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Characterization of Staphylococcus Aureus Skin Infection Using a New in Vivo Proliferation Biosensor”

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Characterization of Staphylococcus Aureus Skin Infection Using a New in Vivo Proliferation Biosensor” Characterization of Staphylococcus aureus skin infection using a new in vivo proliferation biosensor Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg von M. Sc. Elena Anne Seiß (geb. Haas) geb. am 14.03.1988 in Erkelenz Gutachter: Prof. Dr. Andreas J. Müller Prof. Dr. rer. nat. Christiane Wolz eingereicht am: 08.01.2019 verteidigt am: 26.06.2019 ABSTRACT ABSTRACT M. Sc. Elena A. Seiß (née Haas): “Characterization of Staphylococcus aureus skin infection using a new in vivo proliferation biosensor” Distinct bacterial growth rates are decisive for the outcome of infections: First, they impact on the pathogen's susceptibility to both immune effector mechanisms and antibiotic treatment, and can therefore contribute to persistence during chronic or relapsing infections. Second, fast-growing bacteria are a source of more, and different, pathogen-associated molecules than low-proliferating or inactive microbes, thus differentially shaping the immune response. It has however remained difficult to measure bacterial growth rates in the ongoing infection. Here, I established an in vivo biosensor for measuring proliferation of the Gram-positive bacterium Staphylococcus aureus (S. aureus) on a single cell-level in vivo. The method is based on the photoconvertible fluorescence protein mKikume, which can be photoconverted by violet light at any given time point, even during in vivo infection in a non- invasive way. This allows determining bacterial proliferation rates as a function of the recovery from photoconversion. Importantly, I could show that indeed bacterial proliferation, and not protein turnover in nonproliferating bacteria, is mainly responsible for the fluorescence recovery after photoconversion readout of the biosensor. Using intravital 2- photon imaging and quantitative fluorescence microscopy, I saw that upon recruitment of CD45+ leukocytes at the site of infection, mainly consisting of neutrophils, and bacterial uptake, a population-wide dampening of S. aureus growth occurred. In the context of neutrophil-depleted mice, the reduction in bacterial growth rate was partially rescued, but a new cell population appeared, which was not constituted of monocytes, the other cell population recruited upon S. aureus infection, but showed similarities to neutrophils with a downregulated and partially masked GR-1 signal. This residual population might partially compensate the neutrophil depletion phenotype in S. aureus containment. NADPH oxidase constitutes a major antimicrobial effector mechanism of neutrophils. In my experiments, the bacterial growth rate was found to be dampened dependently of NADPH oxidase, suggesting that oxidative burst contributes to the pathogen containment by non- lethal restriction of S. aureus proliferation. Furthermore, a cell-extrinsic mode of action of reactive oxygen species produced by NADPH oxidase seems to be involved in the growth dampening. The possibility to probe, in an ongoing infection, the bacterial growth rate on a single cell- resolved level should provide a powerful tool to investigate the host-pathogen interactions during S. aureus infections in the future. I INTRODUCTION ZUSAMMENFASSUNG M. Sc. Elena A. Seiß (geb. Haas): „Characterization of Staphylococcus aureus skin infection using a new in vivo proliferation biosensor” Die bakterielle Wachstumsrate kann entscheidend für den Verlauf einer Infektion sein: Zum einen beeinflusst sie die Empfänglichkeit für Effektormechanismen des Immunsystems, aber auch die Empfindlichkeit gegenüber Antibiotikabehandlungen. Beides kann persistierende und chronische oder wiederkehrende Infektionen begünstigen. Zum anderen produzieren schnell wachsende Bakterien mehr, und zudem auch unterschiedliche pathogenassoziierte Mustermoleküle als nicht proliferierende Bakterien. Dadurch ist es zu erwarten, dass schnell und langsam wachsende Erreger die Immunantwort unterschiedlich aktivieren. Allerdings war es bislang schwierig und aufwendig, die bakterielle Wachstumsrate während einer Infektion zu bestimmen. In dieser Arbeit habe ich einen in vivo Biosensor etabliert, mit dem das Wachstum des Gram-positiven Bakteriums Staphylococcus aureus (S. aureus) auf der Einzelzellebene in einer laufenden Infektion gemessen werden kann. Die Methode basiert auf dem Fluoreszenzprotein mKikume, das nicht-invasiv mit violettem Licht zu jedem gewünschten Zeitpunkt während der Infektion von roter zu grüner Fluoreszenz photokonvertiert werden kann. Die bakterielle Wachstumsgeschwindigkeit kann dabei über das Wiedererlangen der ursprünglichen grünen Fluoreszenz bestimmt werden. Hierbei konnte ich zeigen, dass das Wachstum des Bakteriums, und nicht der Proteinumsatz in nichtwachsenden Bakterien, entscheidend für das Wiedererlangen der grünen Fluoreszenz von proliferierenden Bakterien ist. Mit Hilfe der intravitalen Zweiphotonenmikroskopie, sowie quantitativer Fluoreszenz- mikroskopie, konnte ich im Laufe einer Infektion eine populationsweite Abschwächung im Wachstum von S. aureus nachweisen. Dies ging einher mit der Einwanderung von CD45+ Leukozyten, hauptsächlich Neutrophile Granulozyten, an den Ort der Infektion. Durch antikörpervermittelte Dezimierung der Neutrophilen wurde die Dämpfung im Wachstum der Bakterien in einer Hautinfektion vermindert. Jedoch wurde in diesem Fall eine neue Immunzellpopulation rekrutiert, welche nicht als Monozyten identifiziert wurden, dem zweiten bei S. aureus Infektion rekrutierten Zelltyp, sondern Merkmale von Neutrophilen Granulozyten aufwiesen. Bei diesen Zellen war das Oberflächenmolekül GR-1 herunterreguliert und teilweise maskiert. Diese Restpopulation könnte den Neutrophilen- Depletionsphänotyp teilweise kompensieren. Die NADPH-Oxidase gehört zu den wichtigsten antimikrobiellen Effektoren von Neutrophilen und beeinflusst, wie ich in der vorliegenden Arbeit zeigen konnte, das II ZUSAMMENFASSUNG bakterielle Wachstum signifikant. Darüber hinaus wirken die durch NADPH-Oxidase produzierten Sauerstoffradikale offenbar Zell-extrinsisch und demnach über den Ort ihrer Produktion hinaus. Dadurch können die Radikale auch in benachbarten Neutrophilen eine Einschränkung der Bakterienproliferation auslösen. Die Möglichkeit, während einer laufenden Infektion Informationen über die bakterielle Wachstumsrate auf Einzelzellebene zu erhalten, könnte sich in Zukunft zu einem wichtigen Werkzeug entwickeln, um die Wechselwirkung zwischen dem Pathogen und dem infizierten Wirt besser zu verstehen. III INTRODUCTION TABLE OF CONTENTS ABSTRACT .......................................................................................................................... I ZUSAMMENFASSUNG ........................................................................................................... II TABLE OF CONTENTS ......................................................................................................... IV ABBREVIATIONS ................................................................................................................ VI 1 INTRODUCTION ............................................................................................................ 1 1.1 Staphylococcus aureus – A commensal out of control ......................................... 1 1.1.1 Pathogenesis and antibiotic resistance ...................................................................... 1 1.1.2 Staphylococcal virulence factors ................................................................................ 5 1.2 The immune response against S. aureus ............................................................ 9 1.2.1 The course of the immune response against S. aureus infections .......................... 10 1.2.2 Hematopoiesis .......................................................................................................... 17 1.2.3 The weapons stockpile of neutrophils ...................................................................... 20 1.2.4 Immune evasion strategies of S. aureus .................................................................. 24 1.2.5 The challenge to develop an anti-staphylococcal vaccine ....................................... 29 1.3 S. aureus as an intracellular pathogen .............................................................. 30 1.4 Approaches to measure bacterial proliferation................................................... 32 1.5 Aims of the study ............................................................................................... 35 2 MATERIALS AND METHODS ......................................................................................... 36 2.1 Material ............................................................................................................. 36 2.1.1 Vectors and constructs ............................................................................................. 36 2.1.2 Kits ............................................................................................................................ 36 2.1.3 Biochemical and chemical reagents ......................................................................... 36 2.1.4 Antibodies ................................................................................................................. 38 2.1.5 Buffers and media..................................................................................................... 40 2.1.6 Technical equipment and software ..........................................................................
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