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Chlamydiales are obligate intracellular gram-negative bacteria that have gained high medical relevance. These important human pathogens cause diverse diseases including trachoma and wide spread sexually transmitted diseases. Chlamydia establishes membrane bound inclusions in the host cell and loots the host for nutritional requirements. Infections are usually recognized by the host immune system and eliminated systematically, by triggering apoptosis. However, the pathogen Chlamydia has evolved various strategies to prevent the detection as well as protect the invaded cell against apoptosis or any other form of cell death. The evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. The present study was aimed at investigating the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Caspases were differentially regulated upon Sn infection. Caspase-3 and -9 were not activated upon Sn infection and apoptosis induction; whereas caspases-8 was activated in Sn infected cells even without apoptosis induction. This indicates that, Sn utilizes death receptor association independent caspase activation for thriving in the host environment. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Proteins (IAP-1/2 and XIAP) and the Akt/PI3K pathway. Sn infection also 20 activated the pro-survival transcription factor NF-кB. Blocking any of these survival pathways sensitized the infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. The NF-кB mutant cells also showed reduced infectivity of Sn, which indicated an essential role of NF-кB in Sn infection. It was interesting to observe that, Acanthamoeba castellanii, a natural host of Sn, survived maintaining its trophozoite forms after infection with Sn upon starvation. The metacaspases, responsible for encystment could be regulated by Sn upon infection. This suggests an early level of gene regulation indicating how the pathogen evolved its ability to inhibit apoptosis in higher organisms. The resistance to apoptosis pathways subverted in Sn-infected cells was similar but not identical to those modulated by Chlamydia. Together, the data supports the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.
Understanding the emergence of species' ranges is one of the most fundamental challenges in ecology. Early on, geographical barriers were identified as obvious natural constraints to the spread of species. However, many range borders occur along gradually changing landscapes, where no sharp barriers are obvious. Mechanistic explanations for this seeming contradiction incorporate environmental gradients that either affect the spatio-temporal variability of conditions or the increasing fragmentation of habitat. Additionally, biological mechanisms like Allee effects (i.e. decreased growth rates at low population sizes or densities), condition-dependent dispersal, and biological interactions with other species have been shown to severely affect the location of range margins. The role of dispersal has been in the focus of many studies dealing with range border formation. Dispersal is known to be highly plastic and evolvable, even over short ecological time-scales. However, only few studies concentrated on the impact of evolving dispersal on range dynamics. This thesis aims at filling this gap. I study the influence of evolving dispersal rates on the persistence of spatially structured populations in environmental gradients and its consequences for the establishment of range borders. More specially I investigate scenarios of range formation in equilibrium, periods of range expansion, and range shifts under global climate change ...
Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens.
The ability to produce toxins is spread among a huge variety of bacterial strains. A very prominent class of bacterial protein toxins is the family of binary AB toxins sharing a common mode of intoxication. A pore forming component B binds and translocates an enzymatic component A into the cytosol of target cells exhibiting a fatal mode of action. These components are supposed to be not toxic themselves but both required for cell toxicity. Anthrax toxin produced by the Gram-positive bacteria Bacillus anthracis is the best studied binary toxin especially since its use as a biological weapon in the context of the attacks of 9/11 in 2001. In contrast to other binary toxins, Anthrax toxin possesses two different enzymatic components, edema factor (EF), a calcium- and calmodulin-dependent adenylat-cyclase and lethal factor (LF), a zinc-dependent metalloprotease. Protective antigen (PA) is the pore-forming component responsible for binding and translocation. Clostridium botulinum possesses in addition to the well known botulinum toxin (Botox) a variety of other toxins, such as the binary C2 toxin. C2 toxin is composed of the binding and translocation moiety C2II and the enzymatic moiety C2I acting as an actin-ADP-ribosyltransferase. In this study, the mode of translocation and the binding kinetics to the enzymatic component were studied in a biophysical experimental setup. In chapter 2, the binding of the N-terminal fractions EFN and LFN to the PA channel are analyzed in artificial bilayer membranes revealing lower binding affinity compared to full-length EF and LF. Other biophysical properties like voltage-dependency and ionic-strength dependency are not influenced. The results suggest that additional forces are involved in the binding process, than those concerning the N-terminus exclusively, as it was supposed previously. As the treatment of an Anthrax infection with antibiotics is often medicated very late due to the lack of early symptoms, tools to prevent intoxication are required. 4-aminoquinolones like chloroquine are known to block the PA channel, thereby inhibiting intoxication but they also lead to severe side-effects. In chapter 3 new promising agents are described that bind to PA in artificial bilayer systems, elucidating common motives and features which are necessary for binding to PA in general. The possible interaction of Anthrax and C2 toxin is investigated by measuring the binding of one enzymatic component to the respective other toxin’s pore (chapter 4). Interestingly, in vitro experiments using the black lipid bilayer assay show that PA is able to bind to C2I resulting in half saturation constants in the nanomolar range. Furthermore, in vivo this combination of toxin components exhibits cell toxicity in human cell lines. This is first-time evidence that a heterologous toxin combination is functional in in vitro and in vivo systems. In contrast, C2II is able to bind to EF as well as to LF in vitro, whereas in in vivo studies almost no toxic effect is detected. In the case of PA, an N-terminal His6-tag attached to the enzymatic subunit increased the binding affinity (chapter 5). A His6-tag attached to not related proteins also led to high binding affinities, providing the possibility to establish PA as a general cargo protein. In chapter 6 a set of different molecules and proteins is summarized, which are either related or not related to binary toxins, PA is able to bind. In first line, the presence of positive charges is found to be responsible for binding to PA which is in accordance to the fact that PA is highly cation selective. Furthermore, we present evidence that different cationic electrolytes serve as a binding partner to the PA channel. In the last decade another toxin has aroused public attention as it was found to be responsible for a rising number of nosocomial infections: Clostridium difficile CDT toxin. The mode of action of the enzymatic subunit CDTa is similar to C2I of C2 toxin, acting as an ADP-ribosylating toxin. The channel forming and binding properties of CDT toxin are studied in artificial bilayer membranes (chapter 7). We found that two different types of channels are formed by the B component CDTb. The first channel is similar to that of iota toxin’s Ib of Clostridium perfringens with comparable single channel conductance, selectivity and binding properties to the enzymatic subunit CDTa. The formation of this type of channel is cholesterol-dependent, whereas in the absence of cholesterol another kind of channel is observed. This channel has a single channel conductance which is rather high compared to all other binary toxin channels known so far, it is anion selective and does not show any binding affinity to the enzymatic component CDTa. The results reveal completely new insights in channel formation properties and the flexibility of a pore-forming component. Additionally, these findings suggest further possibilities of toxicity of the pore forming component itself which is not known for any other binary toxin yet. Therefore, the pathogenic role of this feature has to be studied in detail.
In this thesis the Drosophila mutant loechrig (loe), that shows progressive degeneration of the nervous system, is further described. Loe is missing a neuronal isoform of the protein kinase AMPK γ subunit (AMP-activated protein kinase- also known as SNF4Aγ) The heterotrimeric AMPK controls the energy level of the cell, which requires constant monitoring of the ATP/AMP levels. It is activated by low energy levels and metabolic insults like oxygen starvation and regulates multiple important signal pathways that control cell metabolism. Still, its role in neuronal survival is unclear. One of AMPK’s downstream targets is HMGR (hydroxymethylglutaryl-CoA- reductase), a key enzyme in cholesterol and isoprenoid synthesis. It has been shown that manipulating the levels of HMGR affects the severity of the neurodegenerative phenotype in loe. Whereas the regulatory role of AMPK on HMGR is conserved in Drosophila, insects cannot synthesize cholesterol de novo. However, the synthesis of isoprenoids is a pathway that is evolutionarily conserved between vertebrates and insects. Isoprenylation of target proteins like small G-proteins provides a hydrophobic anchor that allows the association of these proteins with membranes and following activation. This thesis shows that the loe mutation interferes with the prenylation of Rho1 and the regulation of the LIM kinase pathway, which plays an important role in actin turnover and axonal outgrowth. The results suggest that the mutation in LOE, causes hyperactivity of the isoprenoid synthesis pathway, which leads to increased farnesylation of RHO1 and therefore higher levels of phospho-cofilin. A mutation in Rho1 improves the neurodegenerative phenotype and life span. The increased inactive cofilin amount in loe leads to an up regulation of filamentous actin. Actin is involved in neuronal outgrowth and experiments analyzing loe neurons gave valuable insights into a possible role of AMPK and accordingly actin on neurite growth and stability. It was demonstrated that neurons derived from loe mutants exhibit reduces axonal transport suggesting that changes in the cytoskeletal network caused by the effect of loe on the Rho1 pathway lead to disruptions in axonal transport and subsequent neuronal death. It also shows that actin is not only involved in neuronal outgrowth, its also important in maintenance of neurons, suggesting that interference with actin dynamics leads to progressive degeneration of neurons. Together, these results further support the importance of AMPK in neuronal function and survival and provide a novel functional mechanisms how alterations in AMPK can cause neuronal degeneration
Soziale Insekten wie die Honigbiene (Apis mellifera) besitzen ein breites Spektrum an Abwehrmechanismen gegen Pathogenbefall, sowohl auf der Ebene der Kolonie (soziale Immunität) als auch auf der Stufe des Individuums (angeborenes Immunsystem). Die Hauptaufgabe der relativ kurzlebigen Drohnen besteht in der Begattung von Jungköniginnen. Daher stellte sich die Frage, ob auch die Drohnen ähnlich den Arbeiterinnen mit energieaufwendigen Immunreaktionen auf Infektionen reagieren. Wie im Folgenden beschrieben, konnte ich nachweisen, dass Drohnen eine ausgeprägte Immunkompetenz besitzen. Das angeborene Immunsystem setzt sich aus humoralen und zellulären Abwehrreaktionen zusammen. Bei der humoralen Immunantwort werden bestimmte evolutionär konservierte Signalkaskaden aktiviert, an deren Ende die Expression einer Vielzahl von antimikrobiellen Peptiden (AMPs) und immunspezifischen Proteinen (IRPs) steht. Zur Analyse der humoralen Immunantwort wurden von mir zum einen Hemmhoftests durchgeführt, um die gesamte antimikrobielle Aktivität der Haemolymphe nach artifizieller Infektion zu ermitteln und zum anderen spezifische AMPs bzw. IRPs identifiziert. Hierzu wurden die Haemolymphproteine in ein- oder zwei-dimensionalen Polyacrylamidgelen aufgetrennt und ausgewählte Proteinbanden bzw. -spots mittels nano HPLC/Massenspektrometrie analysiert. Die Hauptkomponenten des zellulären Immunsystems sind Wundheilung, Phagozytose, Einkapselung und Nodulation. In meiner Arbeit habe ich zum ersten Mal Noduli bei infizierten Drohnen nachweisen können. Frisch geschlüpfte adulte Drohnen (1d) weisen ein breites Spektrum an Immunreaktionen auf, das sowohl humorale als auch zelluläre Immunantworten umfasst. Nach Infektion mit dem Gram-negativen Bakterium E.coli und verschiedenen bakteriellen Zellwandbestandteilen wie Lipopolysaccharid (LPS), Peptidoglycan (PGN) und 1,3ß-Glucan (Bestandteil von Pilzzellwänden), werden die AMPs Hymenoptaecin, Defensin 1 und Abaecin induziert. Desweiteren exprimieren junge adulte Drohnen eine Reihe hochmolekularer immunspezifischer Proteine (IRPs) wie z.B. Carboxylesterase (CE 1), eine Serinprotease, die möglicherweise an der Prozessierung der Prophenoloxidase beteiligt ist, ein Peptidoglycan-interagierendes Protein (PGRP-S2) und zwei Proteine unbekannter Funktion, IRp42 und IRp30. Parallel zu bekannten bienenspezifischen AMPs wurde ein animales Peptidtoxin (APT) in Drohnenlarven, adulten Drohnen und adulten Hummeln nach E.coli Infektion in der Haemolymphe nachgewiesen. Von dem als OCLP 1 (ω-conotoxin-like protein 1) benannten Peptid war bereits bekannt, dass es in Fischen paralytische und damit toxische Effekte auslöst. Meine Beobachtungen lassen vermuten, dass es sich bei OCLP 1 um ein Peptidtoxin mit antimikrobiellen Eigenschaften und damit um eine neue Klasse von AMPs handelt. Die allgemeine humorale Immunkompetenz scheint während der gesamten Lebensspanne adulter Drohnen (~ 7 Wochen) konstant zu bleiben, wie durch die gleichbleibende antimikrobielle Aktivität im Hemmhoftest gezeigt wurde. Junge Drohnen reagieren auf eine E.coli Infektion mit der Bildung zahlreicher Noduli (~1000 Noduli/Drohn), die vor allem entlang des Herzschlauches zu finden sind. Diese zelluläre Immunantwort nimmt mit dem Alter der Drohnen ab, so dass bei 18 d alten Drohnen nur noch rund 10 Noduli/Drohn gefunden werden. Auf der anderen Seite nimmt die phagozytotische Aktivität bei älteren Drohnen scheinbar zu. In einer Reihe von parallel laufenden Versuchsreihen konnte ich eindrucksvoll zeigen, dass zelluläre Immunreaktionen wie Phagozytose und Nodulation unmittelbar nach bakterieller Infektion einsetzen. Hierbei erreicht die Nodulibildung 8-10 h p.i. eine Plateauphase, wohingegen die humorale Immunantwort erst 6 h p.i. schwach einsetzt, danach stetig zunimmt und noch 72 h p.i. nachweisbar ist. Es ist mir gelungen, eine Methode zur künstlichen Aufzucht von Drohnenlarven zu etablieren. Diese ermöglichte konstante und sterile Versuchsbedingungen zur Untersuchung der Immunreaktionen von Larven. Nach Infektion mit E.coli reagieren Drohnenlarven mit einer starken Aktivierung ihrer humoralen Immunantwort durch die Expression von AMPs, jedoch werden keine hochmolekularen IRPs wie in adulten Drohnen hochreguliert. Zudem ist die Nodulibildung in Larven nur schwach ausgeprägt. Völlig unerwartete Beobachtungen wurden beim Studium der Immunkompetenz von Drohnenpuppen gemacht. Nach Injektion lebender E.coli Zellen in Drohnenpuppen stellte ich eine dramatische Veränderung im Aussehen der Puppen fest. Die Puppen verfärbten sich gräulich schwarz. Genauere Untersuchungen haben dann gezeigt, dass die Drohnenpuppen, wie auch die der Arbeiterinnen, offensichtlich keine zelluläre Abwehrreaktion aktivieren können und die humorale Immunantwort nur sehr schwach ausfällt und viel zu spät einsetzt.
ATP dependent chromatin remodeling complexes are multifactorial complexes that utilize the energy of ATP to rearrange the chromatin structure. The changes in chromatin structure lead to either increased or decreased DNA accessibility. SWI/SNF is one of such complex. The SWI/SNF complex is involved in both transcription activation and transcription repression. The ATPase subunit of SWI/SNF is called SWI2/SNF2 in yeast and Brahma, Brm, in Drosophila melanogaster. In mammals there are two paralogs of the ATPase subunit, Brm and Brg1. Recent studies have shown that the human Brm is involved in the regulation of alternative splicing. The aim of this study was to investigate the role of Brm in pre-mRNA processing. The model systems used were Chironomus tentans, well suited for in situ studies and D. melanogaster, known for its full genome information. Immunofluorescent staining of the polytene chromosome indicated that Brm protein of C. tentans, ctBrm, is associated with several gene loci including the Balbiani ring (BR) puffs. Mapping the distribution of ctBrm along the BR genes by both immuno-electron microscopy and chromatin immunoprecipitation showed that ctBrm is widely distributed along the BR genes. The results also show that a fraction of ctBrm is associated with the nascent BR pre-mRNP. Biochemical fractionation experiments confirmed the association of Brm with the RNP fractions, not only in C. tentans but also in D. melanogaster and in HeLa cells. Microarray hybridization experiments performed on S2 cells depleted of either dBrm or other SWI/SNF subunits show that Brm affects alternative splicing and 3´ end formation. These results indicated that BRM affects pre-mRNA processing as a component of SWI/SNF complexes. 1
Precise control of progression through mitosis is essential to maintain genomic stability and to prevent aneuploidy. The DREAM complex is an important regulator of mitotic gene expression. Depletion of Lin9, one core-subunit of DREAM, leads to reduced expression of G2/M genes and impaired proliferation. In conditional mouse knockout cells (MEFs) Lin9 deletion causes defects in mitosis and cytokinesis and cells undergo premature senescence in order to prevent further proliferation. In this work it could be shown that the senescence phenotype in Lin9 knockout MEFs is independently mediated by the two tumor suppressor pathways p53-p21 and p16-pRB. Studies using the conditional Lin9 knockout mouse model demonstrated an important function of Lin9 in the regulation of mitotic gene expression and proliferation in vivo. Deletion of Lin9 caused reduced proliferation in the intestinal crypts resulting in atrophy of the intestinal epithelium and in rapid death of the animals. In the second part of this work, the pathways leading to p53 mediated G1 arrest after failed cytokinesis were analyzed by using a chemical inhibitor of the mitotic kinase Aurora B. In a high throughput siRNA screen the MAP kinase MAP3K4 was identified as an upstream activator of p53. It could be shown that MAP3K4 activates the downstream stress kinase p38b to induce the p53 mediated cell cycle arrest of tetraploid cells. p38b was required for the transcriptional activation of the p53 target gene p21 in response to Aurora B inhibition. In contrast, phosphorylation, stabilization and recruitment of p53 to the p21 promoter occured independently of p38 signaling. Partial inhibition of Aurora B demonstrated that chromosome missegregation also activates the MAP3K4-p38-p53 pathway, suggesting that subtle defects in mitosis are sufficient for inducing this stress signaling pathway. Although p38 was required for the G1 cell cycle arrest after mitotic failures, long-term co-inhibition of p38 and Aurora B resulted in reduced proliferation probably due to increased apoptosis. Presumably, MAP3K4-p38-p53 signaling is a common pathway that is activated after errors in mitosis or cytokinesis to arrest cells in G1 and to prevent chromosomal instability.
In dieser Arbeit wurden zwei Aspekte der Yersinia β-Laktamasen bearbeitet: (1) Charakterisierung der β-Laktamasen hinsichtlich β-Laktam-Antibiotikaresistenz, Sekretion und Thermostabilität. (2) Untersuchung der Sekretionsfähigkeit von verschiedenen thermostabilen β Laktamasen über das Yersinia T3SS. Im ersten Teil wurden β Laktamase-Deletionsmutanten im Y. enterocolitica Serotyp O:8 Stamm WA-314 hergestellt, um den Einfluss der chromosomalen β Laktamasen auf die in vitro-Resistenz zu untersuchen. Es konnte gezeigt werden, dass WA-314 konstitutiv BlaA produziert und BlaA somit – unter nicht-induzierbaren Bedingungen – der dominante Faktor in der in vitro-Resistenz gegenüber Penicillinen mit erweitertem Wirkungsspektrum (z.B. Ampicillin) und Cephalosporinen der 1. Generation (z.B. Cefazolin) ist. Weiterhin konnte gezeigt werden, dass die zweite chromosomale β Laktamase AmpC (BlaB) unter Zugabe von subinhibitorischen Konzentrationen von Imipenem stark induziert wird. Keine der β Laktamasen ist in der Lage, in vitro-Resistenz gegenüber Carbapenemen und Monobactamen zu vermitteln. Die Konstruktion und Bestimmung der in vitro Antibiotika-Empfindlichkeit der β Laktamase-Deletionsmutanten dient als Grundlage für nachfolgende Untersuchungen im Mausinfektionsmodell. Weiterhin wurden die Transporteigenschaften beider β Laktamasen untersucht. In Gram-negativen Bakterien sind reife β Laktamasen im Periplasma lokalisiert und müssen somit nach der Synthese im Cytosol über die Cytoplasmamembran transportiert werden. Bis auf drei Ausnahmen (β Laktamasen aus Mycobacterium smegmatis, M. tuberculosis und Stenotrophomonas maltophila) sind bisher nur Sec-abhängige β Laktamasen beschrieben worden. Mittels Fusionsproteinen bestehend aus β Laktamase-Signalpeptiden und GFP konnte in dieser Arbeit eindeutig gezeigt werden, dass es sich bei Yersinia BlaA um ein Tat-Substrat handelt, bei Yersinia AmpC hingegen um ein Sec-Substrat. Somit konnte im Rahmen dieser Arbeit zum ersten Mal eine Tat-abhängige β Laktamase bei einer Bakterienart aus der Familie der Enterobacteriaceae nachgewiesen werden. Außerdem konnte gezeigt werden, dass die β Laktamase BlaA nicht diffus im Periplasma, sondern auf bestimmte Bereiche im Periplasma lokalisiert verteilt ist. Allerdings konnte die Art der Lokalisierung bisher nicht genau spezifiziert werden. Die cytosolische Faltung und die Tat-abhängige Translokation von BlaA lassen vermuten, dass eine besondere Thermostabilität von BlaA vorliegt. Deshalb wurde das BlaA-Enzym hinsichtlich seiner Thermostabilität und temperaturabhängigen enzymatischen Aktivität untersucht. Im Vergleich zur E. coli β Laktamase TEM-1 und der hitzestabilen TEM-1-Variante MEGA zeigte BlaA eine erhöhte Thermostabilität und einen starken Anstieg der Aktivität in einem Temperaturbereich zwischen 30 °C und 45 °C. Im zweiten Teil dieser Arbeit wurde geprüft, ob die charakterisierten Yersinia β Laktamasen als Reporterkonstrukte zur Untersuchung des Typ III Sekretionssystems (T3SS) geeignet sind. Y. enterocolitica besitzt ein pYV Virulenzplasmid, auf dem der vollständige Satz der Gene für das Ysc-T3SS und die Effektor-Yops (Yersinia outer protein) lokalisiert sind. Injektion der Yops in eukaryotische Zielzellen ermöglicht das extrazelluläre Überleben der Yersinien im Wirtsorganismus. Bei YopE handelt es sich um ein gut charakterisiertes Effektor-Yop, dessen N Terminus fusioniert an den reifen Teil der β Laktamase TEM-1 bereits vielfach als Reporterkonstrukt eingesetzt wurde. Unter Verwendung des fluoreszierenden β Laktamase-Substrats CCF4-AM kann die Translokation von YopEi-TEM-1 in Zielzellen in Zellkultur-Experimenten und im Mausinfektionsmodell visualisiert werden. In dieser Arbeit sollte deshalb die T3SS-Sekretionsfähigkeit von YopE-β Laktamase-Fusionsproteinen in Abhängigkeit von der „Schmelztemperatur“ (temperaturabhängige Stabilität, TM) untersucht werden. Yop-Substrate werden im ungefalteten Zustand (YscN wirkt dabei vermutlich als ATP-abhängige „Unfoldase“) über das Ysc-„Injektisom“ transloziert. YopEi-TEM-1 wird effizient sekretiert und transloziert (TM (TEM-1) = 50,8 °C). YopE-Fusionsproteine mit thermostabilen TEM-1 Varianten, YopEi-RLT bzw. YopEi-MEGA (TM (RLT) = 60,4 °C; TM (MEGA) = 69,2 °C) werden hingegen nur schwach bzw. nicht sekretiert. Weiterhin konnte gezeigt werden, dass die Sec-abhängige β Laktamase AmpC als YopE-Fusionsprotein (YopEi-AmpC) effizient T3SS-abhängig sekretiert und transloziert werden kann; das native Tat-Substrat BlaA (YopEi-BlaA) kann jedoch weder sekretiert noch transloziert wird. Eine mögliche Erklärung wäre, dass die ATPase YscN nicht in der Lage ist, BlaA und die thermostabilen TEM-1-Varianten zu entfalten und über das T3SS zu sekretieren und zu translozieren. RLT und MEGA können hingegen mithilfe ihrer nativen Signalsequenz über das Sec-System (und somit im ungefalteten Zustand) transloziert werden.
Protection of healthy tissues from infection with systemically administered vaccinia virus strains
(2012)
Oncolytic virotherapy using recombinant vaccinia virus strains is a promising approach for the treatment of cancer. To further improve the safety of oncolytic vaccinia viruses, the cellular microRNA machinery can be applied as the host’s own security mechanism to avoid unwanted viral replication in healthy tissues. MicroRNAs are a class of small single-stranded RNAs which due to their ability to mediate post-transcriptional gene-silencing, play a crucial role in almost every regulatory process in cellular metabolism. Different cancers display unique microRNA expression patterns, showing significant up- or downregulation of endogenously expressed microRNAs. Furthermore, the behavior of cancer cells can be altered by either adding microRNAs known to inhibit cancer cell spread and proliferation or suppressing cancer promoting microRNAs (oncomirs) making microRNAs promising targets for cancer gene therapy. The cell’s own RNAi machinery can also be utilized to control viral replication due to the virus dependence on the host cell replication machinery, a process controlled by microRNAs. GLV-1h68 is a replication-competent recombinant oncolytic vaccinia virus constructed and generated by Genelux Corp., San Diego, CA, USA which carries insertions of three reporter gene cassettes for detection and attenuation purposes and is currently being evaluated for cancer treatment in clinical trials. Though there are hardly any side effects found in GLV-1h68 mediated oncolytic therapy an increased tropism for replication exclusively in cancer cells is desirable. Therefore it was investigated whether or not further cancer cell specificity of a recombinant vaccinia virus strain could be obtained without compromising its oncolytic activity using microRNA interference. Let-7a is a well characterized microRNA known to be expressed in high levels in healthy tissues and strongly downregulated in most cancers. To control vaccinia virus replication rates, four copies of the mature human microRNA let-7a target sequence were cloned behind the stop codon in the 3’end of the vaccinia virus D4R gene, using a GLV-1h68 derivative, GLV-1h190, as parental strain yielding the new recombinant virus strain GLV-1h250. The D4R gene belongs to the group of early transcribed vaccinia genes and encodes an essential enzyme, uracil DNA glycosylase, which catalyzes the removal of uracil residues from double-stranded DNA. A defect in D4R prevents vaccinia virus from entering into the intermediate and late phase of replication, leading to an aborted virus replication. After expression of the microRNA target sequence from the vaccinia virus genome, the endogenously expressed microRNA-let-7a should recognize its target structure within the viral mRNA transcript, thereby binding and degrading the viral mRNA which should lead to a strong inhibition of the virus replication in healthy cells. GLV-1h250 replication rates in cancerous A549 lung adenocarcinoma cells, which show a strong down-regulation of microRNA let-7a, was comparable to the replication rates of its parental strain GLV-1h190 and the control strain GLV-1h68. In contrast, GLV-1h250 displayed a 10-fold decrease in viral replication in non-cancerous ERC cells when compared to GLV-1h190 and GLV-1h68. In A549 tumor bearing nude mice GLV-1h250 replicated exclusively in the tumorous tissue and resulted in efficient tumor regression without adverse effects leading to the conclusion that GLV-1h250 replicates preferentially in cancerous cells and tissues, which display low endogenous let-7a expression levels.