Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Streptococcus pneumoniae (the pneumococcus) is a major human pathogen responsible for significant morbidity and mortality worldwide. Pneumococcal disease, which can include both invasive conditions such as pneumonia, bacteremia and meningitis, as well as less severe conditions such as otitis media, is almost invariably preceded by asymptomatic colonisation of the nasopharynx. To successfully adapt to the different ecological niches it encounters, the pneumococcus is likely to rely on the co-ordinated regulation of key virulence factors. As is the case for many other prokaryotes, this is likely to occur through two-component signal transduction systems (TCSTSs). TCSTSs comprise a histidine kinase (HK) and response regulator (RR). They respond to environmental stimuli, and regulate gene expression by interacting with the transcription machinery. Thirteen complete TCSTSs have been identified in S. pneumoniae, along with a lone RR. This study focused on one of these systems, designated RR/HK06. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1255323 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2006

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Streptococcus pneumoniae (the pneumococcus) is a major human pathogen responsible for significant morbidity and mortality worldwide. Pneumococcal disease, which can include both invasive conditions such as pneumonia, bacteremia and meningitis, as well as less severe conditions such as otitis media, is almost invariably preceded by asymptomatic colonisation of the nasopharynx. To successfully adapt to the different ecological niches it encounters, the pneumococcus is likely to rely on the co-ordinated regulation of key virulence factors. As is the case for many other prokaryotes, this is likely to occur through two-component signal transduction systems (TCSTSs). TCSTSs comprise a histidine kinase (HK) and response regulator (RR). They respond to environmental stimuli, and regulate gene expression by interacting with the transcription machinery. Thirteen complete TCSTSs have been identified in S. pneumoniae, along with a lone RR. This study focused on one of these systems, designated RR/HK06. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1255323 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2006

Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999? / Bibliography: leaves 250-227.

No description available.

Streptococcus pneumoniae (pneumococcus) is one of the leading causes of mortality in Africa. It asymptomatically colonizes the human nasopharynx. The invasive pneumococcal disease occurs when isolates spread to normally sterile sites such as lungs, blood, and the central nervous system. Colonization, though, does not necessarily lead to infection. Some isolates remain in the upper respiratory tract only, without causing any pathogenic symptoms. This thesis hypothesized that invasive and non-invasive isolates differ genetically. We tested this hypothesis by applying a pan-genome approach using whole-genome sequencing short reads of 1477 samples from Malawi, including those obtained from the nasopharynx of carriers (825 samples) and from the blood and cerebrospinal fluid of patients (652 samples). In-silico serotyping identified 56 serotypes in the cohort and statistical analysis showed that despite the vaccination, the prevalence of serotypes 1 and 12F increased amongst patients. Genomes were assembled, and a reference pan-genome for all strains was built. Short reads were aligned to the core genome, and core variants were called. The population structure was determined based on the distribution of variants in the pan-genome. Finally, genes with a significant presence in the invasive isolates were identified. Functional enrichment analysis of potential virulence genes was carried out to address how specific genes may contribute to the pathogenesis. The findings highlighted the features of the pneumococcus pan-genome in Malawi. The core- and accessory-genome were characterized based on the functional analysis of genes. The core components included: Ribosomal subunits. Subunits of F-type ATP synthase. Enzymes that catalyze the attachment of amino acids to tRNA molecules, DNA replication, DNA repair, and homologous recombination. 10.13% of the core and soft-core genes were uncharacterized. In the accessory genome, the study detected the presence of genes from Regions of Diversity (RDs), including Subunits of V-type ATPases and Sodium/solute symporter from RD8a. Enzymes from RD3 catalyzing the capsule synthesis. Subunits of PsrP secY2A2 pathogenicity island from RD10. Genes from RD6 and RD7 involved in transposing mobile genetic elements. Genes from RD2 RD8b, and RD12 participating in communication and competition. Genes from RD4 that assemble pilins into pili and anchor pili to the cell wall. 53.58% of accessory genes were uncharacterized. Most serotypes showed a similar prevalence in carriage and disease groups. However, the significant abundance of serotypes 1, 5, and 12F among patients compared to the carriage group suggested they are highly invasive with a short colonization period. These serotypes exhibited a remarkable genetic distinction from others. Their divergence included the absence and presence of several genes in their genome structure. The lack of genes from a genomic island known as RD8a was the most pronounced difference between serotypes 1, 5, and 12F compared to significantly prevalent serotypes in the nasopharynx. Genes in RD8a are involved in binding to epithelial cells and doing aerobics respiration to synthesize ATP through oxidative phosphorylation. The absence of RD8a from serotypes 1, 5, and 12F may be associated with their short duration in the nasopharynx where they need to bind to epithelial cells and access free oxygen molecules required for aerobic respiration. Given this, the amount of ATP is likely to decline in serotypes 1, 5, and 12F, causing them to harbour more phosphotransferase systems to transport carbohydrates since these transporters use phosphoenolpyruvate as the energy source instead of ATP. In conclusion, serotypes 1, 5, and 12F, the most prevalent and invasive pneumococcal strains in Malawi, showed a considerable genetic distinction from other strains that may be associated with their short colonization period and quickness to infect the blood and cerebrospinal fluid.

Streptococcus pneumoniae is one of the leading causes of morbidity and mortality in children under 5. Low-income countries are disproportionally affected and data in these settings are lacking. Effective strategies to control disease include infant immunisation with pneumococcal protein-polysaccharide conjugate vaccines. However, ongoing surveillance of carriage and disease are important to understand the impact of vaccination within communities. This thesis evaluated nasopharyngeal (NP) carriage in 3 generations, following introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the UK. NP carriage was also compared between rural and urban Nepalese children and a novel method of delayed culture and transport was assessed. Finally, the immunogenicity of a 10-valent pneumococcal conjugate vaccine administered in a 2-dose priming schedule without a booster was compared to a 3-dose priming schedule with a booster in Nepalese infants. Key findings include carriage rates in UK children being similar to pre-PCV7 (7 valent pneumococcal conjugate vaccine) carriage rates at 47% with low carriage rates in seen in adults. PCV7 serotypes accounted for 1.5% of carriage isolates in children, 0% in parents and 15.4% in older adults. In Nepalese children carriage was higher in a rural (69.2%) compared to an urban setting (40.9%) and delayed culture and transport using silica desiccant packets (SDP) provided a reliable, albeit underestimated, estimate of carriage. Finally this author demonstrated that following primary immunisation and boosting, there was no difference in immune responses to serotypes 1, 5 and 14 with a 2 dose priming schedule compared to a 3-dose schedule. At 2-4 years of age a significantly higher proportion of vaccinees in the 2+1 group had ≥0·2µg/mL IgG for serotypes 1, 5, 6B and 18C compared to vaccinees in the 3+0 group.

Streptococcus pneumoniae ist ein Gram-positives Bakterium und ein Kommensale des humanen Nasenrachenraums. Pneumokokken sind andererseits auch die Verursacher schwerer lokaler Infektionen wie der Otitis media, Sinusitis und von lebensbedrohenden invasiven Erkrankungen. So sind Pneumokokken die wichtigsten Erreger einer ambulant erworbenen Pneumonie und sie sind häufige Verursacher von Septikämien und bakteriellen Meningitiden. Die initiale Phase der Pathogenese ist verbunden mit der Besiedelung der mukosalen Epithelzellen des Rachenraumes. Diese Kolonisierung erleichtert die Aufnahme der Bakterien in die Zelle bzw. deren Dissemination in submukosale Bereiche und den Blutstrom. Die Konversion des Kommensalen zu einem invasiven Mikroorganismus ist assoziiert mit der Anpassung des Krankheitserregers an die verschiedenen Wirtsnischen und wird auf der Wirtsseite durch die Zerstörung der transepithelialen Barriere begleitet. Die Anpassung des Erregers ist vermutlich ein in hohem Grade regulierter Prozess. Die Oberfläche von Streptococcus pneumoniae ist mit Proteinen bedeckt, die kovalent oder nicht kovalent mit der Zellwand verknüpft sind. Eine einzigartige Gruppe von Oberflächenproteinen in der Zellwand der Pneumokokken sind die cholinbindenden Proteine (CBPs). Für einige der CBPs konnte bereits die Bedeutung für die Virulenz gezeigt werden. PspC, auch als SpsA oder CbpA bezeichnet, ist ein multifunktionales Oberflächenprotein, das als Adhesin und Faktor H-Bindungsprotein eine wichtige Rolle in der Pathogenese der Pneumokokken hat. PspC vermittelt als Adhesin die Anheftung der Bakterien an die mukosalen Epithelzellen, indem es human-spezifisch an die sekretorische Komponente (SC) des polymeren Immunoglobulinrezeptors (pIgR) bindet. SC ist die Ektodomäne des pIgR und PspC kann ebenso die freie SC binden oder an die SC des sekretorischen IgA Moleküls binden. PspC interagiert auch mit dem löslichen Komplement Faktor H. Die SC und der Faktor erkennen zwei verschiedene Epitope im bakteriellen PspC Protein. Der genaue Mechanismus der jeweiligen Interaktionen unter physiologischen- bzw. wirtspezifischen Bedingungen ist noch nicht vollständig verstanden. In dieser Arbeit wurde die Auswirkung der PspC Interaktion mit dem humanen pIgR (hpIgR) bzw. dem Faktor H auf die Virulenz der Pneumokokken und die Wirtszellantwort, d.h. die induzierten Signalkaskaden in den eukaryotischen Zellen untersucht. Die molekulare Analyse und die Verwendung von spezifischen pharmakologischen Inhibitoren der Signalmoleküle zeigten, dass verschiedene Signalmoleküle an der PspC-pIgR vermittelten Internalisierung beteiligt sind. Die Aktivierung, d.h. die Phosphorylierung der Signalmoleküle wurde in Immunblots demonstriert. Die Studien zeigten, dass das Aktinzytoskelett und die Mikrotubuli für die bakterielle Aufnahme essentiell sind. Es konnte auch zum ersten Mal nachgewiesen werden, dass Cdc42 die entscheidende GTPase für die Invasion der Pneumokokken in die Wirtsepithelzellen, vermittelt über den PspC-hpIgR Mechanismus, ist. Der Einsatz von PI3-kinase und Akt Kinase Inhibitoren reduzierte signifikant die hpIgR-vermittelte Aufnahme der Pneumokokken in die Wirtszelle. Zusätzlich durchgeführte Infektionen von hpIgR exprimierenden Zellen zeigten eine zeitabhängige Phosphorylierung von Akt und der p85α Untereinheit der PI3-Kinase. Damit ist neben der GTPase Cdc42 der PI3K und Akt Signalweg entscheidend für die PspC-pIgR vermittelte Invasion der Pneumokokken. Des Weiteren sind an der Infektion mit Pneumokokken auch die Protein Tyrosin Kinasen Src, ERK1/2 und JNK beteiligt. Dabei wird die Src Kinase unabhängig von der PI3K in hpIgR exprimierenden Zellen aktiviert. Inhibitionsexperimente und genetische Knockdown Versuche mit siRNA bewiesen, dass die Endozytose der Pneumokokken über PspC-pIgR ein Clathrin und Dynamin abhängiger Mechanismus ist. Im weiterenn Teil der Arbeit wurde der Einfluss des PspC gebundenen Faktor H auf die Anheftung an und Invasion in die Epithelzellen analysiert. Die Bindung von Faktor H erfolgte unabhängig vom PspC-Subtyp. Die Bindungsversuche bewiesen, dass die Kapselmenge negativ korreliert mit der Bindung des Faktor H. Der Einsatz von Faktor H aus Maus oder Ratte zeigte keine typische Bindung. Daraus kann abgeleitet werden, dass diese Interaktion humanspezifisch ist. Die Infektionsexperimente demonstrierten, dass Faktor H die Adhärenz und die Invasion der Bakterien in die Nasenrachenraumzellen (Detroit562), alveolären Lungenepithelzellen (A549) und humanen Hirnendothelzellen (HBMEC) steigert. Der Faktor H hat Heparin Bindestellen. Diese Bindestellen vermitteln die Adhärenz der Faktor H gebundenen Pneumokokken mit Epithelzellen. Inhibitionsstudien mit spezifischen monoklonalen Antikörpern, die gegen die short consensus repeats (SCRs) von Faktor H gerichtet waren, konnten die essentielle Bedeutung der SCR19-20 für die Anheftung der Pneumokokken über Faktor H an die Wirtszellen nachweisen. Die Faktor H vermittelte Assoziation der Pneumokokken an polymorphonukleäre Leukozyten (PMNs) erfolgt über das Integrin CD11b/CD18. Die weiteren Inhibitionsstudien zeigten dann auch zum ersten Mal den Einfluss des Aktinzytoskeletts der Wirtszelle auf die Faktor H-vermittelten bakterieller Internalisierung und den dabei bedeutsamen Signaltransduktionswegen in der eukaryotischen Zelle. Dabei wurden insbesondere die Proteintyrosinkinasen und die PI3K als wichtige Signalmoleküle für die Faktor H vermittelte Invasion der Pneumokokken identifiziert. Die in dieser Arbeit erhaltenen Resultate belegen, dass die Faktor H vermittelte Infektion der Zellen mit S. pneumoniae ein konzertierter Mechanismus ist, bei dem Oberflächen-Glycosaminoglycane, Integrine und Signaltransduktionswege der Wirtsepithelzellen involviert sind. Des Weiteren wurde aufgezeigt, dass die PspC-pIgR-vermittelte Invasion in mukosale Epithelzellen unterschiedliche Signalwege wie z.B. den PI3K und Akt Weg induziert und abhängig von Cdc42 und einer Clathrin vermittelten Endozytosemechanismus ist. / Streptococcus pneumoniae (pneumococci) are Gram-positive bacteria and commensals of the nasopharyngeal cavity. Besides colonization, pneumococci are responsible for severe local infections such as otitis media, sinusitis and life-threatening invasive diseases, including pneumonia, sepsis and meningitis. The surface of pneumococci is decorated with proteins that are covalently or non-covalently anchored to the cell wall. The most unique group of cell wall associated proteins in pneumococci are the choline-binding proteins (CBPs). PspC, also known as SpsA or CbpA, is a multifunctional choline-binding protein that plays an essential role in pneumococcal pathogenesis by functioning as an adhesin. PspC promotes adherence of pneumococci to mucosal epithelial cells by interacting in a human specific manner with the free secretory component (SC) or to SC as part of the secretory IgA (SIgA) or polymeric immunoglobulin receptor (pIgR). PspC also interacts specifically with the soluble complement Factor H. Apparently, PspC uses two different epitopes for binding the soluble host protein Factor H and SC of pIgR. However, the mechanism by which these independent interactions facilitate pneumococcal infections under physiological and host specific conditions have not yet been completely elucidated. This study aims to explore the impact of the PspC interaction with human pIgR (hpIgR) or complement regulator Factor H on pneumococcal virulence. Here the cellular and molecular basis of PspC-mediated adherence to and invasion of host epithelial and endothelial cells was demonstrated. The genetic approach, specific pharmacological inhibitors and immunoblot analysis demonstrated the complexity of the induced signal transduction pathways during PspC-hpIgR mediated pneumococcal uptake by host cells. Inhibition studies with specific inhibitors of actin cytoskeleton and microtubules demonstrated that the dynamics of host cell cytoskeleton are essential for pneumococcal uptake by mucosal epithelial cells. Moreover, this study reports for the first time that the small GTPase Cdc42 is essential for pneumococcal internalization into epithelial cells via the PspC-hpIgR mechanism. In addition, in infection experiments performed in presence of specific inhibitors of PI3-kinase/Akt and protein tyrosine kinase (PTKs), hpIgR-mediated pneumococcal uptake by host cells was significantly blocked. Amongst PTKs the Src kinase pathway, ERK1/2 and JNK pathways were implicated during pneumococcal ingestion by hpIgR expressing cells. In addition, inhibition experiments performed in the presence of individual inhibitors or with a combination of inhibitors suggested the independent activation of PI3-kinase/Akt and Src kinase pathways during pneumococcal infections of hpIgR expressing cells. By employing specific inhibitors and siRNA in cell culture infection experiments it was further demonstrated that pneumococcal endocytosis by host epithelial cells via the PspC-hpIgR mechanism depends on clathrin and dynamin. PspC recruits also Factor H to the pneumococcal cell surface. Consequently, the impact of pneumococcal cell surface bound Factor H on adherence to host cells and the molecular mechanism facilitating the uptake of Factor H bound pneumococci by epithelial cells was investigated. Flow cytometry and immunoblots revealed that S. pneumoniae has evolved the ability to recruit both purified Factor H as well as Factor H from human plasma or serum. Moreover, it was demonstrated that the recruitment of Factor H is independent of the PspC-subtypes and that capsular polysaccharide (CPS) interferes with its recruitment. Factor H bound to pneumococci significantly increased bacterial attachment to and invasion of host epithelial cells including nasopharyngeal cells (Detroit562), lung epithelial cells (A549), and human brain-derived endothelial cells (HBMEC). Blocking experiments demonstrated that bacteria bound Factor H interacts via the heparin binding sites on Factor H with eukaryotic cell surface glycosaminoglycans and that this interaction promotes pneumococcal adherence to host cells. In addition, inhibition studies with mAbs recognizing specifically different short consensus repeats (SCR) of Factor H suggested that SCR 19-20 of Factor H are essential for the pneumococcal interaction with host epithelial cells via Factor H. In the presence of Factor H, attachment of pneumococci to human polymorphonuclear leukocytes (PMNs) is enhanced. The integrin CD11b/CD18 was identified as the cellular receptor on PMNs. By using pharmacological inhibitors the impact of host cell cytoskeleton and signalling molecules, such as PTKs and PI3-kinase, for Factor H-mediated pneumococcal internalization into eukaryotic cells was shown. Taken together, the results revealed that Factor-H mediated pneumococcal infection requires a concerted role of host epithelial cell surface glycosaminoglycans, integrins and host cell signalling pathways.

Würzburg, Universiẗat, Diss., 2007.

Würzburg, Universiẗat, Diss., 2007.

Disputats, Københavns Universitet.