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.

This thesis describes the development of chemical tools that inhibit the sialidases NanA and NanB from Streptococcus pneumonia. The primary focus was on the discovery of allosteric inhibitors of NanA and NanB, however, promising inhibitors that act by binding at the active site of these enzymes were also investigated. Chapter 1 gives an overview of the use of chemical tools in the field of chemical biology. It focuses in particular on chemical tools that function by the allosteric regulation of their target proteins. The uses, advantages and methods of discovery of allosteric tools are discussed. Finally this chapter introduces the use of serendipitous binders for the discovery of allosteric sites. In particular, the use of CHES to identify novel allosteric sites on the sialidase NanB is proposed. Chapter 2 describes how the ‘hits' from a series of high throughput screens were reanalysed using a wide range of secondary assays to eliminate any false positives that were contaminating the results. This process removed eight of the eleven ‘hits'. Two of the remaining three compounds were then analysed further in an attempt to characterise their binding mode to NanA and/or NanB using modelling and X-ray crystallographic studies. Whilst, it was not possible to confirm the binding mode by X-ray crystallography modelling studies using the modelling software GOLD generated possible binding modes for these inhibitors. A structure activity relationship study was conducted for both compounds in an attempt to generate more potent inhibitors. Chapter 3 moves from the use of high throughput screens to identify hits against NanA and NanB to the use of the serendipitous binding of N-cyclohexyl-2-aminoethanesulfonic acid in the active site of NanB for the development of selective NanB inhibitors. First taurine was identified as the minimum unit of N-cyclohexyl-2-aminoethanesulfonic acid required to bind to the active site of NanB. Taurine was then used as the basis of an optimisation study. This chapter concludes with the identification of 2-(benzylammonio)ethanesulfonate as the next key intermediate in the development of N-cyclohexyl-2-aminoethanesulfonic acid based active site inhibitors of NanB. Chapter 4 follows on from Chapter 3 with the optimisation of 2-(benzylammonio)ethanesulfonate describing the design and synthesis of a wide range of analogues. From these compounds 2-[(3-chlorobenzyl)ammonio]ethanesulfonate was identified as the most potent and selective inhibitor. Detailed analysis of the binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to NanB gave a rationale for its improved inhibitory activity. The increase in inhibition occurred because on binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to the active site of NanB a well coordinated water molecule was displaced. The displacement of this water caused an increase in the flexibility of the enzyme's 352 loop. A detailed study of the flexibility of this loop in response to various N-cyclohexyl-2-aminoethanesulfonic acid based chemical tools was then conducted. The research in chapters 2 and 3 has recently been published. In Chapter 5 a molecule of N-cyclohexyl-2-aminoethanesulfonic acid that binds serendipitously in a previously unmentioned secondary site is elaborated into a ligand, known as Optactin, that binds strongly and selectively at this secondary site. It was then shown that Optactin inhibited NanB by binding at this secondary site. It was therefore concluded that this secondary site was in fact an allosteric site that could be used for the regulation of NanB. Chapter 6 describes the development of a rationalisation for the inhibition of NanB by Optactin. This study included the X-ray crystallographic analysis of the apo-NanB structure and the NanB-Optactin complex under a range of conditions. This was followed by mechanistic studies that identified the point in the catalytic cycle at which Optactin was inhibiting NanB. This chapter concludes with a hypothesis for the mechanism of inhibition of NanB by Optactin.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O Streptococcus (S.) pneumoniae à considerado como o principal agente causador de morbidade e mortalidade em crianÃas menores de cinco anos de idade. Todas as doenÃas pneumocÃcicas comeÃam com o estabelecimento da colonizaÃÃo do S. pneumoniae na nasofaringe, podendo progredir para doenÃa invasiva se as barreiras naturais forem cruzadas. Nas Ãltimas dÃcadas, o aumento do nÃmero de cepas de S. pneumoniae resistentes à antibiÃticos β-lactÃmicos e a outras classes de antimicrobianos tem dificultado o tratamento da infecÃÃo pneumocÃcica. Atualmente cerca de 13 sorotipos de S. pneumoniae respondem por mais de 85% dos isolados invasivos. A vacina pneumocÃcica polissacarÃdica conjugada 7-valente tem sido amplamente recomendada para crianÃas menores de cinco anos. Os objetivos desse estudo foram determinar a prevalÃncia de S. pneumoniae em crianÃas portadoras, a frequÃncia de isolados de S. pneumoniae de indivÃduos com infecÃÃo sistÃmica, o perfil de sensibilidade a antimicrobianos e os sorotipos mais comuns, em Fortaleza, Brasil. Os isolados de portadores foram recuperados a partir de swabs de nasofaringe de crianÃas usuÃrias de creches, enquanto que os isolados de infecÃÃo sistÃmica foram cedidos pelo LACEN-CE. Foram realizadas as ConcentraÃÃes InibitÃrias MÃnimas (CIM) para penicilina e ceftriaxona para todos os isolados, e levofloxacina apenas para os isolados de nasofaringe. Os pontos de corte das CIM foram determinados de acordo com o CLSI (2007). As sorotipagens dos isolados sistÃmicos foram realizadas pela reaÃÃo de Quellung, enquanto que a genotipagem capsular dos isolados de portadores foi realizada pela tÃcnica de multiplex PCR. De 215 crianÃas usuÃrias de creches, foram isolados S. pneumoniae em 152 (71%). As CIM de 137 isolados de portadores mostraram uma taxa resistÃncia de 71% para penicilina e de 21% para ceftriaxona. NÃo houve resistÃncia nos testes com levofloxacina. Comparado a um estudo similar, realizado hà 10 anos, em Fortaleza, nossos resultados apresentaram um aumento significativo nas taxas de resistÃncia à penicilina e ceftriaxona. De 26 isolados de nasofaringe que apresentaram resistÃncia plena, apenas, seis isolados (23%) tiveram a genotipagem capsular identificada por multiplex PCR. A incidÃncia de isolados invasivos neste estudo por ano, foi de, aproximadamente, 1 caso/100.000 hab. Dos 52 isolados, 42% apresentaram resistÃncia à penicilina e 13,5% à ceftriaxona. Os sorotipos mais comuns dos isolados sistÃmicos foram 19F (12%), 14, 3, 6A (8% cada), 4, 18C e 9V (6% cada), com cobertura estimada, tanto para vacina pneumocÃcica conjugada 7-valente quanto para a 10-valente, de 31,8%. / Streptococcus (S.) pneumoniae is considered the principal causative agent of morbidity and mortality in children younger than five years of age. All pneumococcal diseases are initiated by establishing a S. pneumoniae colonization in nasopharynx, the disease progressing to systemic disease if natural barrier are crossed. During the last decades, the increasing amount of resistant S. pneumoniae strains to beta-lactams and other classes of antimicrobials has modified the treatment of pneumococcal infection. At present, nearly 13 serotypes respond for more than 85% of invasive isolates. The 7-valent polysaccharide-conjugated pneumococcal vaccine has been widely recommended for use in children younger than five years. The aims of this study were to determine the S. pneumoniae carrier in children, the frequence of serotypes from systemic infection patients, the susceptibility profile to antimicrobials in Fortaleza, Brazil. Carrier state isolates were recovered from nasopharyngeal swabs from children attending day-care center facilities, while the isolates from systemic infection fournished by LACEN-CE. Minimal Inhibitory Concentrations (MIC) to penicillin and ceftriaxone were assessed for all isolates, and levofloxacin MIC only from nasopharyngeal isolates. MIC cut-offs were determined according to CLSI standards (2007). Serotyping of systemic isolates was performed by Quellung reaction, while capsular genotyping of carrier isolates was performed by multiplex PCR assay. OF 215 children attending day-care centers, 152 S. pneumoniae isolates were identified (71%). Penicillin MIC showed 71% of resistance, and for ceftriaxone, 21% of resistance. No resistance was found for levofloxacin MIC testing. When compared to a 10-year old similar study in Fortaleza, our results have shown a significant increase of penicillin and ceftriaxone resistance rates. Of 26 isolates tested, only six nasopharyngeal isolates (23%) were positively genotyped by multiplex PCR. The incidence of invasive isolates was 1/100,000 inhab. per year. Of 52 systemic isolates serotyped, 42% were penicillin-resistant, and 13.5% were ceftriaxone-resistant. Systemic serotypes identified were 19F, 3, 6A, 4, 18C and 9V, with a estimated coverage by the 7-valent and 10-v pneumococcal polysaccharide conjugated vaccines of 31.8%.

Pneumolysin, a protein toxin, represents one of the major virulence factors of Streptococcus pneumoniae. This pathogen causes bacterial meningitis with especially high disease rates in young children, elderly people and immunosuppressed patients. The protein toxin belongs to the family of cholesterol-dependent cytolysins, which require membrane cholesterol in order to bind and to be activated. Upon activation, monomers assemble in a circle and undergo conformational change. This conformational change leads to the formation of a pore, which eventually leads to cell lysis. This knowledge was obtained by studies that used a higher concentration compared to the concentration of pneumolysin found in the cerebrospinal fluid of meningitis patients. Thus, a much lower concentration of pneumolysin was used in this work in order to investigate effects of this toxin on primary mouse astrocytes. Previously, a small GTPase activation, possibly leading to cytoskeletal changes, was found in a human neuroblastoma cell line. This led to the hypothesis that pneumolysin can lead to similar cytoskeletal changes in primary cells. The aim of this work was to investigate and characterise the effects of pneumolysin on primary mouse astrocytes in terms of a possible pore formation, cellular trafficking and immunological responses. Firstly, the importance of pore-formation on cytoskeletal changes was to be investigated. In order to tackle this question, wild-type pneumolysin and two mutant variants were used. One variant was generated by exchanging one amino acid in the cholesterol recognising region, the second variant was generated by deleting two amino acids in a protein domain that is essential for oligomerisation. These variants should be incapable of forming a pore and were compared to the wild-type in terms of lytic capacities, membrane binding, membrane depolarisation, pore-formation in artificial membranes (planar lipid bilayer) and effects on the cytoskeleton. These investigations resulted in the finding that the pore-formation is required for inducing cell lysis, membrane depolarisation and cytoskeletal changes in astrocytes. The variants were not able to form a pore in planar lipid bilayer and did not cause cell lysis and membrane depolarisation. However, they bound to the cell membrane to the same extent as the wild-type toxin. Thus, the pore-formation, but not the membrane binding was the cause for these changes. Secondly, the effect of pneumolysin on cellular trafficking was investigated. Here, the variants showed no effect, but the wild-type led to an increase in overall endocytotic events and was itself internalised into the cell. In order to characterise a possible mechanism for internalisation, a GFP-tagged version of pneumolysin was used. Several fluorescence-labelled markers for different endocytotic pathways were used in a co-staining approach with pneumolysin. Furthermore, inhibitors for two key-players in classical endocytotic pathways, dynamin and myosin II, were used in order to investigate classical endocytotic pathways and their possible involvement in toxin internalisation. The second finding of this work is that pneumolysin is taken up into the cell via dynamin- and caveolin-independent pinocytosis, which could transfer the toxin to caveosomes. From there, the fate of the toxin remains unknown. Additionally, pneumolysin leads to an overall increase in endocytotic events. This observation led to the third aim of this work. If the toxin increases the overall rate of endocytosis, the question arises whether toxin internalisation favours bacterial tissue penetration of the host or whether it serves as a defence mechanism of the cell in order to degrade the protein. Thus, several proinflammatory cytokines were investigated, as previous studies describe an effect of pneumolysin on cytokine production. Surprisingly, only interleukin 6-production was increased after toxin-treatment and no effect of endocytotic inhibitors on the interleukin 6-production was observed. The conclusion from this finding is that pneumolysin leads to an increase of interleukin 6, which would not depend on the endocytotic uptake of pneumolysin. The production of interleukin 6 would enhance the production of acute phase proteins, T-cell activation, growth and differentiation. On the one hand, this activation could serve pathogen clearance from infected tissue. On the other hand, the production of interleukin 6 could promote a further penetration of pathogen into host tissue. This question should be further investigated. / Das Protein-Toxin Pneumolysin ist einer der entscheidenden Virulenzfaktoren von Streptococcus pneumoniae. Dieses Protein-Toxin gehört zur Familie der cholesterinabhängigen Zytolysine, die Membrancholesterol für ihre Aktivierung und Bindung benötigen. Nach der Membranbindung ordnen sich die Toxinmonomere kreisförmig an und ändern ihre Konformation, wodurch eine Pore entsteht, die dann zu einer Lyse der Zelle führt. Vor kurzem wurde nach Pneumolysinbehandlung in einer humanen Neuroblastomzelllinie eine Aktivierung kleiner GTPasen gefunden, die für zytoskelettale Veränderungen entscheidend sind (z.B. Zellbewegungen). Deshalb wurde die Hypothese aufgestellt, dass Pneumolysin diese zytoskelettalen Veränderungen auch in primären neuronalen Zellen auslösen könnte. Das Ziel dieser Arbeit war, die Effekte von Pneumolysin auf primäre Mausastrozyten im Hinblick auf Porenbildung, zelluläre Transportprozesse und immunologische Antworten zu untersuchen. Im ersten Teil wird die Bedeutung der Porenbildung auf zytoskelettale Veränderungen untersucht. Hierbei wurden lytische Fähigkeiten, Membranbindung, Membrandepolarisation, Porenbildung im künstlichen Bilayer und Effekte auf das Zytoskelett untersucht. Sowohl der Wildtyp als auch die Varianten zeigten die gleiche Stärke an Membranbindung. Diese Untersuchungen weisen darauf hin, dass die Porenbildung für die Zell-Lyse, Membrandepolarisation und zytoskelettale Veränderungen in Mausastrozyten wichtig ist und führt zu der Schlussfolgerung, dass nicht die Membranbindung, sondern die Porenbildung entscheidend für die beobachteten zytoskelettalen Veränderungen ist. Im zweiten Teil dieser Arbeit wurde der Effekt des Pneumolysin auf zelluläre Transportprozesse untersucht. Erneut zeigten die Pneumolysinvarianten keine Wirkung, während der Wildtyp die Gesamtrate der Endozytose erhöhte. Weiterhin wurde nur der Wildtyp internalisiert. Um einen möglichen Mechanismus für die Internalisierung des Toxins vorschlagen zu können, wurde Pneumolysin als GFP-markiertes Toxin genutzt. Weiterhin wurden einige Marker für unterschiedliche endozytotische Transportprozesse genutzt um eine Ko-lokalisation mit Pneumolysin-GFP zu ermöglichen. Des Weiteren wurden Inhibitoren für zwei Schlüsselproteine endozytotischer Vorgänge, Dynamin und Myosin II, genutzt. Die Ergebnisse dieser Untersuchungen zeigten, dass Pneumolysin wahrscheinlich durch dynamin- und caveolin-unabhängige Pinozytose in die Zelle aufgenommen wird. Dieser Mechanismus führt zu der Bildung von Caveosomen, deren weiterer Transport, und somit das Schicksal des internalisierten Toxins, bis heute noch nicht aufgeklärt ist. Die Beobachtung, dass Pneumolysin die Gesamtrate an Endozytose erhöht, führte zum dritten Teil dieser Arbeit. Wenn das Toxin die Gesamtrate an Endozytose erhöht, stellt sich die Frage, ob dieser Vorgang der Zerstörung des Toxins – also einer Abwehr der Zelle – dient, oder ob diese Internalisierung eine Strategie des Pathogens ist, um tiefer in das Wirtsgewebe einzudringen. Aktuelle Studien belegen, dass Pneumolysin einen Einfluss auf inflammatorische Antworten des Immunsystems hat. Aus diesem Grund wurden unterschiedliche proinflammatorische Zytokine untersucht. Überraschenderweise zeigte sich nur eine Erhöhung des Interleukin 6 nach der Toxinbehandlung. Weiterhin hatten die Endozytoseinhibitoren keinen Effekt auf die Produktion dieses proinflammatorischen Zytokins. Pneumolysin führt also zu einem Anstieg der Interleukin 6 Produktion, diese Produktion ist jedoch unabhängig von der Internalisierung dieses Toxins. Die Produktion dieses Interleukins würde zur Produktion der Akute-Phase Proteine, der Aktivierung der T-Zell Antwort, zu Wachstum und Zelldifferenzierung führen. Einerseits könnte diese Aktivierung die Infektion durch das Pathogen bekämpfen. Andererseits könnte S. pneumoniae die erhöhte Produktion durch PLY an Interleukin 6 nutzen um weiter in das Wirtsgewebe vordringen zu können. Diese Frage sollte noch durch weitere Experimente untersucht werden.

Le but de ce travail consiste à créer un nouveau tag de purification d'affinité permettant la purification de protéines recombinantes sur une matrice DEAESépharose Fast Flow. Dans la nature, certaines protéines de surface de Streptococcus pneumoniae sont liées à la paroi bactérienne par des interactions non convalentes faisant intervenir des molécules de choline présentes sur les acides téichoiques et lipotéichoiques. Ces protéines de surface présentent une organisation modulaire avec le domaine catalytique et le domaine de liaison fonctionnant indépendamment l'un de l'autre. La choline étant un analogue structural du DEAE, l'étude des domaines de liaison à la choline constitue une approche de choix pour concevoir un tag de purification présentant une affinité pour le DEAE-Sépharose. Nous avons plus particulièrement travaillé sur la N-acétyl-L-alanine amidase (LytA) qui dégrade spécifiquement certaines liaisons du peptidoglycan de la paroi de Streptococcus pneumoniae. Son domaine de liaison à la choline C-terminal (ClytA) se compose de six motifs répétés imparfaits, constitué chacun d'une vingtaine de résidus. Deux stratégies ont été développées pour concevoir le tag de purification. D'une part, 126 motifs répétés de 19 domaines de liaison à la choline ont été alignés pour définir une séquence consensus. Cette approche a permis de mettre en évidence les résidus importants conservés parmi les motifs répétés. D'autre part, nous avons construit des protéines de fusion portant des fragments du domaine de liaison ClytA de longueur variable. Des expériences de chromatographies sur matrice DEAESépharose nous ont permis d'isoler un petit fragment de ClytA(L234), présentant toujours une affinité spécifique pour le DEAE Sépharose. Cette affinité est maintenue lorsque le fragment L234 est fusionné à l'extrémité C-terminale d'une autre protéine reporter. Cependant, nos résultats suggèrent que le candidat tag L234 est instable et qu'il conduit à l'insolubilisation de la protéine de fusion lors de la production de celle-ci dans Escherichia coli. Afin d'améliorer la solubilité/stabilité du fragment L234, nous avons développé trois approches bioinformatiques. Cellesci ont permis de définir trois groupes de mutations permettant d'améliorer potentiellement la solubilité et/ou la stabilité du fragment L234. Les tags mutants ont été construits et fusionnés à l'extrémité C terminale de la thiorédoxine. Le premier tag mutant, EDE-L234, est plus soluble que la version non mutante mais présente une perte d'affinité pour le DEAE Sépharose. Le second mutant, NG-L234, ne montre pas d'augmentation de solubilité et perd également une partie de son affinité pour la matrice. Le troisième tag mutant, V1V2V3-L234, présente une augmentation d'affinité pour le DEAE-Sépharose bien que sa solubilité reste inchangée.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Bibliography: leaves [177]-[194] / [194] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1989

Thesis (M.S.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 36 pages. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2005. / "December 2004" Includes bibliographical references (leaves 342-379).