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- Research Center for Infectious Diseases, University of Wuerzburg, Wuerzburg 97080, Germany (1)
- Rudolf-Virchow-Zentrum für Experimentelle Biomedizin der Universität Würzburg (1)
Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a singlechannel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.
Upon synthesis, nascent polypeptide chains are subject to major rearrangements of their side chains to obtain an energetically more favorable conformation in a process called folding. About one third of all cellular proteins pass through the secretory pathway and undergo oxidative folding in the endoplasmic reticulum (ER). During oxidative folding, the conformational rearrangements are accompanied by the formation of disulfide bonds – covalent bonds between cysteine side chains that form upon oxidation. Protein disulfide isomerase (PDI) assists in the folding of substrates by catalyzing the oxidation of pairs of cysteine residues and the isomerization of disulfide bonds as well as by acting as chaperones. In addition to PDI itself, a family of related ER-resident proteins has formed. All PDI family members share the thioredoxin fold in at least one of their domains and exhibit a subset of the PDI activities. Despite many studies, the role of most PDI family members remains unclear. The project presented in this thesis was aimed to establish tools for the biochemical characterization of single members of the PDI family and their role in the folding process. A combination of fluorescence based assays was developed to selectively study single functions of PDI family members and relate their properties of either catalysis of oxidation or catalysis of isomerization or chaperone activity to the rest of the protein family. A binding assay using isothermal titration calorimetry (ITC) was established to complement the activity assays. Using ITC we could show for the first time that members of the PDI family can distinguish between folded and unfolded proteins selectively binding the latter. The unique information provided by this method also revealed a two-site binding of unfolded proteins by PDI itself. In addition to the functional characterization, experiments were conducted to further investigate the oligomeric state of PDI. We could show that the equilibrium between structurally different states of PDI is heavily influenced by the redox state of the protein and its environment. This new data could help to further our understanding of the interplay between oxidases like PDI and their regenerative enzymes like Ero1, which may be governed by structural changes in response to the change in redox status. Another structural approach was the screening of all investigated PDI family members for suitable crystallization conditions. As a result of this screening we could obtain protein crystals of human ERp27 and were able to solve the structure of this protein with X-ray crystallography. The structure gives insight into the mechanisms of substrate binding domains within the PDI family and helps to understand the interaction of ERp27 with the redox active ERp57. In collaboration with the group of Heike Hermanns we could further show the physiological importance of this interaction under oxidative stress. In conclusion, the project presented in this thesis provides novel tools for an extensive analysis of the activities of single PDI family members as well as a useful set of methods to characterize novel oxidoreductases and chaperones. The initial results obtained with the our novel methods are very promising. At the same time, the structural approach of this project could successfully solve the structure of a PDI family member and give information about the interplay within the PDI family.
Platelet activation induces cytoskeletal rearrangements involving a change from discoid to spheric shape, secretion, and eventually adhesion and spreading on immobilized ligands. Small GTPases of the Rho family, such as Rac1 and Cdc42, are known to be involved in these processes by facilitating the formation of lamellipodia and filopodia, respectively. This thesis focuses on the role Rac1 and Cdc42 for platelet function and formation from their precursor cells, the megakaryocytes (MKs), using conditional knock-out mice. In the first part of the work, the involvement of Rac1 in the activation of the enzyme phospholipase (PL) C2 in the signaling pathway of the major platelet collagen receptor glycoprotein (GP) VI was investigated. It was found that Rac1 is essential for PLC2 activation independently of tyrosine phosphorylation of the enzyme, resulting in a specific platelet activation defect downstream of GPVI, whereas signaling of other activating receptors remains unaffected. Since Rac1-deficient mice were protected from arterial thrombosis in two different in vivo models, the GTPase might serve as a potential target for the development of new drugs for the treatment and prophylaxis of cardio- and cerebrovascular diseases. The second part of the thesis deals with the first characterization of MK- and platelet-specific Cdc42 knock-out mice. Cdc42-deficient mice displayed mild thrombo-cytopenia and platelet production from mutant MKs was markedly reduced. Unexpectedly, Cdc42-deficient platelets showed increased granule content and release upon activation, leading to accelerated thrombus formation in vitro and in vivo. Furthermore, Cdc42 was not generally required for filopodia formation upon platelet activation. Thus, these results indicate that Cdc42, unlike Rac1, is involved in multiple signaling pathways essential for proper platelet formation and function. Finally, the outcome of combined deletion of Rac1 and Cdc42 was studied. In contrast to single deficiency of either GTPase, platelet production from double-deficient MKs was virtually abrogated, resulting in dramatic macrothrombocytopenia in the animals. Formed platelets were largely non-functional leading to a severe hemostatic defect and defective thrombus formation in double-deficient mice in vivo. These results demonstrate for the first time a functional redundancy of Rac1 and Cdc42 in the hematopoietic system.
XPD is a 5‘-3‘ helicase of the superfamily 2. As part of the transcription factor IIH it functions in transcription initiation and nucleotide excision repair. This work focus on the role of XPD in nucleotide excision repair. NER is a DNA repair pathway unique for its broad substrate range. In placental mammals NER is the only repair mechanism able to remove lesions induced by UV-light. NER can be divided into four different steps that are conserved between pro- and eukaryotes. Step 1 consists of the initial damage recognition, during step 2 the putative damage is verified, in step 3 the verified damage is excised and in the 4th and final step the resulting gap in the DNA is refilled. XPD was shown to be involved in the damage verification step. It was possible to solve the first apo XPD structure by a MAD approach using only the endogenous iron from the iron sulfur cluster. Based on the apo XPD structure several questions arise: where is DNA bound? Where is DNA separated? How is damage verification achieved? What is the role of the FeS cluster? These questions were addressed in this work. Hypothesis driven structure based functional mutagenesis was employed and combined with detailed biochemical characterization of the variants. The variants were analyzed by thermal unfolding studies to exclude the possibility that the overall stability could be affected by the point mutation. DNA binding assays, ATPase assays and helicase assays were performed to delineate amino acid residues important for DNA binding, helicase activity and damage recognition. A structure of XPD containing a four base pair DNA fragment was solved by molecular replacement. This structure displays the polarity of the translocated strand with respect to the helicase framework. Moreover the properties of the FeS cluster were studied by electron paramagnetic resonance to get insights into the role of the FeS cluster. Furthermore XPD from Ferroplasma acidarmanus was investigated since it was shown that it is stalled at CPD containing lesions. The data provide the first detailed insight into the translocation mechanism of a SF2B helicase and reveal how polarity is achieved. This provides a basis for further anlayses understanding the combined action of the helicase and the 4Fe4S cluster to accomplish damage verification within the NER cascade.
Magnetic Resonance Imaging of Tumors Colonized with Bacterial Ferritin-Expressing Escherichia coli
(2011)
Background: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. Methods and Findings: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. Conclusions: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes
Type 1 diabetes is an autoimmune disease that leads to the destruction of insulin-producing pancreatic beta cells and consequently to hyperglycemia. In the last 60 years, the prevalence of type 1 diabetes has been increasing constantly and is predicted to continue rising. About 80% of the disease risk is attributable to the genetic variation. Thanks to genome wide association studies the number of known disease-associated polymorphisms climbed from five to 53 in the last 10 years. As these studies reveal possible candidate genes but not underlying mechanisms we strove to take the next step and explore the association of two genes suggested by these studies with type 1 diabetes. As a method of choice we decided to use lentiviral RNAi in non obese diabetic (NOD) mice, a widely-used model for type 1 diabetes, introducing a shRNA directed against the target message into the genome of this mouse strain via a lentivirus. This allowed us to study the partial loss-of-function of the target gene within the context of diabetes, directly seeing its effect on autoimmune mechanisms. In this thesis we examined two different genes in this manner, Ctla4 and Clec16a. A type 1 diabetes associated polymorphism in the CTLA4 gene had been found to alter the splicing ratio of its variants soluble CTLA-4 (sCTLA-4) and full length CTLA-4, the associated allele producing less sCTLA-4 than the protective allele. We mimicked this effect by specifically targeting the sCtla4 mRNA via lentiviral RNAi in the NOD model. As a result we could confirm the reduction of sCTLA-4 to accelerate type 1 diabetes development. Furthermore we could show a function of sCTLA-4 in regulatory T cells, more specifically at least partly in their ability to modulate costimulation by antigen presenting cells. The second candidate gene, Clec16a was targeted with the shRNA in a way that was designed to knock down most splice variants. As the gene function and the effect of the associated SUMMARY 10 polymorphism was unknown, we reasoned this method to be feasible to investigate its role in type 1 diabetes. The knockdown of Clec16a in NOD mice resulted in an almost complete protection from diabetes development that could be attributed to T cells dysfunction. However, as expression patterns and a study of the Drospophila orthologue suggested a possible role of CLEC16A in antigen presentation we also examined antigen presenting cells in the thymus and periphery. Although we did not detect any effect of the knockdown on peripheral antigen presenting cells, thymic epithelial cells were clearly affected by the loss of CLEC16A, rendering them more activated and shifting the ratio of cortical to medullary epithelial cells in favor of cortical cells. We therefore suggest a role of CLEC16A in the selection of T cells, that needs, however, to be further investigated. In this thesis we provided a feasible and fast method to study function of genes and even of single splice variants within the NOD mouse model. We demonstrate its usefulness on two candidate genes associated with type 1 diabetes by confirming and unraveling the cause of their connection to the disease.
PTPN22 encodes the lymphoid tyrosine phosphatase Lyp that can dephosphorylate Lck, ZAP-70 and Fyn to attenuate TCR signaling. A single-nucleotide polymorphism (C1858T) causes a substitution from arginine (R) to tryptophan (W) at 620 residue (R620W). Lyp-620W has been confirmed as a susceptible allele in multiple autoimmune diseases, including type 1 diabetes (T1D). Several independent studies proposed that the disease-associated allele is a gain-of-function variant. However, a recent report found that in human cells and a knockin mouse containing the R620W homolog that Ptpn22 protein degradation is accelerated, indicating Lyp-620W is a loss-of-function variant. Whether Lyp R620W is a gain- or loss-of-function variant remains controversial. To resolve this issue, we generated two lines (P2 and P4) of nonobese diabetic (NOD) mice in which Ptpn22 can be inducibly silenced by RNAi. We found long term silencing of Ptpn22 increased spleen cellularity and regulatory T (Treg) cell numbers, replicating the effect of gene deletion reported in the knockout (KO) B6 mice. Notably, Ptpn22 silencing also increased the reactivity and apoptotic behavior of B lymphocytes, which is consistent with the reduced reactivity and apoptosis of human B cells carrying the alleged gain-of-function PTPN22 allele. Furthermore, loss of Ptpn22 protected P2 KD mice from spontaneous and Cyclophosphamide (CY) induced diabetes. Our data support the notion that Lyp-620W is a gain-of-function variant. Moreover, Lyp may be a valuable target for the treatment of autoimmune diseases.
Single-molecule microscopy is one of the decisive methodologies that allows one to clarify cellular signaling in both spatial and temporal dimentions by tracking with nanometer precision the diffusion of individual microscopic particles coupled to relevant biological molecules. Trajectory analysis not only enables determination of the mechanisms that drive and constrain the particles motion but also to reveal crucial information about the molecule interaction, mobility, stoichiometry, all existing subpopulations and unique functions of particular molecules. Efficacy of this technique depends on two problematic issues the usage of the proper fluorophore and the type of biochemical attachment of the fluorophore to a biomolecule. The goal of this study was to evolve a highly specific labeling method suitable for single molecule tracking, internalization and trafficking studies that would attain a calculable 1:1 fluorophore-to-receptor stoichiometry. A covalent attachment of quantum dots to transmembrane receptors was successfully achieved with a techinque that amalgamates acyl carrier protein (ACP) system as a comparatively small linker and coenzyme A (CoA)-functionalized quantum dots. The necessity of optimization of the quantum dot usage for more precise calculation of the membrane protein stoichiometries in larger assemblies led to the further study in which methods maximizing the number of signals and the tracking times of diverse QD types were examined. Next, the optimized techniques were applied to analyze behavior of interleukin-5 β-common chain receptor (IL-5Rβc) receptors that are endogenously expressed at low level on living differentiated eosinophil-like HL-60 cells. Obtained data disclosed that perused receptors form stable and higher order oligomers. Additionally, the mobility analysis based on increased in number (>10%) uninterrupted 1000-step trajectories revealed two patterns of confined motion. Thereupon methods were developed that allow both, determination of stoichiometries of cell surface protein complexes and the acquisition of long trajectories for mobility analysis. Sequentially, the aforementioned methods were used to scrutinize on the mobility, internalization and recycling dynamics characterization of a G protein-coupled receptor (GPCRs), the parathyroid hormone receptor (PTHR1) and several bone morphogenetic proteins (BMPs), a member of the TGF-beta superfamily of receptors. These receptors are two important representatives of two varied membrane receptor classes. BMPs activate SMAD- and non-SMAD pathways and as members of the transforming growth factor β (TGF-β) superfamily are entailed in the regulation of proliferation, differentiation, chemotaxis, and apoptosis. For effective ligand induced and ligand independent signaling, two types of transmembrane serine/threonine kinases, BMP type I and type II receptors (BMPRI and BMPRII, respectively) are engaged. Apparently, the lateral mobility profiles of BMPRI and BMPRII receptors differ markedly, which determinate specificity of the signal. Non-SMAD signaling and subsequent osteoblastic differentiation of precursor cells particularly necessitate the confinement of the BMP type I receptor, resulting in the conclusion that receptor lateral mobility is a dominative mechanism to modulate SMAD versus non-SMAD signaling during differentiation. Confined motion was also predominantly observed in the studies devoted to, entailed in the regulation of calcium homeostasis and in bone remodeling, the parathyroid hormone receptor (PTHR1), in which stimulation with five peptide ligands, specific fragments of PTH: hPTH(1–34), hPTHrP(107–111)NH2; PTH(1–14); PTH(1–28) G1R19, bPTH(3–34), first four belonging to PTH agonist group and the last to the antagonist one, were tested in the wide concentration range on living COS-1 and AD293 cells. Next to the mobility, defining the internalization and recycling rates of the PTHR1 receptor maintained in this investigation one of the crucial questions. Internalization, in general, allows to diminish the magnitude of the receptor-mediated G protein signals (desensitization), receptor resensitization via recycling, degradation (down-regulation), and coupling to other signaling pathways (e.g. MAP kinases). Determinants of the internalization process are one of the most addressed in recent studies as key factors for clearer understanding of the process and linking it with biological responses evoked by the signal transduction. The internalization of the PTH-receptor complex occurs via the clathrin-coated pit pathway involving β-arrestin2 and is initiated through the agonist occupancy of the PTHR1 leading to activation of adenylyl cyclase (via Gs), and phosphatidylinositol-specific phospholipase Cβ (via Gq). Taken together, this work embodies complex study of the interleukin-5 β-common chain receptor (IL-5Rβc) receptors, bone morphogenetic proteins (BMPs) and the parathyroid hormone receptor with the application of single-molecule microscopy with the newly attained ACP-quantum dot labeling method and standard techniques.
Cross-striated muscles enable higher animals to perform directed movements and to create mechanical force. The cells of heart and skeletal muscles consist of myofibrils, serial arrays of the smallest contractile subunits, the sarcomeres. Main components of the sarcomeres are the thin and thick filaments, large protein assemblies consisting of mainly actin (thin filaments) and myosin (thick filaments), whose energy-dependent interaction is responsible for the contraction of sarcomeres and so of the whole muscle. The thin filaments are anchored in the sarcomere bordering Z-discs, while the thick filaments are anchored in the M-bands, traverse structures in the sarcomere center. Electron-microscopic studies revealed that the M-bands consist of regular, lattice-like structures that appear to cross-link the thick filaments. A number of proteins could be identified by immune-fluorescence and biochemical binding studies to be present and interact with each other in the M-bands. These data have been integrated into preliminary models of the M-bands. Detailed knowledge of how these proteins interact with each other in the center of the sarcomeres is, however, largely missing. The current study focuses on the structural characterization of the interactions between the titin, myomesin-1, obscurin and obscurin-like 1 (OBSL1), modular filamentous proteins interacting with each other in the M-bands. The high-resolution crystal structure of the titin M10 – OBSL1 Ig1 complex was solved. The structure and additional biophysical data show that titin and OBSL1 as well as titin and obscurin form stable binary complexes through the formation of a small intermolecular ß-sheet. In contrast to previously characterized intermolecular assemblies of sarcomeric proteins, this sheet is formed between parallel non- homologous ß-strands of the interaction partners. The investigation of disease-related variants of the M10 domain by biophysical methods did not allow to draw unambiguous conclusions on a direct connection between impaired OBSL1/obscurin binding and disease development. Two out of four known M10 variants have effects on the correct domain folding and so interfere with the ability to bind obscurin/OBSL1. The two other known variants displayed however only minor effects on fold and binding affinities. It should therefore be further elucidated whether a direct connection between impaired complex formation and disease development exists. -I- Abstract A direct interaction between titin and myomesin-1 could not be confirmed in vitro. Possible explanations for the different results are discussed. While the consequences of the inability of both proteins to interact are unclear, the further characterization of the putative interacting parts of titin and myomesin-1 led to the discovery of two new potential sites of self-assembly on M-band titin and myomesin-1. The crystal structure of titin M4 showed that this domain can form dimeric assemblies through the formation of a disulfide bridge and an intermolecular metal binding site between residues that are unique to this domain. On myomesin-1, in addition to the described C-terminal interaction site, a potential second site of self-assembly was found in its central Fn3-domain segment. The interacting site was mapped to the predicted Fn3 domain My5. The crystal structure of the domain in its dimeric form showed that the interaction is mediated by a mechanism that has previously not been observed in sarcomeric proteins. Two My5 interact with each other by the mutual exchange of an N-terminal ß-strand which complements the Fn3 fold on the binding partner. This type of interaction can be interpreted as misfolding. However, the position of the interacting domain and its mode of interaction allowed the postulation of a model of how myomesin-1 could be integrated in the M-bands. This model is in good agreement with the electron-microscopic appearance of the M-bands.
The scope of this work is to develop a novel single-molecule imaging technique by combining atomic force microscopy (AFM) and optical fluorescence microscopy. The technique is used for characterizing the structural properties of multi-protein complexes. The high-resolution fluorescence microscopy and AFM are combined (FIONA-AFM) to allow for the identification of individual proteins in such complexes. This is achieved by labeling single proteins with fluorescent dyes and determining the positions of these fluorophores with high precision in an optical image. The same area of the sample is subsequently scanned by AFM. Finally, the two images are aligned and the positions of the fluorophores are displayed on top of the topographical data. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, fluorescence and AFM information can be aligned with an accuracy better than 10 nm, which is sufficient to identify single fluorescently labeled proteins in most multi-protein complexes. The limitations of localization precision and accuracy in fluorescence and AFM images are investigated, including their effects on the overall registration accuracy of FIONA-AFM hybrid images. This combination of the two complementary techniques opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5–10 nm) information about the conformational properties of multi-protein complexes while the fluorescence can indicate spatial relationships of the proteins within the complexes. Additionally, computer simulations are performed in order to validate the accuracy of the registration algorithm.
Pneumolysin, a protein toxin, represents one of the major virulence factors of Streptococcus pneumoniae. This pathogen causes bacterial meningitis with especially high disease rates in young children, elderly people and immunosuppressed patients. The protein toxin belongs to the family of cholesterol-dependent cytolysins, which require membrane cholesterol in order to bind and to be activated. Upon activation, monomers assemble in a circle and undergo conformational change. This conformational change leads to the formation of a pore, which eventually leads to cell lysis. This knowledge was obtained by studies that used a higher concentration compared to the concentration of pneumolysin found in the cerebrospinal fluid of meningitis patients. Thus, a much lower concentration of pneumolysin was used in this work in order to investigate effects of this toxin on primary mouse astrocytes. Previously, a small GTPase activation, possibly leading to cytoskeletal changes, was found in a human neuroblastoma cell line. This led to the hypothesis that pneumolysin can lead to similar cytoskeletal changes in primary cells. The aim of this work was to investigate and characterise the effects of pneumolysin on primary mouse astrocytes in terms of a possible pore formation, cellular trafficking and immunological responses. Firstly, the importance of pore-formation on cytoskeletal changes was to be investigated. In order to tackle this question, wild-type pneumolysin and two mutant variants were used. One variant was generated by exchanging one amino acid in the cholesterol recognising region, the second variant was generated by deleting two amino acids in a protein domain that is essential for oligomerisation. These variants should be incapable of forming a pore and were compared to the wild-type in terms of lytic capacities, membrane binding, membrane depolarisation, pore-formation in artificial membranes (planar lipid bilayer) and effects on the cytoskeleton. These investigations resulted in the finding that the pore-formation is required for inducing cell lysis, membrane depolarisation and cytoskeletal changes in astrocytes. The variants were not able to form a pore in planar lipid bilayer and did not cause cell lysis and membrane depolarisation. However, they bound to the cell membrane to the same extent as the wild-type toxin. Thus, the pore-formation, but not the membrane binding was the cause for these changes. Secondly, the effect of pneumolysin on cellular trafficking was investigated. Here, the variants showed no effect, but the wild-type led to an increase in overall endocytotic events and was itself internalised into the cell. In order to characterise a possible mechanism for internalisation, a GFP-tagged version of pneumolysin was used. Several fluorescence-labelled markers for different endocytotic pathways were used in a co-staining approach with pneumolysin. Furthermore, inhibitors for two key-players in classical endocytotic pathways, dynamin and myosin II, were used in order to investigate classical endocytotic pathways and their possible involvement in toxin internalisation. The second finding of this work is that pneumolysin is taken up into the cell via dynamin- and caveolin-independent pinocytosis, which could transfer the toxin to caveosomes. From there, the fate of the toxin remains unknown. Additionally, pneumolysin leads to an overall increase in endocytotic events. This observation led to the third aim of this work. If the toxin increases the overall rate of endocytosis, the question arises whether toxin internalisation favours bacterial tissue penetration of the host or whether it serves as a defence mechanism of the cell in order to degrade the protein. Thus, several proinflammatory cytokines were investigated, as previous studies describe an effect of pneumolysin on cytokine production. Surprisingly, only interleukin 6-production was increased after toxin-treatment and no effect of endocytotic inhibitors on the interleukin 6-production was observed. The conclusion from this finding is that pneumolysin leads to an increase of interleukin 6, which would not depend on the endocytotic uptake of pneumolysin. The production of interleukin 6 would enhance the production of acute phase proteins, T-cell activation, growth and differentiation. On the one hand, this activation could serve pathogen clearance from infected tissue. On the other hand, the production of interleukin 6 could promote a further penetration of pathogen into host tissue. This question should be further investigated.
Streptococcus pneumoniae is one of the major causes of bacterial meningitis, which mainly affects young infants in the developing countries of Africa, Asia (esp. India) and South America, and which has case fatality rates up to 50% in those regions. Bacterial meningitis comprises an infection of the meninges and the sub-meningeal cortex tissue of the brain, whereat the presence of pneumolysin (PLY), a major virulence factor of the pneumococcus, is prerequisite for the development of a severe outcome of the infection and associated tissue damage (e. g. apoptosis, brain edema, and ischemia). Pneumolysin belongs to the family of pore forming, cholesterol-dependent cytolysins (CDCs), bacterial protein toxins, which basically use membrane-cholesterol as receptor and oligomerize to big aggregates, which induce cell lysis and cell death by disturbance of membrane integrity. Multiple recent studies, including this work, have revealed a new picture of pneumolysin, whose cell-related properties go far beyond membrane binding, pore formation and the induction of cell death and inflammatory responses. For a long time, it has been known that bacteria harm the tissues of their hosts in order to promote their own survival and proliferation. Many bacterial toxins aim to rather hijack cells than to kill them, by interacting with cellular components, such as the cytoskeleton or other endogenous proteins. This study was able to uncover a novel capacity of pneumolysin to interact with components of the actin machinery and to promote rapid, actin-dependent cell shape changes in primary astrocytes. The toxin was applied in disease-relevant concentrations, which were verified to be sub-lytic. These amounts of toxin induced a rapid actin cortex collapse in horizontal direction towards the cell core, whereat membrane integrity was preserved, indicating an actin severing function of pneumolysin, and being consistent with cell shrinkage, displacement, and blebbing observed in live cell imaging experiments. In contrast to neuroblastoma cells, in which pneumolysin led to cytoskeleton remodeling and simultaneously to activation of Rac1 and RhoA, in primary astrocytes the cell shape changes were seen to be primarily independent of small GTPases. The level of activated Rac1 and RhoA did not increase at the early time points after toxin application, when the initial shape changes have been observed, but at later time points when the actin-dependent displacement of cells was slower and less severe, probably presenting the cell’s attempt to re-establish proper cytoskeleton function. A GUV (giant unilamellar vesicle) approach provided insight into the effects of pneumolysin in a biomimetic system, an environment, which is strictly biochemical, but still comprises cellular components, limited to the factors of interest (actin, Arp2/3, ATP, and Mg2+ on one side, and PLY on the other side). This approach was able to show that the wildtype-toxin, but not the Δ6 mutant (mutated in the unfolding domain, and thus non-porous), had the capacity to exhibit its functions through a membrane bilayer, meaning it was able to aggregate actin, which was located on the other side of the membrane, either via direct interaction with actin or in an Arp2/3 activating manner. Taking a closer look at these two factors with the help of several different imaging and biochemical approaches, this work unveiled the capacity of pneumolysin to bind and interact both with actin and Arp2 of the Arp2/3 complex. Pneumolysin was capable to slightly stabilize actin in an actin-pyrene polymerization assay. The same experimental setup was applied to show that the toxin had the capacity to lead to actin polymerization through activation of the Arp2/3 complex. This effect was additionally confirmed with the help of fluorescent microscopy of rhodamine (TRITC)-tagged actin. Strongest Arp2/3 activation, and actin nucleation/polymerization is achieved by the VCA domain of the WASP family proteins. However, addition of PLY to the Arp2/3–VCA system led to an enhanced actin nucleation, suggesting a synergistic activation function of pneumolysin. Hence, two different effects of pneumolysin on the actin cytoskeleton were observed. On the one hand an actin severing property, and on the other hand an actin stabilization property, both of which do not necessarily exclude each other. Actin remodeling is a common feature of bacterial virulence strategies. This is the first time, however, that these properties were assigned to a toxin of the CDC family. Cytoskeletal dysfunction in astrocytes leads to dysfunction and unregulated movement of these cells, which, in context of bacterial meningitis, can favor bacterial penetration and spreading in the brain tissue, and thus comprises an additional role of pneumolysin as a virulence factor of Streptococcus pneumonia in the context of brain infection.
The Nucleotide Excision Repair (NER) pathway is able to remove a vast diversity of structurally unrelated DNA lesions and is the only repair mechanism in humans responsible for the excision of UV induced DNA damages. The NER mechanism raises two fundamental questions: 1) How is DNA damage recognition achieved discriminating damaged from non damaged DNA? 2) How is DNA incision regulated preventing endonucleases to cleave DNA non specifically but induce and ensure dual incision of damaged DNA? Thus, the aim of this work was to investigate the mechanisms leading from recognition to incision of damaged DNA. To decipher the underlying process of damage recognition in a prokaryotic model system, the intention of the first part of this work was to co crystallize the helicase UvrB form Bacillus caldotenax together with a DNA substrate comprising a fluorescein adducted thymine as an NER substrate. Incision assays were performed to address the question whether UvrB in complex with the endonuclease UvrC is able to specifically incise damaged DNA employing DNA substrates with unpaired regions at different positions with respect to the DNA lesion. The results presented here indicate that the formation of a specific pre incision complex is independent of the damage sensor UvrA. The preference for 5’ bubble substrate suggests that UvrB is able to slide along the DNA favorably in a 5’ → 3’ direction until it directly encounters a DNA damage on the translocating strand to then recruit the endonuclease UvrC. In the second part of this work, the novel endonuclease Bax1 from Thermoplasma acidophilum was characterized. Due to its close association to archaeal XPB, a potential involvement of Bax1 in archaeal NER has been postulated. Bax1 was shown to be a Mg2+ dependent, structure specific endonuclease incising 3’ overhang substrates in the single stranded region close to the ssDNA/dsDNA junction. Site directed mutagenesis of conserved amino acids was employed to identify putative active site residues of Bax1. In complex with the helicase XPB, however, incision activity of Bax1 is altered regarding substrate specificity. The presence of two distinct XPB/Bax1 complexes with different endonuclease activities indicates that XPB regulates Bax1 incision activity providing insights into the physical and functional interactions of XPB and Bax1.
Polarity and migration are essential for T cell activation, homeostasis, recirculation and effector function. To address how T cells coordinate polarization and migration when interacting with dendritic cells (DC) during homeostatic and activating conditions, a low density collagen model was used for confocal live-cell imaging and high-resolution 3D reconstruction of fixed samples. During short-lived (5 to 15 min) and migratory homeostatic interactions, recently activated T cells simultaneously maintained their amoeboid polarization and polarized towards the DC. The resulting fully dynamic and asymmetrical interaction plane comprised all compartments of the migrating T cell: the actin-rich leading edge drove migration but displayed only moderate signaling activity; the mid-zone mediated TCR/MHC induced signals associated with homeostatic proliferation; and the rear uropod mediated predominantly MHC independent signals possibly connected to contact-dependent T cell survival. This “dynamic immunological synapse” with distinct signaling sectors enables moving T cells to serially sample antigen-presenting cells and resident tissue cells and thus to collect information along the way. In contrast to homeostatic contacts, recognition of the cognate antigen led to long-lasting T cell/DC interaction with T cell rounding, disintegration of the uropod, T cell polarization towards the DC, and the formation of a symmetrical contact plane. However, the polarity of the continuously migrating DC remained intact and T cells aggregated within the DC uropod, an interesting cellular compartment potentially involved in T cell activation and regulation of the immune response. Taken together, 3D collagen facilitates high resolution morphological studies of T cell function under realistic, in vivo-like conditions.
Bacterial protein toxins belong to the most potent toxins which are known. They exist in many different forms and are part of our every day live. Some of them are spread by the bacteria during infections and therefore play a crucial role in pathogenicity of these strains. Others are secreted as a defense mechanism and could be uptaken with spoiled food. Concerning toxicity, some of the binary toxins of the AB7-type belong to the most potent and dangerous toxins in the world. Even very small amounts of these proteins are able to cause severe symptoms during an infection with pathogen species of the genus Clostridium or Bacillus. Apart from the thread the toxins constitute, they exhibit a unique way of intoxication. Members of the AB7-toxin family consist of a pore-forming subunit B, that acts as a molecular syringe to translocate the enzymatic moieties A into the cytosol of target cells. This complex mechanism does not only kill cells with high efficiency and therefore should be studied for treatment, but also displays a possibility to address certain cells with a specific protein cargo if used as a molecular delivery tool. Concerning both issues, binding and translocation of the channel are the crucial steps to either block or modify the system in the desired way. To gain deeper insight into the transport of binary toxins the structure of the B subunit is of great importance, but being a membrane protein, no crystal could be obtained up to now for either protective antigen (PA) of Anthrax toxin or any other AB7-type binding domain. Therefore, the method of choice in this work is an electro-physical approach using the so-called black-lipid-bilayer system for determination of biophysical constants. Additionally, diverse cell based assays serve as a proving method for the data gained during in vitro measurements. Further information was gathered with specially designed mutants of the protein channel. The first part of this thesis focuses on the translocation process and its possible use as a molecular tool to deliver protein cargo into special cell types. The task was addressed by measuring the binding of different effector proteins related and unrelated to the AB7 toxin family. These proteins were tested in titration experiments for the blockage of the ion current through a membrane saturated with toxin channels. Especially the influence of positively charged His-tags has been determined in detail for PA and C2II. As described in chapter 2, a His-tag transferred the ability of being transported by PA, but not by C2II, to different proteins like EDIN (from S. aureus) in vitro and in cell-based experiments. This process was found to change the well-known voltage-dependency of PA to a huge extend and therefore is related to membrane potentials which play a crucial role in many processes in living cells. Chapter 3 sums up findings, which depict that binding partners of PA share certain common motives. These could be detected in a broad range of substrates, ranging from simple ions in an electrolyte over small molecules to complex protein effectors. The gathered information could be further used to design blocker-substrates for treatment of Anthrax infections or tags, which render PA possible as a molecular syringe for cargo proteins. The deeper insight to homologies and differences of binary toxin components is the core of chapter 4, in which the cross-reactivity of Anthrax and C2-toxin was analyzed. The presented results lead to a better understanding of different motives involved in binding and translocation to and via the B components PA and C2II, as well as the enzymatically active A moieties edema factor (EF), lethal factor (LF) and C2I. In the second part of the thesis, the blockage of intoxication is the center of interest. Therefore, chapter 5 focuses on the analysis of specially designed blocker-substrate molecules for PA. These molecules form a plug in the pore, abolishing translocation of the enzymatic units. Especially, if multi-resistant strains of Anthrax (said to be already produced in Russia as a biological weapon) are taken into consideration, these substrates could stop intoxication and buy time, to deal with the infection. Chapter 6 describes the blockage of PA-channels by anti-His antibody from the trans-side of the porin, an effect which was not described for any other antibody before. Interestingly, even mutation of the estimated target amino acid Histidine 310 to Glycine could not interfere with this ionic strength dependent binding.
There is such vast amount of visual information in our surroundings at any time that filtering out the important information for further processing is a basic requirement for any visual system. This is accomplished by deploying attention to focus on one source of sensory inputs to the exclusion of others (Luck and Mangun 2009). Attention has been studied extensively in humans and non human primates (NHPs). In Drosophila, visual attention was first demonstrated in 1980 (Wolf and Heisenberg 1980) but this field remained largely unexplored until recently. Lately, however, studies have emerged that hypothesize the role of attention in several behaviors but do not specify the characteristic properties of attention. So, the aim of this research was to characterize the phenomenon of visual attention in wild-type Drosophila, including both externally cued and covert attention using tethered flight at a torque meter. Development of systematic quantifiable behavioral tests was a key aspect for this which was not only important for analyzing the behavior of a population of wild-type flies but also for comparing the wild-type flies with mutant flies. The latter would help understand the molecular, genetic, and neuronal bases of attention. Since Drosophila provides handy genetic tools, a model of attention in Drosophila will serve to the greater questions about the neuronal circuitry and mechanisms involved which might be analogous to those in primates. Such a model might later be used in research involving disorders of attention. Attention can be guided to a certain location in the visual field by the use of external cues. Here, using visual cues the attention of the fly was directed to one or the other of the two visual half-fields. A simple yet robust paradigm was designed with which the results were easily quantifiable. This paradigm helped discover several interesting properties of the cued attention, the most substantial one being that this kind of external guidance of attention is restricted to the lower part of the fly’s visual field. The guiding cue had an after-effect, i.e. it could occur at least up to 2 seconds before the test and still bias it. The cue could also be spatially separated from the test by at least 20° and yet attract the attention although the extent of the focus of attention (FoA) was smaller than one lower visual half-field. These observations excluded the possibility of any kind of interference between the test and the cue stimuli. Another interesting observation was the essentiality of continuous visibility of the test stimulus but not the cue for effective cuing. When the contrast of the visual scene was inverted, differences in response frequencies and cuing effects were observed. Syndirectional yaw torque responses became more frequent than the antidirectional responses and cuing was no longer effective in the lower visual field with inverted contrast. Interestingly, the test stimulus with simultaneous displacement of two stripes not only effectuated a phasic yaw torque response but also a landing response. A 50 landing response was produced in more than half of the cases whenever a yaw torque response was produced. Elucidation of the neuronal correlates of the cued attention was commenced. Pilot experiments with hydroxyurea (HU) treated flies showed that mushroom bodies were not required for the kind of guidance of attention tested in this study. Dopamine mutants were also tested for the guidance of attention in the lower visual field. Surprisingly, TH-Gal4/UAS-shits1 flies flew like wild-type flies and also showed normal optomotor response during the initial calibration phase of the experiment but did not show any phasic yaw torque or landing response at 18 °C, 25 °C or 30 °C. dumb2 flies that have almost no D1 dopamine receptor dDA1 expression in the mushroom bodies and the central complex (Kim et al. 2007) were also tested and like THGal4/ UAS-shits1 flies did not show any phasic yaw torque or landing response. Since the dopamine mutants did not show the basic yaw torque response for the test the role of dopamine in attention could not be deduced. A different paradigm would be needed to test these mutants. Not only can attention be guided through external cues, it can also be shifted endogenously (covert attention). Experiments with the windows having oscillating stripes nicely demonstrated the phenomenon of covert attention due to the production of a characteristic yaw torque pattern by the flies. However, the results were not easily quantifiable and reproducible thereby calling for a more systematic approach. Experiments with simultaneous opposing displacements of two stripes provide a promising avenue as the results from these experiments showed that the flies had a higher tendency to deliver one type of response than when the responses would be produced stochastically suggesting that attention increased this tendency. Further experiments and analysis of such experiments could shed more light on the mechanisms of covert attention in flies.
Die chronische Herzinsuffizienz stellt nach wie vor eine der häufigsten Todesursachen weltweit dar. Trotz intensiver Forschung ist es bisher nicht möglich die pathophysiologischen Prozesse aufzuhalten. Es wird nach neuen Strategien gesucht, hier therapeutisch eingreifen zu können. Kleine nicht-kodierende RNAs, sogenannte microRNAs (miRNAs), wurden als wichtige Faktoren bei verschiedenen Herzkrankheiten beschrieben. Die Mehrzahl der bisherigen Studien fokussierte sich dabei auf die am stärksten deregulierten miRNAs im erkrankten Herz. In einer automatisierten Analyse im 96 Well-Format untersuchten wir 230 miRNAs auf ihr Potential, in das Größenwachstum von primären Kardiomyozyten einzugreifen. Aus den miRNAs mit den größten Effekten selektierten wir diejenigen, die eine hohe endogene Expression aufwiesen, und unterzogen sie einem Validierungsprozess. Hier konnten wir die Effekte aller pro- (miR-22, miR-30c, miR-30d, miR-212, miR-365) und anti-hypertrophen (miR-27a, miR-27b, miR-133a) miRNAs bestätigen. Die Mehrzahl dieser miRNAs wurde hiermit erstmalig beschrieben, dass sie eine wichtige Rolle beim Größenwachstum von Kardiomyozyten spielen. Sie wären daher interessante Kandidaten für detaillierte funktionelle Studien mit dem Ziel ihr therapeutisches Potential zu evaluieren. In einem früheren genetischen Screen zur Identifizierung von kardialen, sezernierten Faktoren wurde der Protease Inhibitor 16 (PI16) entdeckt, der sich im insuffizienten Herz durch eine starke Akkumulation auszeichnet. Gegenstand des zweiten Teils dieser Arbeit war es, eine Mauslinie zu generieren, in der PI16 global oder konditionell mit Hilfe des Cre/LoxP-Systems ausgeschaltet werden kann. Nach Elektroporation des Pi16floxneo Targeting Vektors in embryonale Stammzellen und Blastozysteninjektion erhielten wir eine Mauslinie, die Träger der zielgerichteten Modifikation des Pi16 Allels war. Mit der globalen genetischen Deletion des LoxP-flankierten Abschnitts von Exon 3 bis 4 konnten wir die Expression des Pi16 Gens komplett unterbinden. Die PI16 Defizienz führte weder im Herz noch in anderen Organen per se zu pathologischen Veränderungen. Zudem war unbekannt, dass PI16 in der gesunden Maus in der kardialen Fibroblastenfraktion enthalten sowie in den Zilien der Epididymis und der Trachea und im Lumen der Schilddrüse lokalisiert ist. Im insuffizienten Herz bestätigten wir eine Akkumulation von PI16, die sich vor allem auf die fibrotischen Bereiche beschränkte. Das lässt Grund zur Annahme, dass die kardiale Funktion von PI16 erst dann offensichtlich wird, wenn man die defizienten Mäuse zukünftig entsprechenden Stressmodellen aussetzt. Das wird zu einem umfassenden Verständnis der kardialen Funktion von PI16 und dessen Potential als therapeutisches Zielmolekül führen.
Type 1 diabetes affects around 0.5% of the population in developed countries and the incidence rates have been rising over the years. The destruction of beta cells is irreversible and the current therapy available to patients only manages the symptoms and does not prevent the associated pathological manifestations. The patients need lifelong therapy and intensive research is being carried out to identify ways to eliminate autoimmune responses directed against pancreatic beta cells and to replace or regenerate beta cells. The work presented herein aimed at analyzing the role of the Th17 T cell subset, characterized by secretion of the pro- inflammatory cytokine IL-17A, in autoimmune diabetes and also at generating a beta cell reporter mouse line in the NOD background, the most widely- used mouse model for type 1 diabetes. We generated IL- 17A knockdown (KD) NOD mice, using RNAi in combination with lentiviral transgenesis. We analyzed diabetes frequency in IL-17A deficient mice and found that the loss of IL-17A did not protect the transgenic mice from diabetes. Based on these observations, we believe that Th17 cells do not play a critical role in type 1 diabetes through the IL-17A pathway, though they might still be involved in the disease process through alternate pathways. We also generated NOD and NOD-SCID mice with a transgene that drives the beta cell specific expression of a luciferase reporter gene. We used a lentiviral construct, which combined a luciferase sequence and a short- hairpin RNA (shRNA) expression cassette, allowing gene- knockdown under the beta cell specific rat insulin promoter (RIP). These mice will be of use in studying beta cell phenotypes resulting from the knockdown of target genes, using non- invasive bioimaging. We believe that the generation of these reporter mouse lines for diabetes studies will prove valuable in future investigations. Furthermore, the demonstration that the loss of IL-17A does not alter susceptibility to type 1 diabetes should help clarify the controversial involvement of Th17 cells in this disease.
Auf dem Weg vom Primärtumor zur systemischen Metastasierung, der Haupttodesursache von Krebserkrankungen, ist die Einzelzellmigration von Tumorzellen durch dreidimensionales Bindegewebe ein entscheidender Schritt. Die vorliegende Arbeit zeigt Untersuchungen zur Tumorzellmigration und –plastizität in einem 3D-Migrationsmodell. Kleine G-Proteine kontrollieren Zytoskelettfunktionen, insbesondere Aktinpolymerisation und die Bildung von Zellprotrusionen durch Rac sowie Actomyosinkontraktion durch Rho. Durch pharmakologische Inhibitoren von Rac und dem Rho-Effektor ROCK soll deren Bedeutung für Einzelzellmigration in einem dreidimensionalen Modell und vor allem der Effekt auf Morphologie, Plastizität und Migration von Tumorzellen geklärt werden. Nach Inhibition von ROCK zeigen hochinvasive HT1080 Fibrosarkomzellen einen multipolar-dendritischen und sessilen Phänotyp. Nach Hemmung von Rac wird hingegen ein rundlicher, aber ebenfalls apolarer und sessiler Phänotyp induziert. Bei simultaner Inhibition von Rac und ROCK entstehen rundliche, apolare, sessile Zellen mit abortiven Pseudopodien. Wird das Gleichgewicht von Rac und ROCK durch konstitutive Aktivierung von ROCK gestört, so entsteht eine zweigeteilte Population, bestehend aus rundlichen Zellen, die Blebs bilden, und langgezogenen Zellen. Nach Sortierung nach ihrem ß1-Integrinexpressionsniveau zeigten Zellen mit niedriger Integrin-Expression einen rundlichen Migrationstyp mit blasenartigen dynamischen Protrusionen, während Zellen mit hoher Integrin-Expression langgezogen-mesenchymal migrierten. Somit steuern ROCK und Rac gemeinsam und zeitgleich die mesenchymale Einzelzellmigration. Während Rac Protrusion vermittelt, ist ROCK für Kontraktilität und Retraktion verantwortlich. Erst durch Koordination von Rac und Rho/ROCK entsteht somit Polarität und 3D mesenchymale Migration.
Das ANP/GC-A-System spielt durch die Produktion des sekundären Botenstoffs cGMP eine wichtige Rolle bei der Regulation des Blutdruckes und des Blutvolumens. Bei Patienten mit Herzhypertrophie oder Herzinsuffizienz sind die ANP-Plasmakonzentrationen erhöht, aber die GC-A-vermittelten Effekte stark reduziert, was auf einen Defekt des Signalsystems hinweist. Studien an metabolisch markierten GC-A-überexprimierenden HEK 293-Zellen zeigten, dass der GC-A-Rezeptor im basalen Zustand stark phosphoryliert und die homologe bzw. heterologe Desensitisierung wahrscheinlich mit einer Dephosphorylierung verbunden ist. Die Desensitisierung stellt einen Mechanismus dar, der in vivo zu einem Funktionsverlust des Rezeptors beitragen könnte. Im Rahmen dieser Arbeit konnten mittels Massenspektrometrie sieben Phosphorylierungsstellen in der Kinasehomologen Domäne aus FLAG-GC-A exprimierenden HEK 293-Zellen detektiert werden: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 und Thr513. Die massenspektrometrische relative Quantifizierung basierend auf der Multiple-Reaction-Monitoring (MRM)-Methode zeigte bei ANP-induzierter, homologer Desensitisierung eine Dephosphorylierung der Phosphorylierungsstellen Ser497, Thr500, Ser502, Ser506, Ser510 und Thr513, was mit bereits publizierten Daten übereinstimmt, und einen starken Anstieg der Phosphorylierung an Ser487. Nach Inkubation mit Angiotensin II, welches eine heterologe Desensitisierung hervorruft, wurde eine Reduzierung aller Phosphorylierungen verzeichnet, die zudem stärker ausgeprägt war als bei der ANP-abhängigen Desensitisierung. Die Funktion der neu identifizierten Phosphorylierung an Ser487 wurde mittels Mutagenese analysiert. Die Substitution des Serins durch Alanin, welche den unphosphorylierten Zustand nachstellt, resultierte in einer Rezeptoraktivität und desensitisierung vergleichbar zum GC-A Wildtyp-Rezeptor. Wurde hingegen Serin gegen Glutamat getauscht, um den phosphorylierten Zustand zu imitieren, konnte der Rezeptor weder aktiviert noch desensitisiert werden. Diese Ergebnisse bestätigen vorherige Studien, dass die GC-A-Rezeptorantwort auf ANP durch die Phosphorylierungen reguliert wird. Allerdings scheint bei der homologen Desensitisierung die Phosphorylierung an der Position Ser487 eine Rolle zu spielen, da sie die Aktivität des Rezeptors inhibiert. Die Identifizierung und Charakterisierung dieser Phosphorylierungsstelle trägt zum Verständnis des Mechanismus der homologen Desensitierung bei. Zusätzlich konnten einige der beschriebenen Phosphorylierungen in Zellsystemen detektiert werden, die die GC-A endogen exprimieren. Dadurch sind unter physiologischen Bedingungen Analysen der Mechanismen möglich, die bei der Aktivierung und Deaktivierung der GC-A involviert sind und somit wichtige pathophysiologische Konsequenzen haben können.