Thesis advisor: Michelle Meyer / The primary bacterial targets for most antibiotics are well known. To survive the stress of an antibiotic a bacterium must decrease the antibiotic to target binding ratio to escape from harmful effects. This can occur through a number of different functions including down-regulation of the target, mutation of the binding site on the target, and decreasing the intake or increasing the efflux of the antibiotic. However, it is becoming more evident that an antibiotic stress response influences more than just the primary target, and that a wave of secondary responses can be triggered throughout the bacterium. As a result resistance mutations may arise in genes that are indirectly affected by the initial interaction between the antibiotic and target. These indirect responses have been found to be associated with metabolism, regulation, cell division, oxidative stress, and other critical pathways. One technique recently developed in our lab, called transposon insertion sequencing (Tn-seq), can be used to further understand the complexity of these indirect responses by profiling growth rates (fitness) of mutants at a genome-wide level. However, Tn-seq is normally performed with large libraries of pooled mutants and thus it remains unclear how this may influence fitness of some independent mutants that may be compensated by others in the population. Additionally, since the original method has only utilized planktonic culture, it is also not clear how higher order bacterial structures, such as biofilms or microcolonies, influence bacterial fitness. To better understand the dynamics of pooled versus individual mutant culture, as well as the effect of community structure in microcolony development on the influence of fitness, we adapted a droplet microfluidics-based technique to encapsulate and culture single mutants. We were able to successfully encapsulate at least 7 different species of bacterial pathogens, including Streptococcus pneumoniae, and culture them planktonically, or as microcolonies, in either monodisperse liquid or agarose droplets. These experiments, however, raised an important challenge: the DNA yield from one encapsulation experiment is insufficient to generate samples for sequencing by means of the traditional Tn-seq method. This led us to develop a novel Tn-seq DNA library preparation method, which is able to generate functional Tn-seq library molecules from picogram amounts of DNA. This method is not ideal yet because fitness data generated through the new method currently does not correlate well with data from traditional Tn-seq library preparation. However, we have identified one major culprit that should be easily solvable. We expect by modifying the binding site of the primer used for linear amplification of transposon ends that the new preparation method will be able recapitulate results from the traditional Illumina preparation method for Tn-seq. This will enable us to prepare robust Tn-seq samples from very small amounts of DNA in order to probe stress responses in single mutants as well as in microcolonies in a high-throughput manner. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Mycobacterium tuberculosis est la cause d’une des maladies infectieuses les plus fréquentes dans le monde causant la mort de plus de 1,2 millions de personnes chaque année selon l’organisation mondiale de la santé (OMS). Actuellement, la tuberculose à M. tuberculosis émerge chez d’autres espèces comme l’a montré notre revue bibliographique des cas de tuberculose à M. tuberculosis chez les primates non-humains, les éléphants d’Asie, les animaux de ferme, les animaux de compagnie et certains animaux sauvages. Nous avons montré que ces trois espèces survivent dans le sol pendant au moins 12 mois et restent pathogènes dans un modèle souris. Egalement nous avons montré que le sol infecté par M. tuberculosis est une source potentielle de contamination pour les animaux. Nous avons ensuite développé un protocole de culture rapide de M. tuberculosis, incluant un nouveau milieu de culture solide, des conditions optimisées d’incubation, et la détection des microcolonies par autofluorescence. Notre travail de thèse a permis de mettre en place des techniques et protocoles qui révolutionnent la culture et l’isolement de M. tuberculosis en réduisant les délais de culture et des antibiogrammes, un point déterminant pour la lutte contre la tuberculose notamment dans les pays à ressources limitées et les pays à forte émergence de souches de M. tuberculosis de plus en plus résistantes. Ces protocoles sont en cours de transfert pour la routine de laboratoire. / Mycobacterium tuberculosis is the cause of one of the most common infectious diseases in the world killing more than 1.2 million people each year according to the World Health Organization (WHO). Currently, M. tuberculosis tuberculosis emerges in other species like non-human primates, Asian elephants, farm animals and some wild animals. We have shown that three species of M. tuberculosis complex survive in the soil for at least 12 months and are pathogenic in a mouse model and M. tuberculosis-infected soil is a potential source of infection for animals. We then developed a protocol for rapid culture of M. tuberculosis, including a new solid culture medium, optimized conditions of incubation, and detection of microcolonies by autofluorescence. Our thesis has helped develop techniques and protocols that are revolutionizing the culture and isolation of M. tuberculosis by reducing delays culture and susceptibility testing, a crucial point for the fight against tuberculosis, especially in countries limited resources and countries with strong emergence of M. tuberculosis strains more resistant. These protocols are being transferred to the routine laboratory.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

The majority of bacteria are organized in surface-associated communities, the so called biofilms. Crucial processes that drive the formation of such biofilms are the motility of bacteria on a substrate, enabling cells to reach each others vicinity, and attractive cell-cell-interactions, driving the formation of microcolonies. These colonies, aggregates consisting of thousands of cells, are the precursors of biofilms. In this thesis we investigate the role of cell appendages, called type IV pili, in the substrate motion of bacteria and the formation of bacterial microcolonies. Therefore, we study the bacterial dynamics with the help of experiments and theoretical models. We introduce a novel simulation tool in the tradition of Brownian dynamics simulations. In this computational model, that was developed alongside experimental observations, we study how explicit pili dynamics, pili-substrate and pili–pili interactions drive the cell dynamics. First, we apply our model to investigate how individual cells move on a substrate due to cycles of protrusion and retraction of type IV pili. We show that the characteristic features, in particular persistent motion, can solely originate from collective interactions of pili. Next, we perform experiments to study the coalescence of bacterial microcolonies. With the help of experiments and our computational model, we identify a spatially-dependent gradient of motility of cells within the colony as the origin of a separation of time scale, a feature which is in disagreement with the coalescence dynamics of fluid droplets. Additionally, we show that altering the force generation of pili can cause demixing of cells within bacterial aggregates. Finally, we combine our knowledge of the substrate motion of cells and of the pili-mediated interactions of colonies to identify the main processes (aggregation, fragmentation and cell divisions) that drive assembly of colonies. Starting from experiments, we develop a mathematical model and observe excellent qualitative and quantitative agreement to experimental data of the density of colonies of different sizes. In summary, hand in hand with experiments, we develop theoretical frameworks to unravel the role of type IV pili in bacterial surface motility, microcolony dynamics and colony formation.

Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Vývojová biologie (P1520) Studijní obor: Vývojová biologie (1501V000) Oleksandr Chernyavskiy Zdokonalené metody pro snímání obrazových dat a analýzu tkání a buněk pomocí konfokální a multifotonové mikroskopie Improved Methods of Image Acquisition and Analysis of Tissues and Cells by Confocal and Multi-Photon Microscopy Abstrakt disertační práce Školitel: RNDr. Lucie Kubínová CSc Praha, 2015 Abstract The aim of this study was to develop methods and approaches for image acquisition with subsequent image analysis of data, obtained by confocal and two- photon excitation microscopy as well as their combination, enabling new possibilities of visualization and assessment of information on biological tissues and cell structures in 3D and their measurement. We focused on methods that exploited advantages of confocal and multi-photon excitation microscopy. Our further aim was to demonstrate the applicability of non-invasive approach for in vivo applications, usefulness and the relevance of these methods in several special biological applications with emphasis on improved image acquisition, analysis and evaluation of real biological specimens. The present work was not oriented on just one specific biological problem, but rather to methodological...

Neisseria gonorrhoeae verursacht die sexuell übertragbare Krankheit Gonorrhoe und ist ein Typ-IV-Pili (Tfp) exprimierendes Bakterium, das den Urogenitaltrakt besiedelt. Frühe Infektionsstadien piliierter N. gonorrhoeae (P+GC) sind durch die Tfp-vermittelte Adhärenz an Wirtszellen gekennzeichnet, dann erfolgt die Bildung von Mikrokolonien auf Wirtszellepithelien. Hier wird gezeigt, dass die Wirtszellen an der effizienten Bildung der extrazellulären Mikrokolonien beteiligt sind. P+GC die fixierte Wirtszellen infizieren weisen eine verzögerte Mikrokoloniebildung gegenüber einer Infektion lebender Wirtszellen auf. Kortikales Aktin wird zusammen mit Signalproteinen innerhalb der Wirtszellen zu den adhärierten Bakterien rekrutiert, darunter das Hauptstrukturprotein von Caveolae-Membrandomänen, Caveolin-1 (Cav1). Eine Reduzierung der Expression von Cav1 führt zu einer verstärkten Aufnahme von P+GC in die Wirtszellen, wohingegen die Expression von Cav1 in Cav1-negativen Zellen eine Internalisierung verhindert. Internalisierte Bakterien weisen dabei geringere Überlebensraten auf je länger sie in den Wirtszellen verbleiben. Die Rekrutierung von Cav1 ist eine unmittelbare und kontinuierliche zelluläre Antwort auf eine Infektion mit P+GC, welche die Phosphorylierung von Cav1 an Tyrosin 14 bedingt. Zusätzlich erforderte die Cav1-vermittelte Blockierung der Internalisierung der Bakterien und die Verankerung von Cav1 mit dem Zytoskelett eine Tyrosinphosphorylierung von Cav1. Eine Analyse möglicher Interaktionspartner von phosphoryliertem Cav1 zeigte eine direkte Interaktion mit Vav2. Sowohl Vav2 als auch sein Substrat, die kleine GTPase RhoA, blockieren die Aufnahme von Bakterien in die in Wirtszellen. Die Aktivierung von RhoA nach P+GC Infektion erfordert die Expression von Cav1, was auf einen Cav1-Vav2-RhoA Signalweg hindeutet. Darüber hinaus wurden in dieser Arbeit sechs neue, eine SH2-Domäne-beinhaltende Interaktionspartner von phosphoryliertem Cav1 identifiziert. / Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhea and colonizes mucosal epithelia of the human urogenital tract. The early stages of infection with piliated N. gonorrhoeae (P+GC) are characterized by Tfp-mediated adherence to host cells, followed by formation of bacterial microcolonies on the surface of host cells. This study provides evidence that host cell participation is required for the efficient formation of extracellular microcolonies during Neisseria infection. P+GC infecting fixed host cells demonstrate altered motility and delayed microcolony formation compared to infecting living host cells. Cortical actin and various signal transducing proteins are recruited to the site of bacterial attachment within host cells, one of them being the major structural protein of plasma membrane caveolae, Caveolin-1 (Cav1). Down-regulation of Cav1 results in increased uptake of P+GC into host cells whereas expression of the protein in Cav1-negative cells blocks bacterial internalization. Host cell entry results in decreased viability of internalized bacteria over time. Cav1 recruitment is demonstrated to be an immediate and continuous cellular response to P+GC infection that involves Cav1 phosphorylation on its tyrosine 14 residue. Prevention of bacterial uptake mediated by Cav1 as well as tight association of Cav1 with the cytoskeleton also requires tyrosine phosphorylation. A broad analysis of interaction partners of phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, are involved in preventing bacterial uptake and RhoA activation after P+GC infection requires Cav1 expression, thus providing evidence for a Cav1-Vav2-RhoA signaling cascade. Moreover, six novel SH2-domain containing interaction partners of tyrosine phosphorylated Cav1 have been identified, all of which have been implicated in modulating the cytoskeleton.

The majority of bacteria are organized in surface-associated communities, the so called biofilms. Crucial processes that drive the formation of such biofilms are the motility of bacteria on a substrate, enabling cells to reach each others vicinity, and attractive cell-cell-interactions, driving the formation of microcolonies. These colonies, aggregates consisting of thousands of cells, are the precursors of biofilms. In this thesis we investigate the role of cell appendages, called type IV pili, in the substrate motion of bacteria and the formation of bacterial microcolonies. Therefore, we study the bacterial dynamics with the help of experiments and theoretical models. We introduce a novel simulation tool in the tradition of Brownian dynamics simulations. In this computational model, that was developed alongside experimental observations, we study how explicit pili dynamics, pili-substrate and pili–pili interactions drive the cell dynamics. First, we apply our model to investigate how individual cells move on a substrate due to cycles of protrusion and retraction of type IV pili. We show that the characteristic features, in particular persistent motion, can solely originate from collective interactions of pili. Next, we perform experiments to study the coalescence of bacterial microcolonies. With the help of experiments and our computational model, we identify a spatially-dependent gradient of motility of cells within the colony as the origin of a separation of time scale, a feature which is in disagreement with the coalescence dynamics of fluid droplets. Additionally, we show that altering the force generation of pili can cause demixing of cells within bacterial aggregates. Finally, we combine our knowledge of the substrate motion of cells and of the pili-mediated interactions of colonies to identify the main processes (aggregation, fragmentation and cell divisions) that drive assembly of colonies. Starting from experiments, we develop a mathematical model and observe excellent qualitative and quantitative agreement to experimental data of the density of colonies of different sizes. In summary, hand in hand with experiments, we develop theoretical frameworks to unravel the role of type IV pili in bacterial surface motility, microcolony dynamics and colony formation.:1. Introduction 2. Computational model of bacterial motility and mechanics 3. Motility of single bacteria on a substrate 4. Coalescence and internal dynamics of bacterial microcolonies 5. Demixing of bacterial microcolonies 6. Self-assembly of microcolonies 7. Summary and Outlook A. Details of the Simulation model B. Experimental protocols C. Geometric estimation of the parameters of the stochastic model D. Solutions for simplified models of pili-mediated cell motion E. Image analysis of experimental data F. Simulations and data analysis G. The mean squared relative distance (MSRD)