, as shown by metaproteomic, genomic and biochemical analysis. Potentially, this benefits the host, as Codakia digests its symbiont and might thus supplement its diet with organic nitrogen fixed by the symbionts in addition to organic carbon in its nitrogen-poor seagrass habitat.
Bacteria are an integral part of modern biotechnology. They are used to make a variety of products, such as foods, drugs, as well as a multitude of chemicals. In order to increase their production rates molecular biotechnology offers many tuning points, starting from the selection of an applicable host, over its geno- and phenotypical characterization, followed by genetic manipulations for an optimized metabolism and stabilisation of production processes. This work comprises the optimization of Bacillus subtilis as an expression system. It describes the steps taken for selection and genomic characterization of the B. subtilis wild type strain ATCC 6051, the subsequent optimizations of the strain in respect to growth and productivity, as well as the characterization of its behaviour in a variety of cultivation conditions. The B. subtilis strain most commonly found in laboratories around the world is the first sequenced Gram-positive organism B. subtilis 168. Zeigler et al. showed that strain 168 is not a real wild type. Instead it was created through random mutagenesis with X-rays and selected for transformability. This strain has been used as the basis for popular B. subtilis strains in heterologous gene expression such as the extracellular protease deficient WB strains. Growth experiments showed the real wild type strain ATCC 6051 to be superior to its mutated ancestor 168, making it a solid basis for the construction of an optimized B. subtilis expression system. In order to gain a full understanding of the genomic and corresponding physiological differences between the two systems, B. subtilis ATCC 6051 was sequenced and compared to the genome of B. Subtilis 168. Several variations on geno- and phenotypic level could be revealed, that resulted in particular from genes involved in natural competency, the metabolism of amino acids and chemotaxis. This genomically well characterized B. subtilis ATCC 6051 was improved in respect to its application as an expression host. Improvements were achieved through the inactivation of both sporulation and reduction of autolysis, leading to a more robust behaviour during the overproduction and secretion of a reporter enzyme. A positive effect on the activity of an acetoin induced promoter by the addition of second copies for its transcription factors SigmaL and AcoR could be observed. Anaerobic zones and areas with excess glucose caused by insufficient mixing are common conditions in large scale bioprocesses and lead to oscillating conditions for the cells. In turn, this oscillation provokes an excretion of so called overflow metabolites, which can negatively affect the bacterial productivity. Detailed scientific characterizations of industrial scale processes under such oscillating conditions are scarce due to the high costs and logistics involved. A B. Subtilis sporulation mutant was thus examined in respect to its extra- and intracellular metabolites in a scale-down, two-compartment reactor giving hints about conditions the host is exposed to and how it reacts. To improve tolerance thresholds and utilization capacity for such metabolites in B. subtilis, the glyoxylate cycle was transferred from its close relative Bacillus licheniformis into the genome of B. subtilis. This feature enabled our B. subtilis ACE mutant to grow on acetate. The improved strain showed higher tolerance towards excess glucose in a fed-batch as well as higher productivity during the expression of a reporter enzyme in comparison to the wild type. The ACE strain and B. licheniformis showed an increased formation of glycolate during growth with the glyoxylate cycle. This with regard to bacteria undescribed metabolite seems to play a role as a by-product of the glyoxylate cycle. Summarizing, this thesis deals with the characterization and optimization of B. subtilis for growth on overflow metabolites, enhancements of the acoA-expression system and the influence of sporulation and lysis mutants on its activity. Complementary, the host was begun to be characterized in respect to its behaviour in industrial scale processes.
Staphylococcus aureus is a commensal that colonizes the skin and mucosa of 20-30% of the human population without leading to symptoms of diseases. However, it is also the most important cause of nosocomial infections. Those range from minor skin infections to life-threatening diseases such as pneumonia, endocarditis or septicaemia. Development of strains with resistance against many antibiotics complicates the situation further. The variety of strains with their various properties is one reason why no successful vaccine has been introduced to the market, yet. Therefore, efficient strategies for prevention and therapy of these dangerous infections are urgently needed. To accomplish these goals, the understanding of molecular interactions between host and pathogen is indispensable. Within this dissertation, several internalization experiments were performed aiming to investigate the interaction of S. aureus HG001 and human cell lines upon infection on the protein level. In order to obtain sufficient amounts of proteins for comprehensive physiological interpretations, it is necessary to enrich bacteria, secreted bacterial proteins or infected host cells upon internalization. In the framework of this thesis, bacteria which continuously produce green fluorescent protein (GFP) were employed. With that it was possible to sort bacteria from lysed host cells by flow cytometry or to separate host cells carrying bacteria after contact from those which did not. Subsequently, the proteins were proteolytically digested and peptides were analyzed by mass spectrometry in a gel-free proteomics approach. To allow such analyses also for staphylococci which do not produce GFP, such as clinical isolates, an additional protocol was developed. Prior to the infection, bacteria were labeled with fluorescent or para-magnetic nanoparticles. Afterwards bacteria could be separated from host cell debris by fluorescence-based cell sorting or with the help of a strong magnet. In order to cover also important secreted virulence factors of S. aureus HG001, phagosomes and engulfed bacteria and secreted proteins were isolated from infected host cells. Further steps of protocol optimization included improved bacterial cell counting by fluorescence-based flow cytometry, enhanced data analysis by combination of different search algorithms, and comprehensive functional annotation of proteins of the applied strain by sequence comparison with other strains and organisms. First, the proteome adaptation of internalized S. aureus HG001 and the infected A549 host cells was investigated during the first hours of infection. It became clear, that the bacteria replicate inside the host during the first 6.5 h. After internalization the levels of bacterial enzymes involved in protein biosynthesis decreased. Furthermore, bacteria adapted their proteome to the harsh intracellular conditions such as oxygen limitation, cell wall stress, host defense in terms of oxidative stress, and nutrient limitation. After contact to S. aureus HG001, A549 cells produced increased amounts of cytokines (e.g. IL-8, IFN-γ) in comparison to non-treated A549 cells. In addition, activation of the immunoproteasome and hints of early apoptosis activity were observed. Afterwards, the response of S. aureus HG001 to internalization by A549, S9 or HEK 293 cells was compared on the proteome level. It was obvious, that the adaptation to stress and the reduced protein synthesis are conserved mechanisms. Host dependent differences were detected especially in the energy metabolism and the synthesis of some amino acids. Additionally, bacteria showed different intracellular replication patterns depending on the host cell line. A higher percentage of extracellular bacterial proteins was found in isolated phagosomes compared to the sorted samples. Selected low abundant virulence factors could be quantified at two points in time after infection with the help of the sensitive single reaction monitoring (SRM) method. Further, a heterogeneous mixture of several phagosomal maturation steps was present during the first 6.5 h after infection. Finally, the gel-free proteome analyses could be applied to investigate Bordetella pertussis, the cause of whooping cough, during iron limitation and after internalization, and the results were compared to the S. aureus HG001 data.
Cardiovascular diseases are the most common cause of death in industrial nations. The basis of these diseases is a dysfunction in the interaction between the cells the heart is composed of. The main types of cells making up the human heart are cardiomyocytes that build the myocardium and provide the contraction properties, endothelial cells that delimit the blood flowing through the inner chambers and coronary arteries from the myocardial tissue, and fibroblasts, which build the connective tissue. A common process in the development of cardiovascular diseases is the formation of fibrosis due to injury of the endothelium and subsequent infiltration of the cardiac tissue by immune cells, and inflammatory agents like cytokines. Cytokines exert different functions in cardiac cells. Tumor necrosis factor α (TNFα) is an inducer of apoptosis. Transforming growth factor ß (TGFß) is known for activation of proliferation. Other cytokines like C-X-C motif chemokine 11 (CXCL11), interleukin-6 (IL-6), or brain-derived neurotrophic factor (BDNF) have not yet been investigated or their impact on such cells is unknown. Eventually, however, fibrotic scar tissue arises from the transition from fibroblasts to myofibroblasts leading to a stiffening of the cardiac muscle and impaired pump function. In order to prevent the occurrence of these events the balance of proliferation, migration, and differentiation of cardiac cells needs to be controlled very delicately.
The mechanisms controlling these interactions are still not well understood, which is why this work aimed at the elucidation of molecular mechanisms within the three main cell types that might play a role in the regulation of cardiac function. A proteomic approach using mass spectrometry was used to identify alterations in protein levels that could provide hints about the involved pathways and find new players as candidates for more detailed investigation. Initially, the proteomic composition of HL-1 cardiomyocytes, L929 fibroblasts, and human umbilical vein endothelial cells (HUVECs) that were cultivated in standard growth conditions without stress was investigated. Half of the total protein intensity was made up by only 42 to 53 proteins, depending on the cell type. More than a third of all proteins were identified in all three cell types, which may be proteins performing common cell functions. Indeed, the proteins displaying the highest abundance seem to be predominantly involved in such common cellular functions as the regulation of glucose metabolism or the cytoskeleton. More specific functions like heart development and muscle contraction were found enriched in cardiomyocytes as were mitochondrial proteins. The proportion of proteins with extracellular localization and function was higher in fibroblasts and endothelial cells.
Secondly, the impact of cytokines on the proliferative behavior and the proteomic composition of cardiomyocytes and fibroblasts was analyzed. HL-1 cardiomyocytes and L929 fibroblasts were treated with different concentrations of cytokines with a cytotoxic, proliferative, or yet unknown effect on these cells. While HL-1 cells exhibited no macroscopic reaction to any of the cytokines used, cytotoxic/growth inhibitory (TNFα, CXCL11) and proliferative (TGFß, IL6, BDNF) effects were observed for L929 cells. The latter also showed CXCL11-induced upregulated EIF2 signaling, pointing to a higher need of protein synthesis.
The third aim was the examination of proteome adaptations in endothelial cells due to different kinds of stress, as these cells are the first line of defense against inflammatory agents or injury and therefore prone to wounding. The role of the growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in wounding and starvation was another object of this study as they are known for their angiogenic and cell survival supporting properties. Additionally, the impact of the cellular sex on the response to stress and growth factors was examined, because a person’s sex plays an important role in susceptibility, risk factors, and outcome of cardiovascular diseases. This has mainly been attributed to the different hormone levels, especially the higher levels of estrogen in premenopausal women, which exerts cardioprotective properties, but also genetic background was reported to play an important role. Only few studies that examined the molecular properties of HUVECs considered the cellular sex and if so, the genetic bias of unrelated samples was not taken into account. This is why Lorenz and colleagues at the Charité in Berlin collected HUVECs from newborn twins of opposite sex, cultivated them without stress in standard growth medium, exposed them to wounding and serum starvation, and investigated the impact of the growth factors and the sex on migrational behavior and metabolic issues. The current work focused on the alterations of not only the intra- but also the extracellular proteome, because paracrine signaling is crucial for intercellular communication in order to cope with stress. General differences between male and female cells were observed for proteins encoded on the X chromosome with higher levels in females (DDX3X, UBA1, EIF1AX, RPS4X, HDHD1), except for one protein with higher levels in male cells (G6PD). A Y-chromosomal protein was, for the first time, identified in endothelial cells (DDX3Y). Wounding, starvation, and growth factor treatment led to alterations and sex-specific different levels in an unexpectedly high number of proteins, with VEGF showing a stronger impact than bFGF. Many proteins with alterations observed without taking the sex into account, were actually only changed in male or female cells. Some proteins were regulated in opposite directions, or growth factors inhibited their secretion in a sex-specific way by unknown mechanisms. Tissue factor pathway inhibitor 2 (TFPI2) should be emphasized as a protein with sex-specific differences, especially in the extracellular space and with increased levels after starvation and VEGF treatment. These observations suggest a temporal lack in TFPI2 synthesis and secretion in male cells, which might explain the enhanced adaptation of females to wounding.
The results of this work lay the basis for future investigation by providing a database of intra- and extracellular proteome changes due to different environmental circumstances. It strongly suggests the investigation of male and female HUVECs, and other cells, separately to avoid the impact of the sex observed in this work. Essentially, the observations suggest a number of candidate proteins for more detailed investigations of endothelial and cardiovascular diseases.
Clostridium (C.) difficile ist beim Menschen ein bedeutender Erreger von Krankenhaus-assoziierten Durchfallerkrankungen. Die vorliegende Dissertation umfasst die Erhebung und Analyse der ersten epidemiologischen Daten zu C. difficile in deutschen Heim- und Nutztierbeständen und die Entwicklung eines neuartigen DNA-Microarrays, der eine einfache Ribotypisierung von C. difficile ermöglicht. In drei Studien wurden Kotproben von Hunden, Katzen, Ferkeln und Kälbern kulturell auf C. difficile untersucht. Das Bakterium wurde aus 5 von 135 Katzenkot- (3,7%), 9 von 165 Hundekot- (5,4%), 176 von 999 Kälberkot- (17,6%) und 147 von 201 Schweinekotproben (73,1%) isoliert. Neugeborene Ferkel und Kälber waren in den ersten 2 bzw. 3 Lebenswochen signifikant häufiger kulturpositiv für C. difficile als ältere Tiere. Die in den Studien isolierten Stämme wurden 25 Ribotypen zugeordnet; darunter befanden sich 6 bisher nicht beschriebene Varianten. Bei den Ferkelproben dominierten die einander sehr ähnlichen Ribotypen 078 (55% der Isolate) und 126 (20%). Die Ribotypen 033 (57% der Isolate) und 078 (17%) wurden bei Kälbern am häufigsten vorgefunden. Basierend auf ihren Typisierungsprofilen wurden die Ribotypen in einer UPGMA-Analyse (Unweighted Pair Group Method with Arithmetic mean) untereinander verglichen. Von den 25 bei den untersuchten Tieren gefundenen Ribotypen formten 11 einen Cluster, zu dem auch die Ribotypen 033, 078 und 126 gehörten und in dem sich 90% aller in den beiden Nutztierstudien isolierten Stämme wiederfanden. Alle Isolate dieses Clusters waren zudem PCR-positiv für ein binäres Toxin und, im Gegensatz zu allen nicht zu dem Cluster gehörenden Ribotypen, PCR-negativ für den MLVA-Lokus A6Cd. Hierdurch, aber auch durch frühere Studien, in denen gezeigt werden konnte, dass die dem Cluster zugeordneten Ribotypen 033, 045, 078 und 126 den gleichen MLST-Typ (ST-11, Multilocus Sequence Typing) und eine charakteristische Deletion (delta 39 bp) im Toxinregulatorgen tcdC aufweisen, wird die These einer genetische Verwandtschaft unterstützt. In allen untersuchten Tierpopulationen wurden Ribotypen gefunden, die mit C.-difficile-Infektionen des Menschen assoziiert werden. Da Stämme des Ribotyps 078 in deutschen und europäischen Krankenhäusern zunehmend häufiger als Ursache von Durchfallerkrankungen auftreten, wurden alle ermittelten MLVA-Daten von Ribotyp-078-Stämmen humanen und tierischen Ursprungs mit entsprechenden Daten anderer Studien aus 5 europäischen Ländern verglichen. In einer hierbei durchgeführten Minimum-Spanning-Tree-Analyse mit 294 Datensätzen wurde die genetische Abgrenzung von MLVA-Typen unterschiedlicher geographischer Herkunft verdeutlicht und belegt, dass einige aus Tieren und Menschen isolierte C.-difficile-Stämme sehr ähnlichen oder sogar identischen MLVA-Typen entsprechen. Die in den Haus- und Nutztierstudien isolierten C. difficile wurden anschließend mit dem neu entwickelten DNA-Microarray ribotypisiert. Das Sondendesign des Microarrays basiert auf der bei C. difficile modular aufgebauten Intergenic Spacer Region (ISR), welche auch Zielstruktur der herkömmlichen Ribotypisierungsmethoden ist. Die Sonden wurden von in der GenBank-Datenbank publizierten ISR-Modulsequenzen und theoretisch möglichen Modulsequenzkombinationen abgeleitet. Nachdem die Eignung des Arrays in theoretisch und praktisch durchgeführten Experimenten belegt werden konnte, wurde mit 142 repräsentativ ausgewählten C.-difficile-Stämmen eine 48 Ribotypen umfassende Datenbank aus Referenzhybridisierungsmustern erstellt. Diese Referenzmuster wurden anschließend in einer Ähnlichkeitsmatrix-Analyse untereinander verglichen, wobei 27 Referenzmuster eindeutig differenziert werden konnten. Zu den gut unterscheidbaren Ribotypen gehörten u.a. die häufig mit humanen C.-difficile-Infektionen assoziierten Ribotypen 001, 014/020, 027 und 078/126. Nicht unterscheidbar hingegen waren die 11 Ribotypen des oben beschriebenen Clusters, wodurch sich die These ihrer molekularen Verwandtschaft weiter erhärtet. Die Praxistauglichkeit des DNA-Microarrays wurde abschließend in einer Anwendungsstudie überprüft. Hierbei wurden 50 C.-difficile-Stämme, die im Rahmen eines anderen Projektes aus Kotproben von Haustieren und deren Besitzern isoliert wurden, mit herkömmlicher und DNA-Microarray-basierter Ribotypisierung vergleichend untersucht. Berücksichtig man, dass durch den Microarray einige sehr ähnliche Ribotypen derzeit noch nicht unterschieden werden können, wurden alle Isolate dem richtigen Ribotypen bzw. der richtigen Ribotypengruppe zugeordnet. Darüber hinaus wurden 6 für das Microarray unbekannte Ribotypen korrekt als „neu“ und klar voneinander unterscheidbar erkannt. Zusammenfassend trägt das Dissertationsprojekt zum Verständnis über das Vorkommen von C.-difficile-Genotypen in Heim- und Nutztierbeständen bei und präsentiert einen neuartigen DNA-Microarray zur einfachen Ribotypisierung von C. difficile.