Thesis (Ph. D.)--University of Chicago, 1906. / eContent provider-neutral record in process. Description based on print version record.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Bacteria have strain-dependent host attachment mechanisms. Bacteria with pili, also called fimbriae, attach to the host epithelial cells allowing the bacterium time to adhere, form biofilms, and infect the body without being flushed out by the host’s cleansing mechanisms. Haemophilus influenzae type B (Hib) are bacteria that colonize the human nasopharynx by attaching via adhesion pili to the microvilli found in the respiratory tract. To gain insight into the attachment mechanism of Hib bacteria, pilus expression and purification were optimized. To determine the structure of Hib pili, cryogenic electron microscopy (cryo-EM) images were collected of the isolated and purified filaments. Reconstructions were computed and the secondary structure of the Hib subunit (HifA) was predicted and fitted into the reconstruction. The structure of the Hib filament showed the pili to have one-start helical symmetry, with a zigzag shape. The predicted structure of HifA had two regions of interaction with the adjacent subunit, including an N-terminal extension (NTE) and a loop, both extending from the immunoglobulin (Ig)-like domain. These extensive contacts resulted in a stronger interaction between the subunits compared to other types of adhesion pili. Toward the goal of inhibiting pili adherence to the host cells, the effects of salivary peptides were tested. Peptide binding assays were performed and analyzed to determine changes in structure or macromolecular assembly of Hib pili. Our discovery of the presence and absence of pilus three-dimensional bundling and two-dimensional networks can aid in the future prevention of bacterial colonization and infection within the human body thereby preventing Hib related diseases.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Vita. Includes bibliographical references (leaves 156-180). Also issued on the Internet.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Universiteit van Amsterdam, 1989. / Summary in Dutch. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Made available in DSpace on 2015-02-27T17:39:02Z (GMT). No. of bitstreams: 2 81.pdf: 872019 bytes, checksum: b8e894b7b58911018e4a590c0266cbe1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A bactéria Haemophilus da espécie influenzae, pode causar no homem diversas infecções, invasivas ou não. O H. influenzae do tipo b (Hib), antes da introdução da vacina conjugada contra Hib, associava-se a infecções como epiglotite, artrite séptica, bacteriemia, pneumonia septicemia e meningite, principalmente em crianças. As outras espécies tipáveis (a,c,d,e,f) e não tipáveis estavam associadas a infecções do trato respiratório adquiridas na comunidade. Após a introdução dessa vacina, houve redução expressiva das doenças causadas pelo Hib em diversas partes do mundo, inclusive no Brasil. Atualmente, outros tipos da espécie H. influenzae (Hi) que não o Hib estão sendo isoladas não somente em crianças, causando infecções graves. O ressurgimento de casos por Hib, em crianças vacinadas, tem sido reportado como possível falha vacinal. Esses fatos preocupam a Saúde Pública no mundo, demonstrando que mudanças epidemiológicas podem estar ocorrendo devido à seleção de cepas após a introdução da vacina. Neste estudo, mudanças genéticas na região capsular do Hi foram encontradas em cepas isoladas nos períodos pré e pós-vacinal de três estados brasileiros. Através do estudo de susceptibilidade foram encontradas cepas produtoras de β-lactamase (BLA+) e não produtoras resistentes a ampicilina (BLNAR). A análise de redução da susceptibilidade às quinolonas foi avaliada utilizando-se o ácido nalidíxico como marcador, confrontando-se a resistência deste antibiótico com os marcadores moleculares das regiões determinantes de resistência a quinolonas (QRDRs) através do sequenciamento dos genes gyrA e parC que codificam a síntese das proteínas GyrA e ParC. Os resultados obtidos demonstraram que o ácido nalidíxico é o antibiótico mais indicado desse grupo para detectar a redução da sensibilidade. / A bactéria Haemophilus da espécie influenzae, pode causar no homem diversas infecções, invasivas ou não. O H. influenzae do tipo b (Hib), antes da introdução da vacina conjugada contra Hib, associava-se a infecções como epiglotite, artrite séptica, bacteriemia, pneumonia septicemia e meningite, principalmente em crianças. As outras espécies tipáveis (a,c,d,e,f) e não tipáveis estavam associadas a infecções do trato respiratório adquiridas na comunidade. Após a introdução dessa vacina, houve redução expressiva das doenças causadas pelo Hib em diversas partes do mundo, inclusive no Brasil. Atualmente, outros tipos da espécie H. influenzae (Hi) que não o Hib estão sendo isoladas não somente em crianças, causando infecções graves. O ressurgimento de casos por Hib, em crianças vacinadas, tem sido reportado como possível falha vacinal. Esses fatos preocupam a Saúde Pública no mundo, demonstrando que mudanças epidemiológicas podem estar ocorrendo devido à seleção de cepas após a introdução da vacina. Neste estudo, mudanças genéticas na região capsular do Hi foram encontradas em cepas isoladas nos períodos pré e pós-vacinal de três estados brasileiros. Através do estudo de susceptibilidade foram encontradas cepas produtoras de β-lactamase (BLA+) e não produtoras resistentes a ampicilina (BLNAR). A análise de redução da susceptibilidade às quinolonas foi avaliada utilizando-se o ácido nalidíxico como marcador, confrontando-se a resistência deste antibiótico com os marcadores moleculares das regiões determinantes de resistência a quinolonas (QRDRs) através do sequenciamento dos genes gyrA e parC que codificam a síntese das proteínas GyrA e ParC. Os resultados obtidos demonstraram que o ácido nalidíxico é o antibiótico mais indicado desse grupo para detectar a redução da sensibilidade. O presente estudo mostra a necessidade de que os laboratórios de rotina e de pesquisa avaliem e desenvolvam métodos simples e rápidos, capazes de indicar as possíveis mudanças capsulares e o surgimento de cepas resistentes auxiliando a Vigilância Epidemiológica e a Vigilância Sanitária.

H. influenzae ist ein fakultativ anaerobes, Gram-negatives Bakterium und wird in die Familie der Pasteurellacaea eingeordnet. Das Bakterium zeigt Auxotrophien für Hämin unter aeroben Bedingungen und für Nikotinamid-Adenin-Dinukleotid (NAD) bei aeroben wie anaeroben Wachstum. Es können zwei Unterarten unterschieden werden: die bekapselten Stämme, die systemische Erkrankungen hervorrufen und die unbekapselten bzw. nicht-typisierbaren Stämme, die für Oberflächeninfektionen verantwortlich sind. Da bei H. influenzae bisher nur wenig über die NAD-Aufnahme bekannt war, sollten in dieser Arbeit Proteine identifiziert und charakterisiert werden, die an der NAD-Aufnahme beteiligt sind. Das Außenmembranprotein e(P4), kodiert von dem hel-Gen, wurde als eine Komponente des Häminaufnahmesystems beschrieben. In dieser Arbeit wurde eine hel-Deletionsmutante hergestellt, anhand der nachgewiesen wurde, daß e(P4) als saure Phosphatase nicht an der Hämin-, sondern an der NAD-Aufnahme beteiligt ist. Mit Hilfe von verschiedenen Phosphatase-Assays und dem Malat-Enzym-Assay konnten NADP und Nikotinamid-Mononukleotid (NMN) als physiologisch relevante Substrate identifiziert werden. Die biologische Relevanz von e(P4) für die NAD-Aufnahme wurde durch Mutantenanalyse in Wachstumstests und Transport-Assays nachgewiesen. Es wurde gezeigt, daß die hel-Deletionsmutante mit NAD und NMN ein signifikantes Wachstumsdefizit hatte und nicht fähig war, beide Nikotinamid-Nukleotid-Quellen aufzunehmen, während die Nutzung von Nikotinamid-Ribosyl (NR) keinen Unterschied zum Wildtyp aufwies. Um die Frage zu klären, ob die Lokalisation von e(P4) als Lipoprotein an der Außenmembran wichtig für die NMN-Spaltung ist, wurden zwei Mutanten hergestellt, bei denen im hel-Gen der Lipidanker Cystein durch ein Glycin ausgetauscht wurde, um das Protein im Periplasma zu exprimieren. Die Periplasma-Extrakte dieser Cystein-Mutanten zeigten in Phosphatase-Assays keine Aktivität. Um zu untersuchen, ob die Phosphatase-Aktivität die einzige für die NAD-Aufnahme relevante Funktion von e(P4) ist, wurden Phosphatase-Mutanten hergestellt und charakterisiert. Sie exprimierten ein mutiertes e(P4)-Protein, das durch einen Aminosäureaustausch die Phosphatase-Aktivität verloren hatte. Es zeigte sich, daß die Phänotypen der Phosphatase-Mutanten in Bezug auf die Fähigkeit NMN zu spalten, NAD und NMN aufzunehmen und als Faktor V-Quelle zum Wachstum zu nutzen, exakt mit den Phänotypen der Deletionsmutante korrelierten. Es konnten daher keine weiteren Funktionen von e(P4) festgestellt werden. Das periplasmatische Protein NadN wurde als Pyrophosphatase beschrieben, die fähig ist, NAD zu NMN zu hydrolysieren. Weiter wurde eine 5´-Nukleotidase-Aktivität nachgewiesen, mit der NadN NMN zu NR dephosphorylieren kann. Die Relevanz von NadN für die NAD-Aufnahme wurde anhand einer nadN-Knockout-Mutante untersucht. In Wachstumskurven und Transport-Assays zeigte sich, daß die nadN-Mutante nicht fähig war, NAD aufzunehmen und zum Wachstum zu verwenden. Auch die Nutzung von NMN war bei der Mutante eingeschränkt, während mit NR als Faktor V-Substrat kein Unterschied zum Stamm Rd erkennbar war. Durch die Charakterisierung einer hel nadN-Doppelmutante konnte nachgewiesen werden, daß keine weiteren Enzyme außer e(P4) und NadN an der Prozessierung von NAD zu NR beteiligt sind. Es zeigte sich auch, daß nur NR über einen putativen Transporter ins Zytoplasma aufgenommen wird. Das Protein OmpP2 stellt ein Hauptporin der äußeren Membran dar. Durch eine ompP2-Deletionsmutante konnte mit Hilfe von Wachstumskurven und Transport-Assays nachgewiesen werden, daß NAD, NMN und NR durch diese Pore ins Periplasma diffundieren. / H. influenzae is a facultativ anaerobic, Gram-negative bacterium and belongs to the familiy of Pasteurellaceae. This bacterium shows auxotrophies for hemin under aerobic conditions and for nicotinamid adenine dinucleotide (NAD) at aerobic and anaerobic growth. Two subspecies are distinguishable: the encapsulated strains, the causative agents for systemic and invasive diseases and the unencapsulated or nontypeable strains, responsible for inflammation of the respiratory tract. In H. influenzae little is known about the utilization of NAD, therefore this work was aimed to identify and characterize gene products which are involved in the uptake of NAD. The outer membrane protein e(P4), encoded by the gene hel, was described as a component of the hemin uptake system. In this work a hel deletion mutant was constructed to proof that the acid phosphatase e(P4) is not involved in the uptake of hemin but in the uptake of NAD. With the aid of different phosphatase assays and the maleic enzyme assay NADP and NMN could be identified as the physiological relevant substrates. The biological relevance of e(P4) was proofed by mutant analysis in growth tests and transport assays. It was shown that the hel deletion mutant had a significant growth deficiency with NAD and NMN and was not able to take up both nicotinamide nucleotide sources, whereas the utilization of nicotinamide ribosyl (NR) showed no difference compared to the wildtype. To address the question, wether the localization of the lipoprotein e(P4) at the outer membrane is important for the hydrolysis of NMN, two mutants were constructed which had a Cystein-Glycin replacement in the lipid anchor of the hel gene to express the protein in the periplasm. The periplasm extracts of these Cystein mutants showed no activity in phosphatase assays. To investigate wether the phosphatase acitivty of e(P4) is the only relevant function for the uptake of NAD, phosphatase mutants were constructed and characterized. They expressed a mutated e(P4) protein which had lost the phosphatase activity by an amino acid replacement. The phenotypes of the phosphatase mutants correlated exactly with the phenotype of the deletion mutant concerning the ability to cleave NMN, to take up NAD and NMN and to use them as factor V sources. The periplasmic protein NadN was described as a pyrophosphatase which is able to hydrolase NAD to NMN. Further a 5´-nucleotidase function was identified enabling NadN to dephosphorylate NMN to NR. The relevance of NadN for the utilization of NAD was investigated with a nadN knockout mutant. Growth curves and transport assays revealed that the nadN mutant was not able to take up NAD and to use it for growth. The utilization of NMN was reduced, whereas the utilization of NR showed no difference compared to the wildtype. By characterizing a hel nadN double mutant it was shown that no further enzymes than e(P4) and NadN are involved in the processing of NAD to NR and that only NR is taken up into the cytoplasm through a putativ transporter. The protein OmpP2 is the major porin of the outer membrane. Growth curves and transport assays with an ompP2 deletion mutant revealed that NAD, NMN and NR diffuse through this porin into the periplasm.