Methoden der Fluoreszenz-Lokalisationsmikroskopie (engl. single-molecule localization microscopy, SMLM) ermöglichen es Moleküle zu quantifizieren und deren Verteilung zu analysieren. Im Rahmen dieser Arbeit wurden verschiedene Membranmoleküle auf unterschiedlichen eukaryotischen Zellen, aber auch auf Prokaryoten mit dSTORM (engl. direct stochastic optical reconstruction microscopy) oder PALM (engl.: photoactivated localization microscopy) aufgenommen und quantifiziert. Bevor jedoch diese hochauflösende fluoreszenzbasierte Technik für biologische Fragestellungen angewendet werden konnten, mussten zunächst potentielle Artefakt-auslösende Quellen identifiziert und Strategien gefunden werden, um diese zu eliminieren. Eine mögliche Artefakt-Quelle ist eine zu niedrige Photonenzahl, die von Fluorophoren emittiert wird. Werden zu wenige Photonen detektiert, kann die Lokalisation eines Fluorophors weniger präzise bestimmt werden. Dies kann zu einer falschen Abbildung von Strukturen führen oder zu falschen Rückschlüssen über die Verteilung von Molekülen. Eine Möglichkeit die Anzahl der emittierten Photonen zu erhöhen, ist chemische Additive als Triplettlöscher einzusetzen. Sie bewirken, dass die Fluorophore wieder in den Grundzustand relaxieren und somit wieder angeregt werden können. Es wurden verschiedene Additive, die in der Literatur als Triplettlöscher beschrieben sind, getestet. Dazu wurden zunächst ihre Auswirkungen auf den Triplettzustand verschiedener Fluorophore (Alexa Fluor (Al) 488, 532 und 647 und Atto655) mit Hilfe von Fluoreszenzkorrelationsspektroskopie (FCS) untersucht. Cyclooctatetraen (COT) bewirkte dabei eine Abnahme der Triplettausbeute von Al488, Al532 und Al647 um ~ 40-60%, bei Atto655 veränderte sie sich nicht. Obwohl die Ergebnisse der FCS-Messungen darauf hindeuten, dass COT in einer erhöhten Anzahl an emittierten Photonen resultiert, konnte dies bei dSTORM-Messungen nicht bestätigt werden. Hier hatte COT nur einen größeren positiven Effekt auf das Fluorophor Al647 (Zunahme um ~ 60%). Eine Erklärung für diese Widersprüchlichkeit zu den Ergebnissen aus den FCS-Messungen, könnte das Vorhandensein des Schaltpuffers bei dSTORM-Messungen sein. Dieser bewirkt den Übergang der Fluorophore in den Aus-Zustand bzw. entzieht dem Puffer Sauerstoff. Bei der Zugabe von 5 mM Kaliumiodid (KI) nahm die Triplettamplitude bei FCS-Messungen nur bei Al488 ab (um ~ 80%). Eine geringe Steigerung (um ~ 10%) der Intensität von Al488 mit KI konnte bei dSTORM-Messungen mit niedrigen Konzentrationen (~ 0,5 mM) erzielt werden. Bei einer Konzentration von 5 mM sank die Intensität jedoch wieder um 40%. Deuteriumoxid (D2O) soll, anders als die Triplettlöscher, eine Verbesserung der Photonenausbeute dadurch bewirken, dass strahlungslose Relaxationsprozesse minimiert werden. Mit dSTORM-Messungen konnte gezeigt werden, dass Atto655 und Al647 in D2O zwar pro An-Zustand mehr Photonen emittieren als in Schaltpuffer ohne D2O, da die Fluorophore hier jedoch schneller bleichen, letztendlich die gleiche Anzahl an Photonen detektiert werden. Um die Anzahl an emittierten Photonen zu erhöhen, eignet sich also nur COT bei dSTORM-Messungen mit AL647 und KI in sehr geringen Konzentrationen bei Al488. D2O kann eingesetzt werden, wenn eine Probe schnell vermessen werden muss, wie zum Beispiel bei Lebendzellmessungen. Nicht nur eine zu niedrige Photonenzahl, auch eine zu geringe Photoschaltrate kann Artefakte bei dSTORM-Messungen erzeugen. Dies wurde anhand von verschiedenen biologischen Strukturen, die mit unterschiedlichen Anregungsintensitäten aufgenommen wurden, deutlich gemacht. Besonders die Aufnahmen von Plasmamembranen sind anfällig für die Generierung von Artefakten. Sie weisen viele inhomogene und lokal dichte Regionen auf. Wenn nun mehr als ein Emitter pro µm² gleichzeitig an ist, erzeugt das Auswertungsprogramm große artifizielle Cluster. Die hier durchgeführten Messungen machen deutlich, wie wichtig es ist, dSTORM-Bilder immer auf mögliche Artefakte hin zu untersuchen, besonders wenn Moleküle quantifiziert werden sollen. Dafür müssen die unbearbeiteten Rohdaten sorgfältig gesichtet werden und notfalls die Messungen mit einer höheren Laserleistung wiederholt werden. Da dSTORM mittlerweile immer mehr zur Quantifizierung eingesetzt wird und Clusteranalysen durchgeführt werden, wäre es sinnvoll bei Veröffentlichungen die Rohdaten von entscheidenden Aufnahmen der Öffentlichkeit zur Verfügung zu stellen. Die Färbemethode ist ein weiterer Punkt, durch den Artefakte bei der Abbildung von Molekülen mittels SMLM entstehen können. Häufig werden Antikörper zum Markieren verwendet. Dabei sollte darauf geachtet werden, dass möglichst kleine Antikörper oder Antikörperfragmente verwendet werden, besonders wenn Clusteranalysen durchgeführt werden sollen. Anderenfalls leidet die Auflösung darunter, bzw. erhöht sich die Gefahr der Kreuzvernetzung von Molekülen. Im zweiten Teil der vorliegenden Arbeit, wurden Plasmamembran-Ceramide untersucht. Ceramide gehören zu den Sphingolipiden und regulieren diverse zelluläre Prozesse. Verschiedene Stimuli bewirken eine Aktivierung von Sphingomyelinasen (SMasen), die Ceramide in der Plasmamembran synthetisieren. Steigt die Konzentration von Ceramiden in der Plasmamembran an, kondensieren diese zu Ceramid-reichen Plattformen (CRPs). Bisher ist noch wenig über die Verteilung der Ceramide und die Größe der CRPs bekannt. Sie wurden hier über IgG-Antikörper in der Plasmamembran von Jurkat-, U2OS-, HBME- und primären T-Zellen angefärbt und erstmals mit dSTORM hochaufgelöst, um sie dann zu quantifizieren. Unabhängig von der Zelllinie befanden sich 50% aller Ceramidmoleküle in ~ 75 nm großen CRPs. Im Mittel bestanden die CRPs aus ~ 20 Ceramiden. Mit Hilfe einer Titrationsreihe konnte ausgeschlossen werden, dass diese Cluster nur durch die Antikörper-Färbung artifiziell erzeugt wurden. Bei Inkubation der Zellen mit Bacillus cereus Sphingomyelinase (bSMase) stieg die Gesamtkonzentration der Ceramide in der Plasmamembran an, ebenso wie die Ceramidanzahl innerhalb der CRPs, außerdem die Anzahl und Größe der CRPs. Dies könnte zu einer Veränderung der Löslichkeit von Membrankomponenten führen, was wiederum eine Akkumulation bestimmter Rezeptoren oder eine Kompartimentierung bestimmter Proteine erleichtern könnte. Die Anhäufung der Ceramide in den CRPs könnte ebenfalls die lokale Interaktion mit anderen Membranmolekülen erleichtern und dadurch möglicherweise die Reaktivität von Rezeptoren verändern. Mittels Azid-modifizierten Ceramidanaloga und kupferfreier Click-Chemie wurden Plasmamembran-Ceramide auch in lebenden Jurkat-Zellen mit Hilfe konfokaler Laser-Raster-Mikroskopie (CLSM, engl. confocal laser scanning microscopy) und Strukturierter Beleuchtungsmikroskopie (SIM, engl. structured illumination microscopy) untersucht. Dabei konnte gezeigt werden, dass die Fettsäure-Kettenlänge und die Position des Azids bei den Ceramidanaloga eine entscheidende Rolle spielt, wie hoch das detektierte Signal in der Plasmamembran letztendlich ist. Die Versuche machen auch deutlich, dass die klickbaren Ceramidanaloga lebendzellkompatibel sind, sodass sie eine hervorragende Möglichkeit darstellen, zelluläre Reaktionen zu verfolgen. Es wurden hier nicht nur Ceramide in eukaryotischen Zellen analysiert, sondern auch in Bakterien. Neisseria meningitidis (N. meningitidis) sind gramnegative Bakterien, die im Menschen eine Sepsis oder eine Meningitis auslösen können. Es wurde mittels immunhistochemischen Färbungen mit dem anti-Ceramid IgG-Antikörper, aber auch mit den klickbaren Ceramidanaloga, ein Signal in der Membran erhalten, was mit dSTORM hochaufgelöst wurde. In anderen Bakterien wurden ebenfalls schon Sphingolipide nachgewiesen. Studien zu Ceramiden in N. meningitidis wurden bisher jedoch noch nicht veröffentlicht. Im Rahmen dieser Arbeit konnten erstmals Ergebnisse erhalten werden, die darauf hinweisen, dass N. meningitidis ebenfalls Ceramide besitzen könnten. In einem dritten Projekt wurde die Interaktion zwischen NK-Zellen und Aspergillus fumigatus untersucht. Der Schimmelpilz kann eine Invasive Aspergillose in immunsupprimierten Menschen auslösen, was zum Tod führen kann. Verschiedene Studien konnten schon zeigen, dass NK-Zellen eine wichtige Rolle bei der Bekämpfung des Pilzes spielen. Der genaue Mechanismus ist jedoch noch unbekannt. Im Rahmen dieser Arbeit konnte nachgewiesen werden, dass der NK-Zell-Marker CD56 entscheidend für die Pilzerkennung ist. Mit immunhistochemischen Färbungen und LSM-, aber auch dSTORM-Messungen, konnte gezeigt werden, dass die normalerweise homogen verteilten CD56-Rezeptoren auf der Plasmamembran von NK-Zellen aktiv an die Interaktionsstelle zu A. fumigatus transportiert werden. Mit der Zeit akkumulieren hier immer mehr CD56-Proteine, während das Signal in der restlichen Membran immer weiter abnimmt. Es konnte erstmals CD56 als wichtiger Erkennungsrezeptor für A. fumigatus identifiziert werden. In dem letzten bearbeiteten Projekt, wurde die Bindung von Anti-N-Methyl-D-Aspartat (NMDA)-Rezeptor Enzephalitis Autoantikörper an Neuronen untersucht. Bei einer Anti-NMDA-Rezeptor Enzephalitis bilden die Patienten Autoantikörper gegen die NR1-Untereinheit ihrer eigenen postsynaptischen NMDA-Rezeptoren. Da die Krankheit oft sehr spät erkannt wird und die Behandlungsmöglichkeiten noch sehr eingeschränkt sind, führt sie noch oft zum Tod. Sie wurde erst vor wenigen Jahren beschrieben, sodass der genaue Mechanismus noch unbekannt ist. Im Rahmen dieser Arbeit, konnten erste Färbungen mit aufgereinigten Antikörper aus Anti-NMDA-Rezeptor Enzephalitis Patienten an NMDA-Rezeptor-transfizierte HEK-Zellen und hippocampalen Maus-Neuronen durchgeführt und mit dSTORM hochaufgelöst werden. Mit den Messungen der HEK-Zellen konnte bestätigt werden, dass die Autoantikörper an die NR1-Untereinheit der Rezeptoren binden. Es konnten erstmals auch die Bindung der Antikörper an Neuronen hochaufgelöst werden. Dabei wurde sichtbar, dass die Antikörper zum einen dicht gepackt in den Synapsen vorliegen, aber auch dünner verteilt in den extrasynaptischen Regionen. Basierend auf der Ripley’s H-Funktion konnten in den Synapsen große Cluster von ~ 90 nm Durchmesser und im Mittel ~ 500 Lokalisationen und extrasynaptisch kleinere Cluster mit einem durchschnittlichen Durchmesser von ~ 70 nm und ~ 100 Lokalisationen ausgemacht werden. Diese ersten Ergebnisse legen den Grundstein für weitere Messungen, mit denen der Mechanismus der Krankheit untersucht werden kann. / With single molecule localization microscopy (SMLM) quantification of molecules and the analysis of their distribution becomes possible. In this work various plasma membrane molecules of different eukaryotic and prokaryotic cells were imaged with dSTORM (direct stochastic optical reconstruction microscopy) or PALM (photoactivated localization microscopy) and quantified. To use these super-resolution fluorescence microscopy techniques and answer elaborate biological questions, potential sources of artifacts were identified and strategies to circumvent them developed. A possible source of artifacts is an insufficient number of photons emitted by fluorophores. If less photons are detected, determining the localization of one fluorophore is less precise. This can cause a wrong reconstruction of structures or might lead to false conclusions about the distribution of molecules. One possibility to increase the number of photons is to use chemical additives which quench the triplet state of fluorophores. They ensure that the fluorophores relax into the ground state allowing them to become excited again. Different additives, described in literature as triplet quenchers, were tested. The effects of these additives on the triplet state of different fluorophores (Alexa Fluor (Al) 488, 532 und 647 und Atto655) were analyzed with fluorescence correlation spectroscopy (FCS). Cyclooctatetraene (COT) resulted in a decrease of triplet state yield of Al488, Al532 and Al647 by ~ 40-60%, yet the triplet state of Atto655 was unaffected. FCS measurements indicated that COT results in an increased number of emitted photons, but dSTORM measurements could not confirm this finding. Here, COT only revealed a positive effect on the intensity of Al647 (increase by ~ 60%). An explanation for this inconsistency with the FCS results might be the presence of the switching buffer in dSTORM measurements. The buffer is designed to cause a transition of the fluorophores to and stabilize the off-state by removing oxygen from the sample, counteracting the effect of COT. On addition of 5 mM potassium iodide (KI) only Al488 fluorophores showed a decreased triplet state rate (~ 80%) in FCS measurements. This finding was confirmed by dSTORM measurements with low concentrations (~ 0.5 mM) of KI which resulted in a slight intensity increase (~ 10%) of Al488. Higher KI concentration (5 mM) on the other hand showed a reversed effect, resulting in a drop in intensity by ~ 40%. Deuterium oxide (D2O) isn’t a triplet quencher but should minimize non-radiative processes. DSTORM measurements with Atto665 and Al647 revealed, that D2O does not affect the total number of emitted photons per fluorophore. Instead, D2O increased the amount of emitted photons per time. In a nutshell, these results show that dSTORM measurements with Al647 can be improved using COT, and measurements with Al488 by using very low concentrations of KI. If needed, D2O can speed up dSTORM acquisition time considerably, e.g. for life cell measurements. In addition to an insufficient number of collected photons, inappropriate photoswitching rates can induce artifacts in dSTORM measurements as well. This was shown using various biological reference structures. Especially the imaging of plasma membranes is prone to generate artifacts. Plasma membranes exhibit a lot of intrinsically three-dimensional structures with high local emitter densities. In these regions of higher fluorophore densities the likelihood of two close fluorophores emitting at the same time is increased. This in turn can result in large artificial clusters due to misinterpretation by the reconstruction software. Taken together, the performed experiments show how important it is to prove dSTORM images and minimize possibility image artifacts. Thus, raw data movies need to be examined carefully and, if necessary, measurements must be repeated with adapted imaging conditions. Since dSTORM is increasingly used for quantification and cluster analysis it is recommended to publish raw data in the Supporting information of the manuscript. Another source of artifacts when imaging molecules with SMLM is the staining procedure. Usually antibodies are used to label biological structures for dSTORM. In the interest of resolution, small antibodies or just fragments of antibodies should be used, especially if cluster analysis is performed. Otherwise reduced resolution or an increase in cross-linking of molecules might occur. In the second part of this study plasma membrane ceramides were investigated. Sphingolipid ceramides regulate various cellular processes. Different stimuli initiate activation of sphingomyelinases (SMase) which synthesize ceramides at the plasma membrane. A rise in ceramide concentration leads to a condensation of them in ceramide-rich platforms (CRPs). So far, only little is known about the distribution and the size of CRPs. Here, plasma membrane ceramides of Jurkat-, U2OS-, HBME- and primary T-cells were stained with an IgG-antibody, imaged using dSTORM and their distribution quantitatively analyzed. Independent of the analyzed cell line, ~ 50% of all ceramides detected in the plasma membrane formed CRPs with a size of ~ 75 nm. On average one CRP consisted of ~ 20 ceramide molecules. Using a titration series the possibility of artificial cluster generation due to antibody staining was ruled out. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increased the overall ceramide concentration in the plasma membrane, the number of ceramides in the CRPs as well as the quantity and the size of CRPs. This might result in a higher solubility of membrane components in CRPs which in turn could facilitate accumulation or compartmentation of certain proteins. Accumulation of ceramides in the CRPs could also enable local interaction with other molecules and possibly change the reactivity of some receptors. To investigate plasma membrane ceramides in living cells azido-modified ceramides and copper-free click chemistry were used for labeling. Imaging was performed using confocal laser-scanning microscopy (LSM) and structured illumination microscopy. It was shown that the length of fatty acid chains and the position of the azido group of ceramide analogues play a decisive role in the magnitude of the detected signal in the plasma membrane. These results demonstrate that azido-functionalized ceramides are live-cell compatible, making them an excellent tool to follow cellular reactions. In this study, ceramides were not only analyzed in eukaryotic cells but in bacteria as well. Neisseria meningitidis (N. meningitidis) are gram-negative bacteria triggering sepsis or meningitis in humans. Using both immunolabeling with anti-ceramide IgG-antibodies and azido-modified ceramides, ceramides were detected for the first time in the membrane of N. meningitidis by dSTORM. Although sphingolipids were reported to exist in various bacterial membranes, studies about ceramides in N. meningitidis have not yet been published. The results obtained here suggest the presence of ceramides in N. meningitidis. The third part of this thesis addresses the interaction between NK cells and Aspergillus fumigatus. The mold can cause invasive aspergillosis in immunocompromised patients which can lead to death. Various studies have already shown that NK cells play a crucial role in the clearance of the fungal infection. Still, the exact mechanism remains unknown. As part of this work the NK cell marker CD56 was identified as a decisive receptor in recognition of the mold. Using LSM and dSTORM measurements in combination with immunocytochemical staining an active transport of the usually homogenous distributed CD56 receptors to the interaction site of NK cells and fungus was detected. Over time CD56 proteins accumulate at these interaction sites while the signal in the rest of the membrane continuously decreases. For the first time this study was able to identify CD56 as an important recognition receptor for A. fumigatus. In the last project binding of anti-N-Methyl-D-aspartate (NMDA) receptor encephalitis autoantibodies were investigated in neurons. Patients with this form of encephalitis generate autoantibodies against the NR1 subunit of their own postsynaptic NMDA receptors. Since NMDA receptor encephalitis is often diagnosed too late and treatment options are limited the disease often proves to be fatal. Anti-NMDA receptor encephalitis was described quite recently, explaining why the exact mechanism remains still unknown. For this study purified antibodies from anti-NMDA receptor encephalitis patients were used to stain NMDA receptor transfected HEK cells and hippocampal mouse neurons. These samples were subsequently imaged with dSTORM and analyzed. Measurements on HEK cells confirmed that the autoantibodies bind to the NR1 subunit. Using dSTORM, the binding sites of these antibodies at the neurons were imaged for the first time with super-resolution microscopy. The receptors are densely localized in synapses and more equally distributed at lower density in extrasynaptic regions. Based on Ripley’s H function synaptic clusters with a diameter of ~ 90 nm and ~ 500 localizations were determined while the extrasynaptic smaller clusters have a median diameter of ~ 70 nm and ~ 100 localizations per cluster. These first results form the basis for further investigations on the mechanism of anti-NMDA receptor encephalitis.

Das Opc-Protein ist ein Außenmembranprotein von Meningokokken, das über extrazelluläre Matrixproteine mit Integrinen der Wirtszelle interagiert. Opc ist in Menschen immunogen und induziert bakterizide Antikörper. Das Opc-Protein wurde daher als aussichtsreicher Impfstoff-Kandidat angesehen, da es außerdem relativ gut konserviert ist. Allerdings wird das Opc-Protein nicht von allen Meningokokkenstämmen exprimiert. Einerseits fehlt das opc-Gen in einigen klonalen Komplexen (z.B. ST-8, ST-11, ST-53), andererseits ist die Opc-Expression nicht konstitutiv wegen einer phasenvariablen Transkription, die auf einem Poly-Cytidin-Bereich im Promotor des opc-Gens beruht. In dieser Arbeit wurde die Präsenz des opc-Gens und die Opc-Expression in zwei großen Sammlungen deutscher Meningokokkenisolate von invasiven Erkrankungen (n=1141) und gesunden Trägern (n=792) untersucht. Das opc-Gen war bei 71% der invasiven und 77% der Trägerstämme nachweisbar. Der größte Teil der opc-Gen negativen Stämme gehörte zu den klonalen Komplexen ST-8, ST-11, ST-213, ST-231, ST-334 und ST-53. Der Anteil opc-positiver Stämme, die Opc in vitro exprimieren, war bei den invasiven Stämmen kleiner als bei den Trägerstämmen (13% vs. 29%, p<0,001, Chi-square-Test). Der größere Anteil Opc-exprimierender Trägerstämme ist u.a. am ehesten mit der Überrepräsentation von wenig pathogenen klonalen Komplexen (ST-23, ST-35, ST-198) mit einer hohen Opc-Expressionsrate zu erklären. 24 von den 176 invasiven Stämmen mit einer Anzahl von 11 - 14 Cs in der Promotor-Region, die die Opc-Expression begünstigt, zeigten weder im ELISA noch im Westernblot eine Opc-Expression. Bei 14 dieser 24 Stämme wurde als Ursache ein phasenvariabler, intragenischer Poly-Adenin-Bereich identifiziert, der zu einer Leserasterverschiebung führte. Die Vermutung mehrerer Autoren, dass die Opc-Expression mit dem klinischen Bild der Meningitis verknüpft ist, konnte mit der hier genutzten großen Stammsammlung nicht bestätigt werden. Invasive Stämme, die das Opc-Protein exprimierten, wurden genauso häufig von Patienten mit dem klinischen Bild der Meningitis isoliert wie Stämme, die das Opc-Protein nicht exprimierten (46% vs. 47%, Chi-square-Test: p<0,9). Allerdings gibt es eine starke Assoziation der Gegenwart des opc-Gens mit dem klinischen Merkmal Meningitis. Dieser Befund gibt Anlass zu der Hypothese, dass in vitro und in vivo Expression von Opc sich unterscheiden. Zusammenfassend lässt sich festhalten, dass das Opc-Protein nur in 19,8% aller Isolate (invasive und Trägerstämme zusammengenommen) exprimiert wurde. Es zeigte sich eine Tendenz zu häufigerer Opc-Expression in apathogenen Trägerisolaten. Das Vorhandensein des opc-Gens, nicht aber die in vitro Expression konnten mit dem klinischen Merkmal Meningitis assoziiert werden. Zusätzlich wurde ein weiterer Mechanismus der intragenischen Phasenvariation beschrieben. / Presence of opc was associated with meningitis, mostly because ST-11/ST-8 cc meningococci with low meningitis rates were consistently opc negative. On the other hand, lack of opc did not exclude meningitis. Opc was expressed in only 13% of all invasive isolates. In vitro Opc expression was not associated with meningitis. Limitation: Definite conclusion about expression in vivo is not possible with cultured isolates. Evidence for intragenic opc phase-variation was provided.

Thèse de doctorat : Génie des procédés : Vandoeuvre-les-Nancy, INPL : 2005. / Titre provenant de l'écran-titre. Bibliogr.

Thèse de doctorat : Enzymologie moléculaire et Biologie structurale : Nancy 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr.

Neisseria meningitidis is the causative agent of potentially life-threatening meningitis and septicaemia. According to W.H.O., meningococcal disease causes at least 500,000 cases and results in 50,000 deaths worldwide each year (W.H.O., 2008). Neisseria gonorrhoeae is causing the second most common sexually transmitted bacterial infection, with a global incidence of 62 million cases per year. Previous studies have shown surface expressed proteins like pilin, the subunit protein that forms pili (Type IV Fimbriae), in N. meningitidis and N. gonorrhoeae are post-translationally modified by O-glycosylation. This modification has been proposed to be of importance in the pathogenesis of these species. Although the exact function of these post-translational modifications are not fully understood, it is suggested that these modification have a role for immune evasion in the host. In this thesis, an additional outer membrane glycoprotein was identified in pathogenic Neisseria, the nitrite reductase AniA. Mass spectrometry analysis showed that AniA is glycosylated in its C-terminal imperfect (AASAP) repeat region by the pilin glycosylation pathway. This is the first report of a general O-glycosylation pathway in a prokaryote. It was shown AniA is surface exposed. To investigate whether AniA is subject to immune selection, a large collection of N. meningitidis and N. meningitidis clinical isolates were sequence analysed and evaluated. Analysis of published AniA 3D structure revealed that AniA displayed polymorphisms in residues that map to the surface of the protein. This suggests that AniA is under immune selection, and that glycosylation may facilitate immune evasion. Sequencing analyses revealed a frame shift mutation that abolished AniA expression in 34% of N. meningitidis strains surveyed. However, all N. gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology. In summary, the data presented here suggested that the protein may be under immune selective pressure. The addition of a phase variable glycan to this surface protein may serve as an additional immune evasion strategy. Immune selection on surface proteins in these host-adapted pathogens may have been the driving force for the evolution of this general O-glycosylation pathway. Therefore, the discovery that AniA is a glycoprotein has given insights into the pathogenesis and the host-pathogen interactions of these organisms.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 3 uppsatser.

Techn. Univ., Diss., 2010--München.

Made available in DSpace on 2015-12-06T23:47:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2008 / Introdução: Os profissionais de saúde estão expostos a vários riscos no desenvolvimento de suas atividades, no ambiente hospitalar, especialmente os biológicos. As doenças infectocontagiosas são as principais fontes de transmissão de microrganismos de pacientes para profissionais, através de diferentes vias. A doença meningocócica, cuja transmissão é respiratória, causa pânico na população, inclusive entre estes profissionais, devido ao seu caráter epidêmico e letalidade elevada. Objetivos: Determinar a prevalência de portadores de Neisseria meningitidis em profissionais de saúde recém-admitidos em um hospital escola e verificar a colonização da nasofaringe destes profissionais expostos ao microrganismo 12 meses após a admissão, bem como a aplicação das medidas de prevenção e uso de quimioprofilaxia preconizadas. Métodos: Estudo de painel de medidas repetidas, onde os indivíduos selecionados foram avaliados, através de coleta de cultura de nasofaringe, antes da exposição e, num segundo momento, doze meses após a exposição já definida anteriormente. Participaram da pesquisa 117 profissionais de saúde aprovados em concurso púbico e lotados no hospital entre janeiro de 2004 e dezembro de 2005. A análise descritiva das variáveis qualitativas foi apresentada em valores absolutos e relativos e as quantitativas através das medidas de tendência central e de distribuição do conjunto de medidas registradas. Foi utilizado o teste de McNemar para comparar a distribuição de duas variáveis correlacionadas. Resultados: Os 117 profissionais de saúde avaliados apresentaram uma média de idade de 34 anos e 86% eram da categoria de enfermagem. Apenas 3% eram portadores de Neisseria meningitidis no exame admissional. Distribuídos nos setores do hospital, 76% foram selecionados para enfermarias, pronto atendimento e unidade semi-intensiva. Cerca de 66% dos profissionais trabalharam anteriormente em serviços de saúde e 46,1% exerciam, concomitantemente à admissão no hospital, a mesma atividade em outras unidades. Apenas 19% referiram contato recente com pacientes com doença meningocócica e 90% foram orientados a usar máscara ao prestarem assistência a pacientes com esta doença. No intervalo estudado, somente 4% destes profissionais utilizaram rifampicina como quimioprofilaxia, sendo que 68% tiveram contato com pacientes internados com doença meningocócica neste período. Apesar da orientação sobre o uso de máscara, 32% não a utilizaram. Não houve registro de nenhum resultado positivo para Neisseria meningitidis, na cultura realizada nestes profissionais, após um ano de exposição ao microrganismo, no ambiente hospitalar. Conclusões: Não houve colonização dos profissionais de saúde pela Neisseria meningitidis, o que nos permite corroborar as medidas de precaução, padronizadas universalmente, para prevenção da doença meningocócica no ambiente hospitalar / Introduction: Healthcare professionals are frequently exposed to a variety of hazards and risks whilst carrying out their activities in hospital environments, especially with regards to biological risks. The contagious diseases are the main source of transmission of infectious agents from patients to professionals through a wide and different variety of ways. The meningococcal disease, which is transmitted from respiratory secretions, causes panic in the population including these very professionals due to epidemic and lethal character. Objectives: To determine the prevalence of carriage of Neisseria meningitidis amongst the healthcare professionals whom have been recently admitted in a hospital and to verify the colonization in the nasopharynx of these very professionals exposed to the agent 12 months after their admission as well as the use of preventive measures. Methods: A panel study of repeated measurements where the selected individuals had been evaluated through the collection of cultures from the nasopharynx before exposure and, in a second instance, 12 months after the exposure previously defined. Between January 2004 and December 2005, 117 healthcare professionals, whom were successfully approved in a public admission exam and were duly assigned to a hospital, have participated in the study. The descriptive analysis of the qualitative variables was presented in absolute and relative values and the qualitative variables were presented through the measures of central tendency and distribution tendency from the set of registered measures. The McNemar Test was used in order to compare the distribution of the two correlated variables. Results: The evaluated 117 healthcare professionals presented an average age of 34 years of age and 86% were from the Nursing Category. Only 3% were carriers of N. meningitidis at the admission exam. When distributed throughout the hospital sections, 76% were selected for wards, emergency wards and semi intensive units. Approximately 66% of the professionals had previous experience in the health settings and 46.1% performed, concomitantly to the admission in the hospital, the same activity in other units. Only 19% mentioned recent contact with patients suffering from meningococcal disease and 90% were duly orientated to use a mask whenever assisting patients suffering from this disease. During the studied interval, only 4% of these professionals used rifampicin as chemoprophylaxis although 68% had actually had contact with interned patients with meningococcal disease within the said period. Independently from the orientation regarding the use of masks, 32% of them did not use it. There were no registrations of a positive result for Neisseria meningitidis in the culture carried out on these professionals after one year of exposure to the agent within the hospital environment. Conclusion: There was no colonization in the healthcare professionals by N. meningitidis thus we may corroborate the universally standardized preventive measures for the meningococcal disease in a hospital environment. / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2015-07-08T12:28:14Z (GMT). No. of bitstreams: 2 17.pdf: 1549058 bytes, checksum: d1d338fb698a95bb0d0ea23528f8cf74 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A doença meningocócica permanece como um problema de saúde pública, estando relacionada a altas taxas de morbidade e mortalidade, principalmente em crianças e lactentes. A rápida evolução da doença requer uma intervenção terapêutica específica e precoce, e enfatiza a necessidade de disponibilizar vacinas que possam ser utilizadas na prevenção dessa doença. Estratégias utilizando proteínas de membrana externa de Neisseria meningitidis como antígeno estão sendo desenvolvidas para a produção de uma vacina universal contra o sorogrupo B, que pode, inclusive, conferir proteção contra outros sorogrupos. Duas vacinas se encontram atualmente em ensaios clínicos. A vacina recombinante rfHbp possui duas variantes da proteína fHbp (V1 e V2) como antígeno, e a vacina 4CMenB é uma vacina multicomponente que inclui três proteínas (fHbp, NHBA e NadA) combinadas com OMVs da cepa epidêmica da Nova Zelândia. Embora estes antígenos sejam relativamente conservados, os mesmos já demonstraram variabilidade em diferentes regiões. O objetivo deste estudo foi avaliar a prevalência e diversidade genética destes antígenos em isolados clínicos de N. meningitidis sorogrupos B e C circulantes no Brasil. A caracterização dos isolados pelo MLST revelou seis complexos clonais principais (CCS ST-41/44, ST-1136, ST-32/ET-15, ST-8/A4, ST-11/ET-37 E ST 103). Todos os isolados apresentaram o gene de fHbp, onde a variante 1 foi predominante para sorogrupo B e a variante 2 para o sorogrupo C. O gene NHBA também foi identificado em todos os isolados. Apenas 29,73% dos isolados apresentaram o gene nadA. Nenhuma das cepas analisadas apresentou o mesmo subtipo de PorA presente na OMV que compõe a vacina, não sendo interessante a inclusão desta vesícula em uma possível vacina brasileira. Apenas três variantes de VR3 de PorA foram identificadas, demonstrando a baixa variabilidade desta região da proteína e a possível aplicação em uma vacina. / Meningococcal disease remains a public health problem causing high case-fatality rates worldwide, especially among young children and young adults. The rapid evolution of the disease requires specific and fast therapeutic actions and the use of prophylaxis through vaccination is the best choice to halt the spread of the disease. New vaccine approaches using surface exposed proteins from Neisseria meningitidis have been developed for the design of a universal vaccine against serogroup B, which could also elicit protection against other serogroups. Two vaccines using this strategy are currently through clinical trials. The recombinant vaccine rfHbp with two fHbp antigenic variants (V1 and V2) and the multicomponent vaccine 4CMenB including three proteins (fHbp, NHBA and NadA) combined with an OMV from the New Zealand vaccine strain. Although these are highly conserved antigens, some genetic variation has been already observed within their genes. The main objective of this study was to evaluate the prevalence and genetic diversity of these antigens among N. meningitidis serogroup B and C isolates circulating in Brazil. The characterization of such isolates by MLST analysis, revealed the presence of six main clonal complexes (CCS ST-41/44, ST-1136, ST-32/ET-15, ST-8/A4, ST-11/ET-37 E ST 103). The gene fHbp was amplified from all isolates and Variant 1 was predominant among serogroup B and Variant 2 among serogroup C. Gene NHBA was also amplified from all isolates. Gene nadA was only amplified form 29.73% of all isolates. We didn´t find any strain matching the same PorA subtype of the OMV used in the 4CMenB vaccine, thus the inclusion of such bacterial structure in a future vaccine to be used in Brazil would be useless. Only three PorA VR3 variants were detected showing the low diversity of this protein region and the possible inclusion of this structure on a future vaccine. We have detected a possible indication of capsule “switching” among some isolates which shows the need of a wide surveillance of meningococcal strains that can escape the protection of serogroup C conjugate vaccines. The rfHbp vaccine suggested that it could show a higher efficiency when compared with 4CMenB, but it is important to cover possible scape mechanisms showed by several strains.

Made available in DSpace on 2015-07-08T12:28:20Z (GMT). No. of bitstreams: 2 14.pdf: 2053160 bytes, checksum: 6a9f2af0a7daaa1f0f06673bc00622fc (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A doença meningocócica (DM) é uma enfermidade aguda, grave, de evolução rápida com taxas de mortalidade de 15 a 20% e que atinge principalmente as faixas etárias mais jovens, considerada um grave problema de saúde pública mesmo em países desenvolvidos. A DM está associada à colonização da nasofaringe de humanos, único hospedeiro, por uma bactéria Gram negativa, Neisseria meningitidis. O método considerado padrão ouro para a tipificação molecular da Neisseria meningitidis é o MLST, que consiste no sequenciamento de 7 genes Housekeeping , o MLST é uma técnica complexa que exige uma demanda de custo e tempo, tornando-se inviável sua implementação em laboratórios públicos que recebem amostras de Nm, uma estratégia que poderá contornar este obstáculo seria a PCR em tempo real que com poucas reações e em poucos dias fornecerá o perfil de MLST de isolados com alta porcentagem de confiabilidade. O objetivo do estudo é detectar os complexos clonais do MLST causadores dos grandes surtos de DM através de sondas específicas pela PCR em Tempo Real Qualitativa. A análise pelo MLST de 98 isolados estudados caracterizou todos os 15 isolados do sorogrupo B como integrantes do complexo clonal ST-32/ET-5 além de outras 11 cepas do sorogrupo C. O ST-103 foi o complexo clonal predominante entre as cepas do sorogrupo C com 43 isolados, outros STs foram encontrados para o sorogrupo C,ST-11 (n=11), ST-41/44 (n=2), ST-8/4A (n=9), ST-35 (n=1), ST-269 (n=1) e outros 5 isolados não foram associados a nenhum complexo clonal. A análise pela PCR em Tempo Real forneceu dados importantes como a caracterização de grande número dos isolados com 100% de certeza e de outros isolados com probabilidade acima de 75%. A PCR em Tempo Real qualitativa mostrou ser uma ferramenta rápida e sensível no auxílio a estudos epidemiológicos para controle de surtos regionais quando há a necessidade de ações rápidas por parte das autoridades de saúde pública. / Meningococcal disease (MD) is an acute illness, severe, rapidly evolving with mortality rates of 15 to 20% and that primarily affects younger age groups, considered a serious public health problem in developed countries. DM is associated with colonization of the nasopharynx of human host by Gram negative bacteria, Neisseria meningitidis (Nm). The method considered the "gold standard" for molecular typing of Neisseria meningitidis is the MLST, which consists in sequencing of 7 genes "Housekeeping", the MLST is a complex technique that requires a time and cost consuming, making it impractical implementation in public laboratories that receive Nm samples, a strategy that could circumvent this obstacle would be the real-time PCR with few reactions in a few days coud provide the MLST profile of isolates with a high percentage of reliability. The objective in this study is to detect the MLST clonal complexes causing major outbreaks of DM through specific probes for Real Time PCR Qualitative method. The analysis by MLST of 98 isolates studied featured all 15 isolates of serogroup B as clonal complex ST-32/ET-5 plus 11 other strains of serogroup C. The ST-103 was the predominant clonal complex among strains of serogroup C isolates with 43. Others STs were found for serogroup C, ST-11 (n=11), ST-41/44 (n=2), ST-8/4A (n=9), ST-35 (n=1), ST-269 (n=1) and other five isolates were not associated with any clonal complex. The analysis by Real Time PCR provided important data such as the characterization of large numbers of isolates with 100% certainty and other isolates with probability above 75%. The Real-Time PCR using the qualitative method proved to be a highly useful tool to aid in rapid and epidemiological studies for outbreak control is necessary when regional rapid actions of public health authorities.