Ma, Hok Lun. / Thesis submitted in: December 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 233-273). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.v / Tables of contents --- p.vi / Acknowledgement --- p.xvi / List of figures --- p.xvii / List of tables --- p.xxi / List of abbreviations and symbols --- p.xxviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Respiratory illnesses in children --- p.1 / Chapter 1.1.1 --- Worldwide burden of childhood pneumonia --- p.1 / Chapter 1.1.2 --- Further mortality related to childhood pneumonia --- p.4 / Chapter 1.2 --- Etiology agent of childhood respiratory illnesses --- p.5 / Chapter 1.2.1 --- Difficulties in determining etiological agent --- p.5 / Chapter 1.2.2 --- Overall situation of etiological agents in childhood pneumonia --- p.6 / Chapter 1.2.3 --- Relationship between age and pathogens --- p.9 / Chapter 1.2.4 --- "Relationship between serotypes, carriage and invasiveness" --- p.11 / Chapter 1.2.4.1 --- Carriage and Invasiveness --- p.12 / Chapter 1.2.4.2.1 --- Carriage of S. pneumoniae and H. influenzae in children in Hong Kong --- p.12 / Chapter 1.2.4.2.2 --- "Serotypes, carriage and invasiveness in S. pneumoniae" --- p.14 / Chapter 1.2.4.2.3 --- "Serotypes, carriage and invasiveness in H. influenzae" --- p.17 / Chapter 1.3 --- Epidemiology of antibiotic-resistant pathogens --- p.18 / Chapter 1.3.1 --- Molecular typing methods --- p.18 / Chapter 1.3.2 --- Spread of antibiotic-resistant pathogens --- p.20 / Chapter 1.3.2.1 --- Spread of antibiotic-resistant S. pneumoniae --- p.26 / Chapter 1.3.2.1.1 --- Spread of penicillin-resistant S. pneumoniae --- p.26 / Chapter 1.3.2.1.1.1 --- Spread of Spanish-23F-1 --- p.27 / Chapter 1.3.2.1.1.2 --- Spread of Spanish-6B-2 --- p.28 / Chapter 1.3.2.1.1.3 --- Spread of antibiotic-resistant S. pneumoniae clones in Hong Kong --- p.28 / Chapter 1.3.2.1.2 --- Spread of cephalosporin-resistant S. pneumoniae --- p.29 / Chapter 1.3.2.1.3 --- Spread of macrolide-resistant S. pneumoniae --- p.30 / Chapter 1.3.2.1.4 --- Spread of fluoroquinolone-resistant S. pneumoniae --- p.31 / Chapter 1.3.2.2 --- Spread of antibiotic-resistant H. influenzae --- p.32 / Chapter 1.3.2.2.1 --- Spread of β-lactam-resistant H. influenzae --- p.32 / Chapter 1.3.2.2.2 --- Spread of macrolide-resistant H. influenzae --- p.33 / Chapter 1.3.2.2.3 --- Spread of fluoroquinolone-resistant H. influenzae --- p.34 / Chapter 1.4 --- Mechanism of antibiotic-resistance in respiratory pathogens --- p.36 / Chapter 1.4.1 --- Mechanism of antibiotic-resistance in S. pneumoniae --- p.37 / Chapter 1.4.1.1 --- Mechanism of penicillin- and cephalosporin-resistance in S. pneumoniae --- p.37 / Chapter 1.4.1.1.1 --- Penicillin-binding protein (PBP)-mediated mechanism --- p.37 / Chapter 1.4.1.1.2 --- PBP-independent mechanisms --- p.49 / Chapter 1.4.1.1.2.1 --- "Murine peptide branching genes, murMN operon" --- p.49 / Chapter 1.4.1.1.2.2 --- "Two-component system, CiaRH" --- p.50 / Chapter 1.4.1.1.2.3 --- "Putative glycosyltransferase, CpoA" --- p.52 / Chapter 1.4.1.1.3 --- RNA and protein expression studies on S. pneumoniae for β-lactam-resistance --- p.52 / Chapter 1.4.1.1.3.1 --- RNA expression in penicillin-sensitive S. pneumoniae --- p.53 / Chapter 1.4.1.1.3.2 --- Protein expression in penicillin-resistant S. pneumoniae --- p.53 / Chapter 1.4.1.2 --- Mechanism of macrolide- and lincosamide- resistance in S. pneumoniae --- p.54 / Chapter 1.4.1.3 --- Mechanism of tetracycline-resistance in S. pneumoniae --- p.55 / Chapter 1.4.1.4 --- Mechanism of fluoroquinolone-resistance in S. pneumoniae --- p.55 / Chapter 1.4.2 --- Mechanism of antibiotic-resistant in H. influenzae --- p.56 / Chapter 1.4.2.1 --- Mechanism of β-lactam-resistance in H. influenzae --- p.56 / Chapter 1.4.2.1.1 --- β-lactamase-producing H. influenzae --- p.56 / Chapter 1.4.2.1.2 --- β-lactamase-negative ampicillin-resistant (BLNAR) H. influenzae --- p.58 / Chapter 1.4.2.1.2.1 --- Relationship between amino acid substitutions in PBP3 and β-lactam- resistance --- p.58 / Chapter 1.4.2.1.2.2 --- Relationship between amino acid substitutions in AcrR and β-lactam-resistance --- p.60 / Chapter 1.4.2.2 --- Mechanism of macrolide-resistance in H. influenzae --- p.61 / Chapter 1.4.2.3 --- Mechanism of fluoroquinolone-resistance in H. influenzae --- p.64 / Chapter 1.5 --- Impact of vaccination --- p.65 / Chapter 1.5.1 --- H. influenzae type b vaccination --- p.65 / Chapter 1.5.1.1 --- Efficacy of Hib conjugate vaccine --- p.66 / Chapter 1.5.1.2 --- Herd immunity related to Hib conjugate vaccine --- p.66 / Chapter 1.5.2 --- Pneumococcal vaccination --- p.66 / Chapter 1.5.2.1 --- Vaccine efficacy and herd immunity of pneumococcal vaccines --- p.67 / Chapter 1.5.2.2 --- Development of conjugate vaccines with higher valency --- p.67 / Chapter 1.5.2.3 --- Serotype replacement --- p.67 / Chapter 1.5.2.4 --- Development of pneumococcal vaccines with new targets --- p.69 / Chapter 1.6 --- Objectives of this study --- p.70 / Chapter Chapter 2 --- Materials and methods --- p.72 / Chapter 2.1 --- Collection and Identification of microorganisms --- p.72 / Chapter 2.1.1 --- Collection of S. pneumoniae and H. influenzae --- p.72 / Chapter 2.1.2 --- Identification of S. pneumoniae and H. influenzae --- p.73 / Chapter 2.2 --- Serotyping of S. pneumoniae and H. influenzae --- p.74 / Chapter 2.2.1 --- Serotyping by polymerase chain reaction (PCR) --- p.74 / Chapter 2.2.1.1 --- Preparation of crude DNA extract --- p.74 / Chapter 2.2.1.2 --- Screening for common serotypes by multiplex PCR --- p.74 / Chapter 2.2.1.3 --- Composition of PCR Mix --- p.77 / Chapter 2.2.1.4 --- Serotyping PCR conditions --- p.81 / Chapter 2.2.1.5 --- Gel Electrophoresis --- p.81 / Chapter 2.2.2 --- Serotyping by serum agglutination --- p.82 / Chapter 2.3 --- Antimicrobial susceptibility testing --- p.83 / Chapter 2.4 --- Clonal analysis of penicillin- and cephalosporin-resistant S. pneumoniae --- p.87 / Chapter 2.4.1 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.87 / Chapter 2.4.1.1 --- Preparation of agarose plugs for PFGE --- p.87 / Chapter 2.4.1.2 --- Lysis of bacteria in agarose plugs --- p.89 / Chapter 2.4.1.3 --- Digestion of chromosomal DNA by restriction enzyme --- p.89 / Chapter 2.4.2 --- Multi-locus sequence typing (MLST) --- p.90 / Chapter 2.4.2.1 --- PCR amplification of house-keeping genes in MLST --- p.90 / Chapter 2.4.2.1.1 --- Preparation of DNA from agarose plugs --- p.92 / Chapter 2.4.2.1.2 --- Composition of PCR Mix --- p.92 / Chapter 2.4.2.1.3 --- MLST PCR conditions --- p.92 / Chapter 2.4.2.1.4 --- Gel Electrophoresis of MLST PCR products --- p.92 / Chapter 2.4.2.1.5 --- MLST PCR products purification --- p.93 / Chapter 2.4.2.2 --- Sequencing of housekeeping genes in MLST --- p.93 / Chapter 2.4.2.3 --- Sequencing analysis and sequence type (ST) determination in MLST --- p.94 / Chapter 2.4.3 --- Extended panel of antibiotic susceptibility testing on S. pneumoniae with known STs --- p.94 / Chapter 2.5 --- Analysis on potential penicillin- and cephalosporin-resistance mechanisms in S. pneumoniae --- p.96 / Chapter 2.5.1 --- Sequencing of potnetial penicillin- and cephalosporin- resistance determinants in S. pneumoniae --- p.96 / Chapter 2.5.1.1 --- Primer design of penicillin-binding protein (PBP) genes --- p.96 / Chapter 2.5.1.2 --- Primer design of non-PBP resistance determinants --- p.100 / Chapter 2.5.1.3 --- PCR amplification and sequencing of resistant determinants --- p.100 / Chapter 2.5.1.4 --- Sequence analysis --- p.100 / Chapter 2.5.2 --- Study on efflux mechanism of S. pneumoniae --- p.103 / Chapter 2.5.2.1 --- Modification of macrodilution for efflux assay --- p.103 / Chapter 2.5.2.2 --- Cefotaxime MIC determination with efflux inhibitors --- p.104 / Chapter 2.5.2.3 --- Determination of appropriate CCCP concentration --- p.105 / Chapter 2.5.2.4 --- Growth curve with efflux inhibitor --- p.105 / Chapter 2.5.3 --- Heteroresistance assay of S. pneumoniae --- p.106 / Chapter 2.5.4 --- "RNA expression study on penicillin- and cefotaxime-resistance determinants (pbp2x, pbpla and pbp2a) of S. pneumoniae" --- p.107 / Chapter 2.5.4.1 --- Growth of S. pneumoniae for RNA extraction --- p.107 / Chapter 2.5.4.2 --- RNA extraction and DNase digestion --- p.107 / Chapter 2.5.4.3 --- cDNA synthesis and real-time PCR --- p.108 / Chapter 2.6 --- Analysis on cephalosporin- and macrolide-resistance mechanisms in H. influenzae --- p.111 / Chapter 2.6.1 --- β-lactamase production of H. influenzae --- p.111 / Chapter 2.6.1.1 --- Nitrocefin Hydrolysis --- p.111 / Chapter 2.6.1.2 --- Screening for the presence of p-lactamase gene (blaTEM-1 and blaROB-1) by multiplex PCR --- p.111 / Chapter 2.6.2 --- PCR detection and sequencing of β-lactam- and macrolide- resistance determinants in H. influenzae --- p.113 / Chapter Chapter 3 --- Results of S. pneumoniae and H. influenzae children study --- p.116 / Chapter 3.1 --- Patient demographics of children study --- p.116 / Chapter 3.2 --- Serotype distributions --- p.117 / Chapter 3.2.1 --- Serotypes / serogroup distribution in S. pneumoniae --- p.117 / Chapter 3.2.2 --- Serotype distribution in H. influenzae children study --- p.120 / Chapter 3.3 --- Antibiotic susceptibilities and resistance antibiograms --- p.122 / Chapter 3.3.1 --- Antibiotic susceptibilities of S. pneumoniae --- p.122 / Chapter 3.3.2 --- Relationship between antibiotic resistance profiles and serotypes in S.pneumoniae --- p.126 / Chapter 3.3.3 --- Antibiotic susceptibilities of H. influenzae --- p.135 / Chapter 3.3.4 --- Antibiotic resistance profiles of H. influenzae --- p.138 / Chapter 3.4 --- Clonal analysis of penicillin- and cephalosporin-resistant S.pneumoniae --- p.139 / Chapter 3.4.1 --- Pulsed-field gel electrophoresis (PFGE) of S. pneumoniae --- p.139 / Chapter 3.4.2 --- Multi-locus sequence typing of S. pneumoniae --- p.141 / Chapter 3.5 --- Analysis of the penicillin- and cephalosporin-resistance determinants in S. pneumoniae --- p.143 / Chapter 3.5.1 --- "Sequence analysis of major pbp genes (pbp2x, pbpla and pbp2a)" --- p.143 / Chapter 3.5.2 --- "Sequence analysis of other potential penicillin- and cephalosporin- resistance determinants (pbp 1 b, pbp2b, pbp3, cpoA, ciaRH and murMN)" --- p.152 / Chapter 3.5.3 --- Sequence analysis of putative promoter sequences of pbp genes --- p.167 / Chapter 3.5.4 --- Efflux Inhibition Assay --- p.171 / Chapter 3.5.5 --- Heteroresistance Assay --- p.177 / Chapter 3.5.6 --- "RNA expression study on penicillin- and cephalosporin resistance determinants (pbp2x, pbpla and pbp2a)" --- p.179 / Chapter 3.6 --- Analysis of β-lactam-resistance determinants in H. influenzae --- p.185 / Chapter 3.6.1 --- β-lactamase production and blaTEM-1 promoter study --- p.185 / Chapter 3.6.2 --- "Sequence analysis of β-lactam-resistance determinants (ftsl, acrR genes, AcrAB-TolC efflux pump)" --- p.188 / Chapter 3.6.2.1 --- Sequence analysis offtsl --- p.188 / Chapter 3.6.2.2 --- Analysis of acrR and AcrAB-TolC efflux pump --- p.189 / Chapter 3.7 --- "Analysis of macrolide-resistance determinants in H, influenzae (AcrAB-TolC efflux pump, 23SrRNA, Ribosomal proteins L4 and L22)" --- p.199 / Chapter Chapter 4 --- Discussion on S. pneumoniae and H. influenzae children study --- p.204 / Chapter 4.1 --- Carriage rate of S. pneumoniae children collection --- p.204 / Chapter 4.2 --- Serotype distribution --- p.205 / Chapter 4.2.1 --- Serotype distribution and potential vaccine coverage in S. pneumoniae --- p.205 / Chapter 4.2.2 --- Serotype distribution in H. influenzae --- p.209 / Chapter 4.3 --- Antimicrobial resistance --- p.210 / Chapter 4.3.1 --- Antimicrobial resistance in S. pneumoniae --- p.210 / Chapter 4.3.2 --- Antimicrobial resistance in H. influenzae --- p.214 / Chapter 4.4 --- "Clonal analysis of high-level β-lactam-resistant S, pneumoniae" --- p.217 / Chapter 4.5 --- "β-lactam-resistance mechanisms in S, pneunomiae" --- p.220 / Chapter 4.6 --- Antimicrobial resistance mechanisms in H. influenzae --- p.224 / Chapter 4.6.1 --- β-lactam-resistance mechanism in β-lactamase-producing H. influenzae --- p.224 / Chapter 4.6.1.1 --- Variations in blaTEM-1 promoters in β-lactamase-producing H.influenzae --- p.224 / Chapter 4.6.1.2 --- β-lactam-resistance in β-lactamase-nonproducing H. influenzae --- p.225 / Chapter 4.6.2 --- Macrolide-resistance mechanisms in H. influenzae --- p.228 / Chapter Chapter 5 --- Conclusion and future studies --- p.230 / Chapter 5.1 --- "S, pneumoniae children study" --- p.230 / Chapter 5.2 --- H. influenzae children study --- p.231 / Chapter 5.3 --- Future studies --- p.232 / Bibliography --- p.233 / Appendix I 一 Sequence alignments and Tables --- p.274 / Appendix II 一 Materials and Methods --- p.313

No description available.

Streptococcus pneumoniae ist der häufigste Erreger bakterieller Meningitiden. Eine Pneumokokken-Meningitis führt trotz Ausschöpfung aller heute verfügbaren Behandlungsmöglichkeiten in 25 % der Fälle zum Tod. Steigende Antibiotikaresistenzen und die Limitation verfügbarer Vakzine auf einige Serotypen von S. pneumoniae erfordern neue Ansätze in der antimikrobiellen Therapie. Cholin-bindende Proteine (CBPs) sind gemeinsames Merkmal aller Pneumokokkenstämme und für die Virulenz dieses Bakteriums essenziell. Durch Zugabe potenter Anticholinergika können die CBPs von der Bakterienoberfläche abgelöst und damit inhibiert werden. In dieser Arbeit wurde untersucht, ob durch Inhibition von CBPs während des Wachstums der Pneumokokken deren Phagozytose durch Mikrogliazellen in vitro gesteigert werden kann. Während ihres Wachstums wurden die Bakterien dazu mit potenten Anticholinergika inkubiert und am Ende ihrer exponentiellen Wachstumsphase auf murine Mikrogliazellen gegeben. Nicht alle eingesetzten Anticholinergika konnten die Inhibition der CBPs – angezeigt durch die Bildung langer Kokkenketten – bewirken, obwohl für sie alle eine hohe Affinität zu den CBPs in früheren Arbeiten nachgewiesen worden war. Ipratropium, das in höheren Konzentrationen das Pneumokokkenwachstum inhibiert, induzierte in den von uns eingesetzten niedrigen Konzentrationen weder die Bildung von Ketten noch führte es zu einer erhöhten Phagozytoseleistung. Mit DMAE funktionalisiert zeigten PAMAM-Dendrimere der 1. Generation ebenfalls keine Inhibition der CBPs: Es bildeten sich weder Kokkenketten noch zeigte sich eine erhöhte Bakterienaufnahme der Mikroglia. Im Gegensatz dazu stellte sich unter Einfluss von PPI-g2-DMAE neben ausbleibender Kettenbildung ein dosisabhängiger phagozytosehemmender Effekt dar. Einzig durch Co-Inkubation mit dem mit Cholin funktionalisierten PPI-Dendrimer der 2. Generation gelang die Inhibition der CBPs mit resultierender Bildung langer Ketten. Die Phagozytoseleistung zeigte eine dosisabhängige Steigerung sowohl für eine CoInkubation während der gesamten exponentiellen Wachstumsphase als auch nach Co-Inkubation während ihrer letzen 2 Stunden. Dennoch konnte im Sepsismodell der Maus durch intraperitoneale Injektion dieses Dendrimers 15 min vor Infektion mit S. pneumoniae kein protektiver Effekt erzielt werden: Zwischen den mit Dendrimeren behandelten Tieren und denen der Kontrollgruppe zeigten sich keine Unterschiede in Überlebenszeit und Sterblichkeit, dem krankheitsbedingten Gewichtsverlust, dem klinischen Score und der durch Ausplattieren von Milzhomogenaten ermittelten Keimkonzentration im Blut infektionsbedingt verstorbener Tiere. Die von uns eingesetzten Konzentrationen von Ipratropium scheinen für eine Inhibition der CBPs nicht ausgereicht zu haben. Der bislang nicht genau geklärte wachstumsinhibitorische Effekt, der sich in unseren Versuchen bereits ab 5 mM bemerkbar machte, könnte jedoch durch Inhalation von Ipratropium gezielt zur Prophylaxe von Pneumokokken-Pneumonien genutzt werden. Bei an Dendrimere gekoppelten, eigentlich potenten Liganden der CBPs konnte beobachtet werden, dass sie als Teil des Dendrimers ihre Affinität gegenüber den CBPs nicht nur deutlich verändern, sondern auch unerwartete Effekte (Verminderung der Phagozytose) hervorrufen können. Wegen der raschen Elimination scheint die einmalige Gabe eines potenten Dendrimers zur Inhibition der CBPs in vivo nicht auszureichen und erklärt das Versagen im Sepsismodell. Neuere Untersuchungen zur Distribution im Hirnparenchym nach intraventrikulärer oder subarachnoidaler Injektion lassen hoffen, dass durch Gabe subtoxischer Dosen die von uns beobachtete Phagozytosesteigerung in vivo reproduzierbar ist. Durch Inhibition der CBPs ist es möglich, die Virulenz aller Serotypen des Pneumokokkus stark zu reduzieren. Potente Inhibitoren könnten sowohl als Therapeutikum als auch zur Infektionsprophylaxe eingesetzt werden, ohne dass es dabei zur Ausbildung von Resistenzen kommt, da zeitgleich mehrere Virulenzfaktoren inhibiert werden. Es ist daher von großem medizinischen Interesse, Inhibitoren der CBPs zu entwickeln, die in subtoxischen Dosen eine hohe Affinität zu den CBPs aufweisen und diese inhibieren.

No description available.

La broncho-pneumopathie chronique obstructive (BPCO) est une maladie inflammatoire chronique des voies aériennes dont le facteur de risque principal est l’exposition chronique à la fumée de cigarette. L’histoire de la maladie est fréquemment associée à une colonisation bactérienne des voies aériennes et ponctuée d’épisodes aigus d’exacerbation de la maladie associés à une morbi-mortalité importante. Ces exacerbations sont principalement d’origine infectieuses et plus particulièrement, associées à une bactérie dans 50 % des cas, majoritairement Haemophilus influenzae, Streptococcus pneumoniae et Moraxella catarrhalis. Le contrôle de ces infections bactériennes implique en particulier une réponse immunitaire de type Th17 efficace. Cette immunité Th17 médiée principalement par les cytokines IL-17A et IL-22 est impliquée dans la physiopathologie de la BPCO mais n’a été que peu étudiée dans le contexte des exacerbations. Notre hypothèse est que la réponse Th17 aux pathogènes est altérée dans la BPCO, mécanisme qui serait impliqué dans la susceptibilité aux infections respiratoires observée chez les patients.Différentes approches ont été utilisées pour tester cette hypothèse. Tout d’abord, une approche ex vivo, à partir de cellules mononucléées circulantes (PBMC) de patients atteints de BPCO comparées à celles issues de sujets sains non fumeurs et de sujets fumeurs sans obstruction bronchique, montrant un défaut de production par les PBMC des cytokines IL-17A et IL-22 mais également des cytokines IL-6 et IL-23 produites par les cellules présentatrices d’antigènes (CPA) et impliquées dans l’activation de cette immunité Th17, en réponse à une activation par S. pneumoniae. Ensuite, une approche in vitro, avec un modèle de cellules dendritiques dérivées de monocytes (MDDC) exposées à la fumée de cigarette. Ces MDDC présentaient un défaut de maturation, de production de cytokines pro-Th17 et de leur capacité à activer une réponse Th17 lymphocytaire en réponse à S. pneumoniae. Enfin, une approche in vivo, utilisant un modèle murin de souris exposées de façon chronique à la fumée de cigarette confirmant ces résultats avec un défaut de réponse IL-17A et IL-22 mais également de production des cytokines pro-Th17 IL-1β et IL-23 par les CPA en réponse à S. pneumoniae. Dans ce modèle, l’apport d’IL-22 permettait d’améliorer la clairance bactérienne et de réduire les lésions pulmonaires, suggérant des possibilités thérapeutiques pour améliorer la prise en charge de ces exacerbations infectieuses.Ces trois approches permettent d’apporter des arguments forts en faveur d’un défaut de réponse Th17 au cours des exacerbations bactériennes de la BPCO, hypothèse confortée par d’autres travaux de notre équipe montrant dans le modèle murin d’exposition chronique à la fumée de cigarette la présence d’un défaut de production d’IL-22 dans la réponse à Haemophilus influenzae. Ces travaux qui doivent maintenant être d’une part, confirmés par une étude clinique incluant des patients atteints de BPCO en exacerbation, et d’autre part, complétés pour préciser l’impact sur les cellules lymphoïdes innées productrices de ces cytokines et sur la réponse de l’épithélium bronchique à l’infection, ouvrent la voies à des perspectives thérapeutiques dans la prise en charge de ces exacerbations bactériennes. / Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the airways mainly due to chronic exposure to cigarette smoke. Evolution of the disease is often associated with bacterial colonization of the airways and punctuated by acute exacerbation of the disease with a frequent related morbi-mortality. These exacerbations are mainly due to infection and almost 50 % are associated with bacteria, often Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis. Th17 immune response is particularly involved in control of bacterial infection and is principally mediated by IL-17A and IL-22 cytokines. This Th17 inflammation is involved in COPD physiopathology but there is paucity of data focusing on this immune response during COPD exacerbations. Our hypothesis is that Th17 immune response to pathogens is defective in COPD leading to airways infection susceptibility.We have tested our hypothesis by different approaches. First, the ex vivo responses to Streptococcus pneumoniae of peripheral blood mononuclear cells (PBMC) from COPD patients, healthy non smokers and healthy smokers were compared showing decreased production of IL-17A and IL-22 but also of pro-Th17 cytokines IL-6 and IL-23 which are produced by antigen presenting cells (APC). Second, we used an in vitro model of monocyte-derived dendritic cells (MDDC) exposed to cigarette smoke showing a defective MDDC maturation, pro-Th17 cytokines production and ability to promote T-cells Th17 response, in response to S. pneumoniae. Finally, an in vivo murine model of mice chronically exposed to cigarette smoke showing defective production of IL-17A and IL-22 but also of pro-Th17 cytokines IL-1β and IL-23 produced by APC, in response to S. pneumoniae. In this model, supplementation with IL-22 restored bacterial clearance and limited lung alterations suggesting therapeutic opportunities to improve infectious COPD exacerbation management.Altogether, these results strengthen our hypothesis of a defective Th17 immune response during bacterial COPD exacerbations. They are comforted by other studies in our team showing a defective IL-22 production in response to Haemophilus influenzae in our in vivo model of mice chronically exposed to cigarette smoke. Now we have to confirm these results in a clinical trial including COPD patients in exacerbation and to further explore the impact on innate lymphoid cells, which produced these cytokines, and on the innate immune response of epithelial cells to infection, in order to develop new infectious COPD exacerbation therapeutics.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-20T21:34:48Z No. of bitstreams: 1 Larissa Carneiro Rocha.pdf: 1632038 bytes, checksum: 82deed6171da78f07c96b948629b2896 (MD5) / Made available in DSpace on 2012-07-20T21:34:48Z (GMT). No. of bitstreams: 1 Larissa Carneiro Rocha.pdf: 1632038 bytes, checksum: 82deed6171da78f07c96b948629b2896 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A doença falciforme (DF) possui prevalência mundial elevada e manifestação clinicamente variável, sendo que as infecções constituem risco elevado e causa de óbito nas crianças diagnosticadas com anemia falciforme (HbSS). A colonização da nasofaringe pode ser fator importante para a ocorrência de doença local ou sistêmica. O Streptococcus pneumoniae é um patógeno de importância epidemiológica mundial e causador de infecções entre os pacientes com DF. A prevalência da colonização pelo pneumococo em nasofaringe varia de acordo com a população estudada e condições ambientais. O Staphylococcus aureus também pode colonizar a nasofaringe, além de causar infecção de pele e tecidos moles, pneumonia, septicemia e infecções ósteo-articulares. Diferentes biomarcadores têm sido associados à modulação clínica na DF e eles são comumente associados à hemólise e inflamação. O presente estudo teve como objetivo estabelecer o perfil de biomarcadores em indivíduos com DF associando-os ao perfil de colonização nasofaríngea e orofaríngea, com ênfase para os marcadores de infecção e hemólise que possam estar associados ao prognóstico clínico dda doença. As análises bioquímicas foram realizadas para a avaliação do perfil lipídico, hepático, inflamatório e hemolítico; marcadores clássicos de biologia molecular, como a talassemia α, os haplótipos ligados aos genes da globina beta e os polimorfismos no gene da mieloperoxidase foram também investigados. Desta forma, foi desenvolvido um estudo de corte transversal, com casuística composta por 154 pacientes com DF em idade pediátrica e em estado estável da doença, sendo 68,2% (105/154) HbSS e 31,8% (49/154) com doença SC (HbSC), todos provenientes do estado da Bahia. As crianças HbSS apresentaram diferenças significativas na grande maioria das variáveis laboratoriais analisadas e associadas ao metabolismo lipídico, renal, hepático e à hemólise e inflamação, quando comparadas ao grupo HbSC e ao controle saudável. A colonização em nasofaringe/orofaringe pelo S. pneumoniae esteve presente em 14 (9,6%) pacientes e pelo S. aureus em 81(56,6%) pacientes. Quanto ao perfil de sensibilidade dos isolados de pneumococos da população estudada, não foi observado o aumento da resistência pneumocócica à penicilina. A avaliação de modelos de análise multivariada demonstrou que a presença de colonização nasofaríngea e orofaríngea esteve associada à ocorrência de infecção juntamente com a contagem de leucócitos, sendo que o genótipo exibido pelo paciente foi fator de risco para a ocorrência de pneumonia. Os mesmos modelos apontaram o envolvimento dos polimorfonucleares neutrófilos na ocorrência de vaso-oclusão. Os resultados demonstram que os pacientes colonizados em nasofaringe pelo S. pneumoniae e pelo S. aureus apresentaram elevação dos valores de HCM, VCM, AST, ALT e Ferritina; investigações rotineiras de biomarcadores clássicos associados ao estudo da colonização de nasofaringe e orofaringe podem ter papel importante no acompanhamento da evolução clinica de indivíduos com DF, uma vez que os achados significativos sugerem que a presença de colonização tem papel importante na modulação dos eventos hemolítico, inflamatório e infeccioso presentes na doença. / The sickle cell disease (SCD) has a high prevalence worldwide and a variable clinical manifestation and infections are considered an event of high risk and cause of death in children diagnosed with sickle cell anemia (HbSS). The colonization of nasopharynx and oropharynx can be an important factor for the occurrence of local or systemic disease. The Streptococcus pneumoniae is a pathogen of epidemiological importance worldwide and cause of infection among SCD patients. The Staphylococcus aureus may also colonize the nasopharynx and may be cause of infection of skin and soft tissue infections, pneumonia, sepsis and osteo-articular infections. Different biomarkers have been associated with clinical modulation in SCD and they are commonly associated with hemolysis and inflammation. This study aimed to establish a profile of biomarkers in individuals with SCD in association with the profile of oropharyngeal and nasopharyngeal colonization, with emphasis on infection and hemolysis markers that may be associated with clinical prognosis. Biochemical analysis were performed to evaluate the lipid profile, liver, hemolytic and inflammatory markers and classical molecular biology, such as α-thalassemia, the haplotypes linked to the beta globin genes and polymorphisms in the myeloperoxidase gene were also investigated. Thus, it was developed a cross-sectional study and the casuistic was composed of 154 children with SCD in a steady-state, with 68.2% (105/154) HbSS and 31.8% (49/154) with SC disease (HbSC), all from the state of Bahia. HbSS children showed significant differences in almost every mean values analyzed for variables associated with lipid metabolism, kidney, liver and hemolysis and inflammation when compared to HbSC and control group. In the present study there was colonization in the nasopharynx / oropharynx by S. pneumoniae in 14 (9.6%) patients and by S. aureus in 81 (56.6%) patients. The profile of sensitivity of pneumococcal isolated from the studied population did not show an increase in pneumococcal resistance to penicillin. The evaluation of models of multivariate analysis showed that the presence of oropharyngeal and nasopharyngeal colonization was associated with the occurrence of infection and white blood cell count, and the patient genotype was a risk factor for the occurrence of pneumonia. The same models indicated the involvement of polymorphonuclear neutrophils in the occurrence of vaso-occlusion. The results presented in this study demonstrate that patients colonized in the nasopharynx by S. pneumoniae and S. aureus have elevated values of MCH, MCV, AST, ALT and ferritin, suggesting that a routine investigation of biomarkers associated with the classic study of the colonization of the nasopharynx and oropharynx may play a role in monitoring the clinical course of patients with SCD, once that significant findings described here suggest that the presence of colonization plays an important role in modulation of hemolytic events, inflammatory state, and infectious presented by patients.

Streptococcus pneumoniae, le pneumocoque, est une bactérie pathogène qui entraîne le décès de plus d'un million et demi de personnes dans le monde chaque année. Les β-lactamines sont très utilisées pour traiter les infections à pneumocoques. Ces antibiotiques inhibent la synthèse du peptidoglycane, une molécule géante constituant un réseau de chaînes glycopeptidiques qui englobe la cellule, lui confère sa forme et lui permet de maintenir son intégrité face à la pression osmotique. Le mécanisme d'action des β-lactamines est bien connud'un point de vue biochimique. En revanche, la réponse physiologique empêchant la multiplication des bactéries traitées est mal connue. Au cours de ma thèse, j'ai étudié les mécanismes moléculaires de la morphogenèse du pneumocoque par des approches de biochimie et de microbiologie. Un modèle de morphogenèse est proposé intégrant mes résultats à la littérature et permettant de formuler des hypothèses sur la réponse physiologique de S. pneumoniae aux β-lactamines. / Streptococcus pneumoniae, the pneumococcus, is a bacterial pathogen that causes more than 1.5 million deaths each year in the world. β-Lactams are widely used to treat patients with pneumococcal infections. These antibiotics inhibit the synthesis of the peptidoglycan, a giant molecule constituting a mesh of aminosugar strands encasing the cell. This main constituent of the cell wall allows cells to maintain their integrity under the turgor pressure, and endows bacteria with their shape. The action of β-lactams is well understood from a biochemical point of view. However, a complete understanding of the physiological response of treated bacteria remains elusive. In this thesis, I investigated the molecular mechanisms of the morphogenesis of S. pneumoniae using methods of biochemistry and microbiology. A morphogenesis model is built based on my results and the literature, which permits to emit hypotheses concerning the response of the pneumococcus to β-lactams.

Afin de faire face à différents types de stress et s'adapter à de nouveaux environnements, les bactéries ont développé de nombreux mécanismes génétiquement régulés. La compétence pour la transformation naturelle est un processus qui favorise le transfert horizontal de gènes. Si les espèces phylogénétiquement éloignées partagent des mécanismes conservés d'intégration et de remaniement de l'ADN, les circuits de régulation de la compétence ne sont toutefois pas universels mais adaptés au mode de vie de chaque espèce. Chez les bactéries Gram-positives, les cascades de régulation de Streptococcus pneumoniae et Bacillus subtilis sont les mieux documentées. Si de nombreux modèles mathématiques ont été établis pour étudier différents aspects de la régulation des compétences chez B. subtilis, un seul modèle à échelle de population a été développé pour S. pneumoniae, il y a plus de dix ans, sur la base d'hypothèses contestées par de nouvelles données expérimentales. Nous avons développé, chez S. pneumoniae, un modèle fondé sur la connaissance de la régulation de la compétence qui intègre les éléments biologiques essentiels connus à ce jour. La cohérence structurelle de la topologie du réseau est confirmée par le formalisme des réseaux de Petri. Le réseau est ensuite transformé en un ensemble d'équations différentielles ordinaires pour étudier son comportement dynamique. La cinétique des protéines a été estimée en utilisant des données de luminescence et l'estimation des paramètres a été contrainte à partir des connaissances disponibles. Après avoir testé des modèles alternatifs, nous avons proposé l'existence d'un produit de gène tardif supplémentaire pouvant inhiber l'action de ComW, l'activateur du facteur sx. Nous apportons également un nouvel éclairage sur cette cascade de régulation en prédisant la cinétique de composantes du système qui pourraient être impliquées dans des comportements spécifiques. Ce modèle consolide les connaissances expérimentales acquises sur la régulation de la compétence chez S. pneumoniae. De plus, il peut être appliqué aux autres espèces de streptocoques appartenant aux groupes mitis et anginosus puisqu'ils partagent le même circuit régulateur. À l'échelle populationnelle, la transition vers l'état de compétence se produit d'abord dans une sous-population de cellules et se propage ensuite dans toute la population par contact physique cellule à cellule. En permettant la simulation du comportement d'une cellule individuelle, le modèle pourra servir de module dans la conception d'un modèle d'une population bactérienne composée de cellules hétérogènes. / Bacteria have evolved many types of genetically induced mechanisms to face different types of stresses and to adapt to new environments. Competence for natural transformation is one such process that promotes horizontal gene transfer. If phylogenetically distant species share conserved uptake and processing apparatus, competence regulatory circuits are not universal but adapted to every species' lifestyle. In Gram-positive bacteria, Streptococcus pneumoniae and Bacillus subtilis regulatory cascades are the best documented. If many mathematical models have been established to study different aspects of competence regulation in B. subtilis, only one population-scaled model has been developed for S. pneumoniae, a decade ago, based on hypotheses that are challenged by new experimental data. We develop, in S. pneumoniae, a knowledge-based model of the competence regulation at cell level that integrates the enriched biological knowledge acquired to date. The structural consistency of the network topology is confirmed using Petri net formalism. The network is further turned into a set of ordinary differential equations to study its dynamics behavior. Protein kinetics are estimated using time-series luminescence data and other parameter estimations are constrained according to available knowledge. We point out some gap in competence shut-off knowledge, and, after testing alternative models, we predict the requirement of a yet unknown late com gene product inhibiting the action of ComW, the ?x factor activator. We also bring new insights into this regulatory cascade by predicting the system components that might be involved in specific experimental behavior. Our model consolidates the experimental knowledge acquired on competence regulation in S. pneumoniae. Moreover, it can be applied to the other streptococci species belonging to the mitis and anginosus groups since they shared the same regulatory circuit. In the population, the competence shift happens first in a subpopulation of cells and spreads into the whole population through cell to cell contact. Allowing simulation of individual cell behavior, our model will provide a brick for the design of a population-scale model composed of heterogeneous cells.

A proteína A de superfície de pneumococo (PspA) é encontrada na superfície de todas as cepas de Streptococcus pneumoniae e candidata promissora para novas vacinas pneumocócicas. Foi desenvolvido um processo de purificação da PspA4Pro cujas etapas iniciais foram: ruptura da biomassa celular, precipitação do homogenato obtido com o detergente brometo de cetiltrimetilamônio (CTAB) e remoção do precipitado por centrifugação. Foram avaliadas cromatografias de troca iônica (aniônica, catiônica), afinidade por metais, interação hidrofóbica e mista de troca catiônica e hidrofóbica. Utilizando precipitação com CTAB, cromatografia de troca aniônica, crioprecipitação em pH4,0 e cromatografia de troca catiônica atingiu-se a pureza requerida de PspA4Pro (>95%) com recuperação entre 14% e 33%. O processo alcançou níveis aceitáveis de endotoxina no produto final e a PspA4Pro purificada foi reconhecida por anticorpos anti-PspA4, manteve sua atividade e sua estrutura secundária. / Pneumococcal surface protein A (PspA) is found in all Streptococcus pneumoniae strains and is a promising candidate to be used in new pneumococcal vaccines. A purification process for PspA4Pro which inicial steps were: cell disruption, precipitation of the homogenate with the cationic detergent cetyltrimethylammonium bromide (CTAB) and pellet removal by centrifugation. The chromatographic techniques tested were ion exchange (anionic and cationic), immobilized metal affinity, hydrophobic interaction and mix mode with hydrophobic and cationic ligands. Using CTAB precipitation, anion exchange chromatography, crioprecipitation in pH4.0 and cation exchange chromatography the PspA reached the required purity (>95%) with recovery between 14% and 33% . The process reached acceptable levels of endotoxin in the final product and the purified PspA4Pro was recognized by anti-PspA4 antibodies and manteined its activity and secondary structure.

La transformation naturelle bactérienne permet aux micro-organismes d'échanger des informations génétiques pour promouvoir leurs réponses adaptatives pour faire face aux changements environnementaux. De l'ADN extracellulaire est incorporé et recombiné au génome de l'hôte. Ce processus augmente la plasticité des bactéries. Chez S. pneumoniae, un pathogène majeur chez l'Homme engendrant des infections pouvant être mortelles, la transformation bactérienne accentue la transmission de gènes de résistance aux antibiotiques. Chez les bactéries à Gram positif, l'opéron comF encode l'expression de deux protéines. L'une est démontrée comme étant essentielle à la transformation, est décrite pour être membranaire. La seconde n'a pas été étudiée. Cependant ces protéines n'ont pas été étudiées d'un point de vue structural ou fonctionnel. Des mutagenèse et le double hybride bactérien ont permis de mettre en évidence que ses protéines sont indispensables pour l'expression de la compétence et interagissent avec de nombreuses protéines du transformasome. De plus, l'expression des deux protéines de manière hétérologue prouve qu'elles sont solubles et forment des oligomères. L'analyse structurale de ComFA, atteste de la conformation atypique de cette helicase trimerique et hexamerique. En outre, l'activité ATPasique simple brin DNA-dépendant de cette protéine est démontrée. Finalement un complexe protéique a été révélé entre ComFA et ComFC dont l'étude microscopique à hautes résolutions prouve l'apparition d'un anneau via l'assemblage de deux hexamères. Ces résultats suggèrent que ComFA est le moteur tirant l'ADN dans la cellule. Quant à ComFC, elle semble aider à la stabilisation de ComFA. / Bacterial natural transformation allows microorganisms to exchange genetic information to promote their adaptive responses to cope with environmental changes. The extracellular DNA is incorporated and recombined with the genome of the host. This phenomenon increases the plasticity of Gram positive and negative bacteria. S. pneumoniae is a major pathogen for humans, which is causing infections that can be deadly. In this specie, bacterial transformation increases the transmission of antibiotic resistance.In Gram-positive bacteria, comF operon encodes the expression of two proteins. One of them, shown to be essential for natural transformation, is expected to be a membrane protein. The second is not described. However, up to now neither protein has been studied from a structural or functional point of view. Mutagenesis technique and double hybrid bacterial assay allowed to show that both proteins are essential for the expression of the competence and interact with many proteins of the transformasome. In addition, heterologous expresion of both proteins have shown their solubility and the formation of oligomers. Structural analysis of ComFA demonstrates the unique conformation of this hexameric and trimeric helicase. Furthermore, the ATPase single stranded DNA-dependent activity of this protein could be detected. Finally, a protein complex is formed between ComFA and ComF, and high-resolution microscopic study proves the occurrence of a ring via a two-hexamers. These results suggest that ComFA is the engine pulling the DNA in the cell. As for ComFC, this protein seems to help stabilizing of ComFA.