Streptococcus pneumoniae ist ein gram-positives Bakterium und ein Krankheitserreger des Menschen. Ein Charakteristikum des Bakteriums ist, dass es Cholin-Reste in seine dicke Zellwand einbaut. Das Ziel meiner Doktorarbeit war herauszufinden, inwiefern diese Cholin-Reste zur Virulenz des Bakteriums während experimenteller Sepsis und Meningitis beitragen. Dabei konnte ich feststellen, das cholinierte Wildtyp-Bakterien hoch virulent waren, ungestört im Wirt wachsen konnten und letztendlich zu einer massiven Überaktivierung des Wirts-Immunsystems (gemessen anhand der Zytokine IL-1beta, IL-6, IL-12, TNFalpha) sowie zum Tode der Versuchtiere führten. Im Gegensatz dazu waren cholin-freie Bakterien nicht in der Lage eine permanente Infektion im Wirt zu etablieren. So wuchsen sie nur anfangs und wurden vom Wirts-Immunsystem kontrolliert und beseitigt, sodass alle Tiere überlebten. Die Injektion von cholin-freien und cholinierten, hoch aufgereinigten Zellwänden, führte zu der Schlussfolgerung, dass Cholin in der Zellwand ein Immunevasionsmechanismus der Bakterien sein muss. Ausserdem waren cholin-freie Bakterien in der Lage einen protektiven, serotyp-spezifischen Immunschutz im Wirt zu induzieren. / Streptococcus pneumonia is a major human pathogen. Since it is a gram positive bacterium it is characterized by the production of a thick cell wall. Being auxotrophic for choline, the pneumococcus attaches this aminoalcohol to its teichoic acids thus decorating its surface with choline-residues. The aim of this work was to investigate the role that these choline residues play in the virulence of the bacterium. By using an isogenic choline-containing and choline-free pair of S. pneumoniae I was able to demonstrate that surface bound choline is essential for the virulence of the bacterium in animal models of experimental sepsis and meningitis. In either model choline-containing bacteria were able to persistently grow within the host system, continuously stimulate the production of proinflammatory cytokines (IL-1beta, IL-6, IL-12, TNFalpha) and eventually led to the death of all infected animals within 24h. In contrast, choline-free bacteria showed only transient growth within the host and induced only moderate and limited expression of cytokines. Consequently, the bacterium was virtually avirulent and all animals survived. Intracisternal application of highly purified cholinated and choline-free cell wall preparations, induced a comparable activation of the immune system. These findings led to the conclusion that choline residues contribute to an immunevasion strategy that allows the bacteria to grow despite an ongoing immune response. Although being avirulent choline-free bacteria were able to induce serotype specific immunity in the animals.

Streptococcus pneumoniae ist die häufigste Ursache für ambulant erworbene Pneumonien weltweit. Daher müssen die Wirts-Pathogen-Interaktionen erforscht werden, um neue Therapiestrategien zu entwickeln. In dieser Studie habe ich 1. den Typ I Interferon (IFN)-stimulierenden Signalweg des angeborenen Immunsystems in Pneumokokken-infizierten Wirtszellen sowie 2. dessen Bedeutung in der Pneumokokkenpneumonie untersucht. Humane und murine Makrophagen, aber nicht alveolare Epithelzellen, produzierten Typ I IFNs nach Infektion mit S. pneumoniae. Dieses war abhängig vom Virulenzfaktor Pneumolysin und erforderte sowohl die Phagozytose der Bakterien als auch die Ansäuerung der Endosomen. Die Induktion der Typ I IFNs wird durch einen zytosolischen Signalweg vermittelt, welcher wahrscheinlich DNA erkennt und sowohl das Adapterprotein STING als auch den Transkriptionsfaktor IRF3 aktiviert. Typ I IFNs, welche von infizierten Makrophagen gebildet wurden, regulierten die Expression von IFN-stimulierten Genen (ISGs) und Chemokinen in Makrophagen und co-kultivierten alveolaren Epithelzellen in vitro und in Mauslungen in vivo. In einem murinen Pneumoniemodell hatten die Typ I IFNs jedoch einen negativen Effekt für den Wirt. Mäuse mit einem Defekt im Typ I IFN-Rezeptor oder mit einem Knockout im Typ I und Typ II IFN-Rezeptor hatten eine signifikant geringere Bakterienlast in der Lunge und eine verminderte Reduktion der Körpertemperatur und des Körpergewichtes als wild-typ Mäuse. Diese Effekte waren nicht durch Änderungen in der Zellrekrutierung oder durch Änderungen der Zytokin-/Chemokinexpression erklärbar. Zusammenfassend lässt sich feststellen, dass Typ I IFNs durch Pneumokokken induziert werden, aber dass sie trotz einiger positiver Effekte auf die Expression von ISGs einen negativen Gesamteffekt in einem murinen Pneumoniemodell aufweisen. Ein detailliertes Verständnis der Typ I IFN-Antwort während der Pneumokokkeninfektion kann die Entwicklung neuer Therapiestrategien unterstützen. / Streptococcus pneumoniae is the leading cause of community-acquired pneumonia world-wide. A detailed understanding of the host-pathogen interactions is required in order to foster the development of new therapeutic strategies. Here, I (I) characterized an innate immune recognition pathway that senses pneumococcal infection and triggers the production of type I interferons (IFNs), and (II) examined the role of type I IFNs in pneumococcal pneumonia in mice. Human and murine macrophages, but not alveolar epithelial cells, produced type I IFNs after infection with S. pneumoniae. This induction was dependent on the virulence factor pneumolysin, the phagocytosis of the bacteria, and the acidification of the endosome. Moreover, it appeared to be mediated by a cytosolic DNA-sensing pathway involving the adaptor molecule STING and the transcription factor IRF3. Type I IFNs produced by S. pneumoniae-infected macrophages positively regulated the expression of IFN-stimulated genes (ISGs) and chemokines in macrophages and co-cultured alveolar epithelial cells in vitro and in mouse lungs in vivo. However, in a murine model of pneumococcal pneumonia, type I IFN signaling was detrimental to the host defense. Mice deficient in the type I IFN signaling or double deficient in type I and type II IFN signaling had a significantly reduced bacterial load in the lung and a diminished reduction of body temperature and body weight compared to wild-type mice. The decreased susceptibility of the knockout mice was unlikely to be attributable to alterations in cell recruitment or cytokine/chemokine production. In conclusion, type I IFNs are induced during pneumococcal infection. However, despite their positive effects on the expression of some ISGs and chemokines, they negatively affect the outcome of pneumococcal pneumonia in an in vivo mouse model. Targeting the type I IFN system could potentially be an effective way in enhancing the immune response in patients with S. pneumoniae pneumonia.

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.

O Streptococcus pneumoniae (pneumococo) constitui um dos mais importantes patógenos bacterianos do trato respiratório, podendo causar infecções invasivas e não invasivas, levando a altas taxas de morbimortalidade, particularmente em crianças menores de cinco anos de idade. A bactéria ganha acesso ao hospedeiro através da colonização da nasofaringe, que representa um importante reservatório para a transmissão deste patógeno na comunidade, contribuindo para a disseminação horizontal de pneumococo entre os indivíduos de uma população. As doenças respiratórias causadas por pneumococo constituem em uma das prioridades atuais em Saúde Pública, recebendo atenção destacada das organizações internacionais da área da saúde, como a Organização Mundial da Saúde. No presente trabalho procura-se conhecer e avaliar a ocorrência da pneumonia adquirida na comunidade (PAC) antes e após a implantação no Calendário Vacinal da Vacina Pneumocócica-10 Valente Conjugada em 2010, na área de abrangência da Superintendência Regional de Saúde (SRS) de Alfenas/MG. Foi realizado um estudo ecológico com componente temporal que incluiu registros de crianças menores que um ano de idade, vacinadas e não vacinadas com a vacina antipneumocócica 10-valente conjugada, no período pré e pós inclusão da vacina no PNI nos municípios da Superintendência Regional de Saúde (SRS) de Alfenas/MG, sendo a vacinação o fator de exposição e a ocorrência de PAC o desfecho, utilizando dados anuais secundários por município para cálculo da cobertura vacinal e das taxas de morbidade por pneumonia em menores de um ano no período de 2007 a 2013. Considerando se os 26 municípios da SRS de Alfenas, houve redução significativa do número de casos de PAC em crianças abaixo de um ano de idade, cuja Razão de Prevalência foi de 0,81 (IC95%: 0,74 0,89; p<0,05). Mesmo com um tempo reduzido de uso, a vacina pneumocócica conjugada 10 valente apresentou um impacto relevante na redução de PAC em crianças, ajustada por cobertura vacinal no período pós vacinação (2011-2013), sendo estatisticamente significativa na maioria dos municípios, o que sugere a efetividade da vacina PCV-10 na prevenção de casos da doença em crianças menores de um ano de idade. / The Streptococcus pneumoniae (pneumococcus) is one of the most important bacterial pathogens of the respiratory tract, may cause invasive and non-invasive infections, leading to high morbidity and mortality rates, particularly in children under five years of age. The bacteria gain access to the host through the nasopharyngeal colonization, which is an important reservoir for the transmission of this pathogen in the community, contributing to the horizontal spread among individuals in a population. Respiratory diseases caused by pneumococcus are in one of the current priorities in Public Health, receiving outstanding attention of international organizations in the health field, such as the World Health Organization. In the present study we aimed to understand and evaluate the occurrence of community acquired pneumonia (CAP) before and after implantation in 10- valent pneumococcal conjugate vaccine in 2010, on the coverage area of the Regional Health Service (SRS) of Alfenas / MG. An ecological study with temporal component was conducted which included records of children under one year old, vaccinated and not vaccinated with 10-valent pneumococcal conjugate vaccine, before and after period inclusion of the vaccine in PNI, in the municipalities of SRS of Alfenas / MG, with vaccination the exposure factor and the occurrence of CAP the outcome, using annual data side by municipality to calculate vaccination coverage and pneumonia morbidity in children under one year old, in the period 2007 to 2013. Considering the 26 municipalities of SRS Alfenas, there was a significant reduction in the number of CAP cases in children under one year old. The prevalence ratio was 0.81 (95%CI: 0.74 - 0.89; p<0.05). Even with a short period of use, the 10-valent pneumococcal conjugate vaccine had a significant impact on the reduction of CAP in children, adjusted for immunization coverage in the post vaccination period (2011-2013) and was statistically significant in most municipalities, which suggests the effectiveness of PCV-10 vaccine in preventing cases of the disease in children under one year of age.

Introdução: Atualmente a incidência anual de pneumonia adquirida na comunidade nos países em desenvolvimento é de 150,7 milhões de casos entre crianças menores de 5 anos de idade , dos quais 11 a 20 milhões (7-13%) necessitam de internação hospitalar devido à gravidade. O tratamento geralmente é empírico mas o Streptococcus pneumoniae é o principal agente etiológico bacteriano.É necessário manter monitoramento dos sorotipos e padrão de resistência para melhor orientação terapêutica. Metodologia: Estudo de coorte retrospectivo com inclusão de 107 crianças com diagnóstico clínico e radiológico de pneumonia e com isolamento de Streptococcus pneumoniae em sangue e ou líquido pleural no período de janeiro de 2003 a outubro de 2008. Realizado determinação de concentração inibitória mínima (MIC) para penicilina e antibiograma para outros antimicrobianos. A sensibilidade para penicilina utilizada foi conforme Clinical and Laboratory Standards Institute (CLSI ) de 2008. Realizado sorotipagem de 96 cepas de pneumococos (89,7%) e analisados os dados da população em estudo e da evolução clínica. Resultados:Cerca de 47,5% das internações na enfermaria foram por pneumonia ou broncopneumonia e a média de positividade em cultura para pneumococo (sangue e ou líquido pleural) foi de 2,5%. Houve uma sazonalidade bem definida da pneumonia pneumocócica. Cerca de 70% ocorreram nos meses de junho a outubro. A mediana de idade foi de 23 meses (82,2%<5anos); predomínio do sexo masculino (58,9%);utilização de antibioticoterapia nos dois meses prévios à internação de 23,4%; freqüência em creche no menores de 2 anos de 36,4%; apenas um caso com vacinação heptavalente completa; doença associada em 44,9% sendo a mais freqüente a sibilância( 77,1%); tempo de febre e de sintomas respiratórios antes da admissão foi de 4 dias;necessidade de oxigenoterapia não invasiva em 70,1% com tempo médio de utilização de 4 dias;necessidade de ventilação mecânica em 19,6%, mediana do tempo de internação de 9 dias.Em 62% houve complicações sendo as mais freqüentes: empiema (53%) e efusão pleural não complicada (42%). As crianças com empiema tiveram mais pneumonia necrotizante, abscesso pulmonar, sepse, pneumotórax, necessidade de decorticação e ainda maior mortalidade (todas com p<0,05). As crianças com complicações tiveram mais dias de sintomas respiratórios antes da admissão (3x5dias), mais tempo de febre após o início de antibiótico (1x4,5dias), necessitaram de oxigenoterapia não invasiva(58,5x77,3%) e ventilação mecânica (7,3x27,3%) por tempo maior e permaneceram por mais tempo internados (5x12 dias). Das 107 cepas de pneumococo, 100 (93,5%) foram sensíveis à penicilina e 7 (6,5%) de sensibilidade intermediária. Todas as cepas testadas foram sensíveis para rifampicina e vancomicina e ainda mantiveram boa sensibilidade para clindamicina, cloranfenicol, ceftriaxone, eritromicina e levofloxacina. Cinco cepas foram multiresistentes. Notou-se que a média geométrica das concentrações inibitórias mínimas (GMC) para penicilina foram maiores nas crianças com complicações. Os sorotipos mais freqüentes foram: 14(36,5%), 1(16,7%) , 5(14,6%) e 6B(6,3%). O sorotipo 14 apresentou a maior GMC para penicilina e houve um aumento progressivo no decorrer dos anos de estudo. A cobertura dos sorotipos pela vacina heptavalente seria de 53,1% e esta cobertura menor se deve principalmente ao sorotipo 1 e 5, que corresponde a 31,3% dos casos. A cobertura dos sorotipos associados à resistência seria de 94,2%. A cobertura pela vacina 10-valente seria de 86,5% e com a 13-valente seria de 96,9%. Três casos que evoluíram para óbito (2,8%) tinha mediana de idade de 18 meses, todos do sexo masculino, todos com concentração inibitória mínima para penicilina menor ou igual 1g/mL, todos evoluíram com empiema e sepse. Dois foram do sorotipo 5 e um do sorotipo 14. Conclusões: Aproximadamente 2,5% das crianças internadas com diagnóstico de pneumonia foram diagnosticadas como pneumonia pneumocócica.Verificamos uma sazonalidade bem definida.Houve complicações em 62% dos casos. As mais freqüentes foram : empiema e a efusão pleural não complicada. Evidenciou-se uma GMC para penicilina maior nas crianças com complicações comparadas às crianças com ausência de complicações. Os sorotipos mais freqüentes foram 14,1 ,5 e 6B sendo que os sorotipos 1 e 5 totalizam 31,3%. A cobertura pela vacina heptavalente dos sorotipos isolados seria de 53,1%. A sensibilidade para penicilina dos pneumococos isolados de pneumonia foi de 93,5%. Assim, a opção terapêutica continua sendo a penicilina. / Introduction: Currently, the annual incidence of the acquired pneumonia in the developing country communities are around 150.7 million cases, among childrens under 5 years of age, and 11 to 20 million (7-13%) of those require hospitalization due to their gravity. In general, the treatments used to be empirical, however, it is important to be noted that Streptococcus pneumoniae is far the major bacterial etiologic agent. It is necessary to keep monitoring the serotypes and the pattern of resistance in order to improve the therapy guidance. Methodology: Retrospective cohort study with inclusion of the 107 childrens with clinical and radiological diagnosis of the pneumonia, and the isolation of Streptococcus pneumoniae in the blood and/or pleural fluid during the period of January 2003 to October 2008. It was performed determination of the minimum inhibitory concentration (MIC) related to the penicillin and other antibiotics. The sensitivity analysis to the penicillin was based on Clinical and Laboratory Standards Institute (CLSI), 2008, recommendations. They were performed serotyping in 96 pneumococcal strains (89.7%) and they were analyzed datas referred to the considered population and their clinical course. Results: About 47.5% of admissions in the ward were caused by pneumonia or bronchopneumonia, and the average positive occurrences in the pneumococcal (blood and / or pleural) culture were 2.5%. It was noted a clear seasonality phenomena of the pneumococcal pneumonia. About 70% of the cases occurred during months of June to October. The median age was 23 months (82.2%<5 years); with predominance of males (58.9%); in the 23,4% of the cases the antibiotic therapy was used during two months prior to the admission; the daycare frequency of the childs less than 2 years were 36.4%; only one case with complete vaccination heptavalent; associated disease was detected in the 44.9% of the cases and the most frequent was wheezing (77.1%); time of fever and respiratory symptoms before admission were 4 days; the need for noninvasive oxygen therapy occurred in 70.1% being 4 days of the average time of the use; the need for mechanical ventilation occurred in 19.6%; the median period of stay were 9 days. In 62% of the cases there were the most frequent complications: empyema (53%) and non-complicated pleural effusion (42%). The childrens with empyema had more necrotizing pneumonia, lung abscess, sepsis, pneumothorax, need for decortication, and even higher mortality (all with p<0.05). The childrens with complications had more days of respiratory symptoms before admission (3x5days), more time of the fever after initiation with antibiotic (1x4, 5days), they need noninvasive oxygen therapy (58,5 x77, 3%) and mechanical ventilation (7 , 3x27, 3%) for more time and remained hospitalized during longer period(5x12 days). Among 107 pneumococcal strains, 100 (93.5%) were susceptible to penicillin and 7 (6.5%) presented intermediate sensitivity. All strains tested were sensitive to rifampicin and vancomycin, and they maintained good sensitivity to clindamycin, chloramphenicol, ceftriaxone, erythromycin and levofloxacin. Five strains were multi-resistant. It was noted that the geometric mean of minimum inhibitory concentrations (GMC) to penicillin were higher in children with complications. The most frequent serotypes were: 14 (36.5%), 1 (16.7%), 5 (14.6%) and 6B (6.3%). The serotype 14 presented the highest GMC for penicillin and it was verified a progressive increase during the years of the study. The coverage of serotypes by the heptavalent vaccine would be cover 53.1% and this less coverage is represented by serotype 1 and 5, which corresponds to 31.3% of the cases. The coverage of serotypes associated with resistance would be 94.2%. The coverage of the 10-valent vaccine would be 86.5% and for 13-valent would be 96.9%. Three cases that carried to died (2.8%) had median age of 18 months, all they male, all they with minimum inhibitory concentration for penicillin <= 1g/mL, all they progressed to empyema and sepsis. Two of them were serotype 5 and one of them was serotype 14. Conclusions: Approximately 2.5% of children were admitted with diagnosis of pneumonia were diagnosed as pneumococcal pneumonia. It was verified a clear seasonality phenomena. They were observed complications in 62% of the cases. The most frequent were: empyema and non-complicated pleural effusion cases. It was confirmed a higher GMC for penicillin in children with complications compared to the children without complications. The most frequent serotypes were 14, 1, 5 and 6B and the serotypes 1 and 5 accounted 31.3%. The coverage of heptavalent vaccine for the isolated serotypes would be 53.1%. The sensitivity to the penicillin of the isolated pneumococcal was 93.5%. Therefore, the therapy option remains being the penicillin.

A proteína A de superfície de pneumococo (PspA) é indispensável para a virulência da bactéria e foi escolhida para a elaboração de uma nova vacina conjugada contra S. pneumoniae. Para tanto foi desenvolvido um processo industrial de produção e purificação do fragmento recombinante da PspA clado 3 em E. coli. Cultivos descontínuos alimentados foram estabelecidos com glicose ou glicerol em reator de 5L, obtendo-se 62g/L de células secas e 3g/L de PspA3. As células foram lisadas por homogeneizador contínuo de alta pressão com eficiência de 96,7%. A centrifugação foi definida como etapa de clarificação. A sequência cromatográfica troca aniônica seguida de afinidade por Ni+2 rendeu os melhores resultados de pureza (81%) e recuperação (70%). A cromatografia de troca catiônica foi selecionada como terceira etapa do processo, definindo assim um processo de produção e purificação escalonável que possibilitou a obtenção de PspA3 com alto grau de pureza (90%). / The pneumococcal surface protein A (PspA) is indispensable for virulence of S. pneumoniae and it was the first choice as carrier for a new conjugated vaccine against S.pneumoniae. Hence, the purpose of this work was to develop an industrial production and purification process of a recombinant fragment PspA clade 3 (rfPspA3) in E. coli. Fed-batch cultivations in 5 L bioreactors with defined medium were carried out using glucose or glycerol as carbon sources. It was obtained 62 g/L of dry cell weight and 3 g/L of rfPspA3. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. Centrifugation was defined for the clarification step. The sequence with Q- followed by IMAC-Sepharose yielded the best purity and recovery of rfPspA3 (81 and 70%, respectively). Cation exchange was chosen for the last chromatography. In conclusion, an industrial production and purification process was developed and rfPspA3 was obtained with high purity (90%).

Streptococcus pneumoniae é uma bactéria encapsulada causadora de doenças infecciosas como pneumonia, bacteremia e meningite, infecções essas que estão entre as principais causas de morte entre crianças, idosos e imunodeprimidos, indivíduos que constituem o grupo de risco para tais infecções. A vacinação tem sido a mais eficaz forma de conter tais infecções. A vantagem das vacinas conjugadas em comparação às polissacarídicas é a capacidade de indução de uma resposta imune T-dependente o que garante proteção mesmo ao grupo de risco para infecções por S. pneumonia. A proposta do projeto foi estabelecer um protocolo para obtenção de um conjugado constituído pelo polissacarídeo capsular de S. pneumonia sorotipo 1 (PS1) e pela proteína de superfície pneumocócica A (PspA). A síntese do conjugado empregou uma metodologia inédita para o sorotipo 1. A avaliação da resposta imune humoral induzida pelo conjugado mostrou a indução de IgG anti-PS1 gerada pelas imunizações com o conjugado PS1-PspA. / Streptococcus pneumoniae is an encapsulated bacteria causing infectious diseases such as pneumonia, bacteremia and meningitis, these infections are among the leading causes of death among children, elderly and immunocompromised, who constituting individuals of risk group. The vaccination has been the more effective form to counter these infection. The advantage of conjugated vaccines compared to vaccines polysaccharide, is the ability to induce a T-dependent immune response which provides protection even at risk groups for infection by S. pneumoniae. The project proposal was establish a protocol for obtaining a conjugate consisting of the capsular polysaccharide of S. pneumoniae serotype 1 (PS1) and the pneumococcal surface protein A (PspA). The synthesis of conjugate employed a new methodology for serotype 1. The evaluation of humoral immune response induced by the conjugate showed anti-PS1 IgG induction generated by immunization with the PS1-PspA.

Longtemps décrit comme un simple tissu de réserve énergétique, le tissu adipeux blanc est, depuis l’identification de la leptine en 1994, considéré comme un véritable organe endocrine. En effet, ce tissu secrète de nombreuses hormones et cytokines agissant de manière paracrine et endocrine pour contrôler le métabolisme énergétique. Par ailleurs, en plus des préadipocytes et des adipocytes, le tissu adipeux blanc contient également des cellules immunes innées et adaptatives ; lui conférant ainsi un rôle important dans le développement et le contrôle de l’immunité. Cependant, le rôle joué par le tissu adipeux blanc dans les infections - notamment pulmonaires - reste encore peu étudié. C’est dans ce cadre général que s’est inscrit ce travail de Thèse. La susceptibilité accrue des individus obèses (expansion du tissu adipeux blanc) à l’infection par le virus de la grippe (influenza) est largement étayée dans la littérature. Nous avons évalué l’impact de l’infection par le virus influenza sur le tissu adipeux blanc, chez des souris minces et des souris obèses. Nos résultats montrent que, de manière inattendue, le virus est détecté dans les tissus adipeux, sous-cutané (inguinal) et viscéral (périgonadique), de souris infectées par voie intra-nasale (détection du génome viral par RT-qPCR). La présence de virus dans le tissu adipeux est associée à l’augmentation de la sécrétion de cytokines pro- et anti-inflammatoires, à la diminution de l’expression de gènes impliqués dans la lipolyse et la lipogénèse, et à l’augmentation de l’expression des gènes impliqués dans l’induction d’une réponse immune anti-virale. De manière intéressante, l’infection par le virus influenza est associée au brunissement du tissu adipeux sous-cutané chez les souris minces. Chez les souris obèses, l’infection par le virus de la grippe n’induit pas l’effet dépôt spécifique observé chez la souris mince et ne montre pas de brûnissement au niveau du tissu adipeux sous-cutané 7 jours p.i. In vitro, nous montrons que le virus influenza peut infecter les préadipocytes et les adipocytes (lignée murine et cellules primaires humaines). Cependant, alors que le virus effectue la totalité de son cycle dans l’adipocyte, le préadipocyte libère très peu, voire pas, de nouveaux virions infectieux (PCR, transcriptomique, technique de plages de lyse, microscopie confocale et électronique). Ainsi nos résultats, très originaux, identifient le tissu adipeux blanc comme un nouveau tissu cible de l’infection par le virus de la grippe, in vivo. Au sein de ce tissu, les préadipocytes et les adipocytes sont potentiellement infectés par le virus, comme le montrent nos données in vitro, les adipocytes seuls permettant la production de nouvelles particules infectieuses.Contrairement à l’infection grippale, les données épidémiologiques et/ou expérimentales concernant la susceptibilité des obèses à l’infection par la bactérie Streptococcus pneumoniae sont contradictoires, du fait de l’utilisation de différents modèles d’obésité d’origine génétique et de sérotypes de pneumocoques. Dans ce projet, nous avons utilisé un modèle d’obésité d’origine nutritionnelle ; le modèle de souris nourries par un régime enrichi en lipides. Nous montrons que les souris obèses infectées (sérotype Sp1) développent un syndrome de type méningite, mortel, tandis que les souris minces contrôlent l’infection. Si les réponses pulmonaires à l’infection sont comparables entre les souris minces et obèses (dénombrement des colonies bactériennes, histologie, PCR, ELISA, cytométrie en flux), le nombre de bactéries dans le cerveau est significativement plus élevé chez les souris obèses, associé à une altération de la perméabilité de la barrière hématoencéphalique [...] / Since the identification of leptin in 1994, the white adipose tissue (WAT) is no longer considered to solely be an inert tissue storing fat. As an endocrine organ, the adipose tissue synthesizes and secretes several hormones and cytokines involved in the control of whole-body metabolism. In addition, besides preadipocytes and adipocytes, WAT also contains innate and adaptive immune cells; thereby contributing to the development and control of immunity. However, the role played by the adipose tissue during infections - notably during pulmonary infections, such as those resulting from influenza virus or Streptococcus pneumoniae infections - has barely been investigated. This defines the general scope of this work. Epidemiological and experimental data convincingly report that obese individuals are more susceptible to influenza infection. During this project, we therefore questioned whether influenza infection may impact on adipose tissues, both subcutaneous (inguinal, SCAT) and visceral (perigonadal, EWAT) depots, in lean and high-fat diet-induced obese mice. We unexpectedly showed that influenza virus is detected in SCAT and EWAT (quantification of the viral genome by RtqPCR) and that this was associated with drastic changes in the tissue such as: increased secretion of pro- and anti-inflammatory cytokines, decreased expression of genes involved in lipogenesis and lipolysis, as well as increased expression of genes related to the induction of anti-viral immunity. Interestingly enough, influenza infection was associated with the development of brown-like adipocytes in the SCAT, only in lean animals. Moreover, we demonstrated in vitro that preadipocytes and adipocytes (murine cell-line and human primary cells) are permissive to infection, yet with different outcomes. Indeed, only adipocytes allowed the release of new infectious particles (RtqPCR, transcriptomics, quantification of infectious particules on MDCK cells, confocal and transmission electron microscopy). Altogether, our findings revealed, for the first time, that the white adipose tissue, an organ at the crossroads of metabolism and immunity, is deeply affected by influenza infection and might thus be undervalued in influenza pathophysiology.In opposite to influenza infection, the impact of obesity on the outcome of Streptococcus pneumoniae (S.p.) infection remains uncertain, due to the different models (genetically-based obesity, and bacterial strains) used. During this work, we investigated S. pneumoniae (Sp1 strain, sublethal dose) infection in lean and high-fat diet-induced obese mice. We showed that obese mice died from sublethal S. pneumoniae infection, compared to lean animals. The increased mortality induced by infection did not result from impaired pulmonary response but rather from the development of a meningitis-like syndrom likely resulting from an increased bacterial dissemination through the bloodbrain barrier into the brain. We propose that the model of dietary obesity induced by consumption of fat-enriched diet, may be envisaged as a novel and valuable experimental model of memingitis to study Streptococcus pneumoniae travel through the blood-brain barrier and the subsequent immune consequences.

Introdução: Atualmente a incidência anual de pneumonia adquirida na comunidade nos países em desenvolvimento é de 150,7 milhões de casos entre crianças menores de 5 anos de idade , dos quais 11 a 20 milhões (7-13%) necessitam de internação hospitalar devido à gravidade. O tratamento geralmente é empírico mas o Streptococcus pneumoniae é o principal agente etiológico bacteriano.É necessário manter monitoramento dos sorotipos e padrão de resistência para melhor orientação terapêutica. Metodologia: Estudo de coorte retrospectivo com inclusão de 107 crianças com diagnóstico clínico e radiológico de pneumonia e com isolamento de Streptococcus pneumoniae em sangue e ou líquido pleural no período de janeiro de 2003 a outubro de 2008. Realizado determinação de concentração inibitória mínima (MIC) para penicilina e antibiograma para outros antimicrobianos. A sensibilidade para penicilina utilizada foi conforme Clinical and Laboratory Standards Institute (CLSI ) de 2008. Realizado sorotipagem de 96 cepas de pneumococos (89,7%) e analisados os dados da população em estudo e da evolução clínica. Resultados:Cerca de 47,5% das internações na enfermaria foram por pneumonia ou broncopneumonia e a média de positividade em cultura para pneumococo (sangue e ou líquido pleural) foi de 2,5%. Houve uma sazonalidade bem definida da pneumonia pneumocócica. Cerca de 70% ocorreram nos meses de junho a outubro. A mediana de idade foi de 23 meses (82,2%<5anos); predomínio do sexo masculino (58,9%);utilização de antibioticoterapia nos dois meses prévios à internação de 23,4%; freqüência em creche no menores de 2 anos de 36,4%; apenas um caso com vacinação heptavalente completa; doença associada em 44,9% sendo a mais freqüente a sibilância( 77,1%); tempo de febre e de sintomas respiratórios antes da admissão foi de 4 dias;necessidade de oxigenoterapia não invasiva em 70,1% com tempo médio de utilização de 4 dias;necessidade de ventilação mecânica em 19,6%, mediana do tempo de internação de 9 dias.Em 62% houve complicações sendo as mais freqüentes: empiema (53%) e efusão pleural não complicada (42%). As crianças com empiema tiveram mais pneumonia necrotizante, abscesso pulmonar, sepse, pneumotórax, necessidade de decorticação e ainda maior mortalidade (todas com p<0,05). As crianças com complicações tiveram mais dias de sintomas respiratórios antes da admissão (3x5dias), mais tempo de febre após o início de antibiótico (1x4,5dias), necessitaram de oxigenoterapia não invasiva(58,5x77,3%) e ventilação mecânica (7,3x27,3%) por tempo maior e permaneceram por mais tempo internados (5x12 dias). Das 107 cepas de pneumococo, 100 (93,5%) foram sensíveis à penicilina e 7 (6,5%) de sensibilidade intermediária. Todas as cepas testadas foram sensíveis para rifampicina e vancomicina e ainda mantiveram boa sensibilidade para clindamicina, cloranfenicol, ceftriaxone, eritromicina e levofloxacina. Cinco cepas foram multiresistentes. Notou-se que a média geométrica das concentrações inibitórias mínimas (GMC) para penicilina foram maiores nas crianças com complicações. Os sorotipos mais freqüentes foram: 14(36,5%), 1(16,7%) , 5(14,6%) e 6B(6,3%). O sorotipo 14 apresentou a maior GMC para penicilina e houve um aumento progressivo no decorrer dos anos de estudo. A cobertura dos sorotipos pela vacina heptavalente seria de 53,1% e esta cobertura menor se deve principalmente ao sorotipo 1 e 5, que corresponde a 31,3% dos casos. A cobertura dos sorotipos associados à resistência seria de 94,2%. A cobertura pela vacina 10-valente seria de 86,5% e com a 13-valente seria de 96,9%. Três casos que evoluíram para óbito (2,8%) tinha mediana de idade de 18 meses, todos do sexo masculino, todos com concentração inibitória mínima para penicilina menor ou igual 1g/mL, todos evoluíram com empiema e sepse. Dois foram do sorotipo 5 e um do sorotipo 14. Conclusões: Aproximadamente 2,5% das crianças internadas com diagnóstico de pneumonia foram diagnosticadas como pneumonia pneumocócica.Verificamos uma sazonalidade bem definida.Houve complicações em 62% dos casos. As mais freqüentes foram : empiema e a efusão pleural não complicada. Evidenciou-se uma GMC para penicilina maior nas crianças com complicações comparadas às crianças com ausência de complicações. Os sorotipos mais freqüentes foram 14,1 ,5 e 6B sendo que os sorotipos 1 e 5 totalizam 31,3%. A cobertura pela vacina heptavalente dos sorotipos isolados seria de 53,1%. A sensibilidade para penicilina dos pneumococos isolados de pneumonia foi de 93,5%. Assim, a opção terapêutica continua sendo a penicilina. / Introduction: Currently, the annual incidence of the acquired pneumonia in the developing country communities are around 150.7 million cases, among childrens under 5 years of age, and 11 to 20 million (7-13%) of those require hospitalization due to their gravity. In general, the treatments used to be empirical, however, it is important to be noted that Streptococcus pneumoniae is far the major bacterial etiologic agent. It is necessary to keep monitoring the serotypes and the pattern of resistance in order to improve the therapy guidance. Methodology: Retrospective cohort study with inclusion of the 107 childrens with clinical and radiological diagnosis of the pneumonia, and the isolation of Streptococcus pneumoniae in the blood and/or pleural fluid during the period of January 2003 to October 2008. It was performed determination of the minimum inhibitory concentration (MIC) related to the penicillin and other antibiotics. The sensitivity analysis to the penicillin was based on Clinical and Laboratory Standards Institute (CLSI), 2008, recommendations. They were performed serotyping in 96 pneumococcal strains (89.7%) and they were analyzed datas referred to the considered population and their clinical course. Results: About 47.5% of admissions in the ward were caused by pneumonia or bronchopneumonia, and the average positive occurrences in the pneumococcal (blood and / or pleural) culture were 2.5%. It was noted a clear seasonality phenomena of the pneumococcal pneumonia. About 70% of the cases occurred during months of June to October. The median age was 23 months (82.2%<5 years); with predominance of males (58.9%); in the 23,4% of the cases the antibiotic therapy was used during two months prior to the admission; the daycare frequency of the childs less than 2 years were 36.4%; only one case with complete vaccination heptavalent; associated disease was detected in the 44.9% of the cases and the most frequent was wheezing (77.1%); time of fever and respiratory symptoms before admission were 4 days; the need for noninvasive oxygen therapy occurred in 70.1% being 4 days of the average time of the use; the need for mechanical ventilation occurred in 19.6%; the median period of stay were 9 days. In 62% of the cases there were the most frequent complications: empyema (53%) and non-complicated pleural effusion (42%). The childrens with empyema had more necrotizing pneumonia, lung abscess, sepsis, pneumothorax, need for decortication, and even higher mortality (all with p<0.05). The childrens with complications had more days of respiratory symptoms before admission (3x5days), more time of the fever after initiation with antibiotic (1x4, 5days), they need noninvasive oxygen therapy (58,5 x77, 3%) and mechanical ventilation (7 , 3x27, 3%) for more time and remained hospitalized during longer period(5x12 days). Among 107 pneumococcal strains, 100 (93.5%) were susceptible to penicillin and 7 (6.5%) presented intermediate sensitivity. All strains tested were sensitive to rifampicin and vancomycin, and they maintained good sensitivity to clindamycin, chloramphenicol, ceftriaxone, erythromycin and levofloxacin. Five strains were multi-resistant. It was noted that the geometric mean of minimum inhibitory concentrations (GMC) to penicillin were higher in children with complications. The most frequent serotypes were: 14 (36.5%), 1 (16.7%), 5 (14.6%) and 6B (6.3%). The serotype 14 presented the highest GMC for penicillin and it was verified a progressive increase during the years of the study. The coverage of serotypes by the heptavalent vaccine would be cover 53.1% and this less coverage is represented by serotype 1 and 5, which corresponds to 31.3% of the cases. The coverage of serotypes associated with resistance would be 94.2%. The coverage of the 10-valent vaccine would be 86.5% and for 13-valent would be 96.9%. Three cases that carried to died (2.8%) had median age of 18 months, all they male, all they with minimum inhibitory concentration for penicillin <= 1g/mL, all they progressed to empyema and sepsis. Two of them were serotype 5 and one of them was serotype 14. Conclusions: Approximately 2.5% of children were admitted with diagnosis of pneumonia were diagnosed as pneumococcal pneumonia. It was verified a clear seasonality phenomena. They were observed complications in 62% of the cases. The most frequent were: empyema and non-complicated pleural effusion cases. It was confirmed a higher GMC for penicillin in children with complications compared to the children without complications. The most frequent serotypes were 14, 1, 5 and 6B and the serotypes 1 and 5 accounted 31.3%. The coverage of heptavalent vaccine for the isolated serotypes would be 53.1%. The sensitivity to the penicillin of the isolated pneumococcal was 93.5%. Therefore, the therapy option remains being the penicillin.

La paroi des bactéries à Gram positif se compose du peptidoglycane (PG) et des acides téichoïques (TA). Leur étude a révélé de nouveaux mécanismes de régulation chez le pathogène humain Streptococcus pneumoniae. Nous avons montré que la O-acétylation intervient précocement dans la biosynthèse du PG, participe à sa maturation et à la division cellulaire. Nous avons développé une approche innovante basée sur la chimie click pour le marquage in vivo des TAs, et révélé que leur synthèse est septale et corrélée à celle du PG. Le PG et les TAs contribuent aussi à réguler l'activité enzymatique de l'autolysine majeur du pneumocoque LytA: la O-acétylation du PG protège les cellules en division de l'autolyse par LytA et les TAs, sur lesquels elle se fixe, régulent sa localisation de surface. Pour conclure, ce travail souligne le rôle coopératif du PG et des TAs dans la synthèse de la paroi, la division cellulaire et la régulation de composants de la surface bactérienne. / Gram-positive bacteria cell wall (CW) is composed by peptidoglycan (PG) and teichoic acids (TA). We studied both CW components and revealed new regulation mechanisms in the human pathogen Streptococcus pneumoniae. We showed that PG O-acetylation occurs in the early steps of PG biosynthesis, promotes the formation of mature PG and plays a role in cell division. We developed an innovative click chemistry-based approach to label TA in live cells, opening the way to explore mechanistic issues of pneumococcal TA biosynthesis. We revealed that TA synthesis occurs at the division site and is correlated with PG synthesis. Finally, we showed that both PG and TA polymers contribute to regulate the major autolysin LytA which binds TA and cleaves the PG: the O-acetylation of PG protects dividing cells from LytA-induced autolysis while TA finely regulates LytA surface localization. In conclusion, our work highlights the cooperative role of PG and TA in CW biosynthesis, cell division and regulation of surface components.