Streptococcus pneumoniae é um importante agente causador de pneumonia, meningite e septicemia. O alto custo e a cobertura limitada da vacina conjugada atual reforçam a necessidade de se desenvolver uma vacina mais abrangente e acessível. A proteína de superfície de pneumococo A (PspA) é imunogênica e protetora em modelos animais; porém, devido à sua diversidade - há 6 clados e 3 famílias de PspA - uma vacina baseada em PspA deverá incluir fragmentos das duas famílias prevalentes (1 e 2). Neste estudo, foram produzidos fragmentos contendo a região N-terminal de PspA das famílias 1 e 2, e proteínas híbridas - contendo fusões destes fragmentos. Os anticorpos gerados contra os híbridos reconheceram PspAs nativas das duas famílias, foram capazes de se ligar a bactérias íntegras, e de aumentar a deposição de complemento em sua superfície. Finalmente, a imunização de camundongos com os híbridos foi capaz de proteger contra desafio com pneumococos contendo PspAs diversas, mostrando que estes seriam candidatos promissores na composição de uma vacina anti-pneumocócica. / Streptococcus pneumoniae is an important cause of pneumonia, meningitis and septicaemia. The high cost and limited coverage of the available conjugate vaccine reinforce the need for cost effective strategies, with broader coverage. Pneumococcal Surface Protein A (PspA) is immunogenic and protective in animal models; however, due to its diversity - there are six clades and threee families of PspA - a PspA based vaccine should include fragments of each major family (1 and 2). In the present study, we have produced fragments of the N-terminal region of PspAs families 1 and 2, and hybrid proteins - containing fusions of these fragments. Sera made against the hybrids induced antibodies that recognized PspAs from both families; these sera were also able to bind pneumococcal strains bearing diverse PspAs, and to increase complement deposition on their surface. Finally, immunization of mice with PspA hybrids was protective against challenge with pneumococci bearing diverse PspAs, showing that these hybrids should constitute promising candidates in an anti-pneumococcal vaccine.

A aterosclerose é um processo sistêmico crônico, caracterizado pela presença de lesões, espessamento da parede arterial com acúmulo de lipídeos, e resposta inflamatória e fibroproliferativa. Muitos processos associados à oxidação da LDL, ao aumento de expressão de fatores prótrombóticos, moléculas de adesão próinflamatórias, citocinas e fatores quimiotáticos estão envolvidos na fisiopatologia da doença aterosclerótica. Trabalhos anteriores mostraram a presença de Mycoplasma pneumoniae (Mic) em placas lipídicas vulneráveis, associada à liberação de micropartículas (MPs) capazes de interagir com o ambiente e influenciar o processo aterogênico. Assim, a H&S Ciência e Biotecnologia, desenvolveu o complexo PTCTS que tem em sua composição nanopartículas naturais provenientes de plantas medicinais (PTC) associadas à enzima transialidase (TS) para combater as MPs e o Mic, além de ter efeito antilipemiante e antiaterosclerótico. O presente estudo teve como objetivo verificar os efeitos dos diferentes componentes do PTCTS administrados por via oral em relação à eficácia, toxicidade e mecanismo de ação na terapia antiaterosclerótica em coelhos hipercolesterolêmicos. Foram estudados coelhos da raça Nova Zelândia separados randomicamente em 6 grupos de tratamento: Controle Negativo (CNeg n=6), Controle Positivo (CPos n=6), Partículas orgânicas (PTC n=6), Transialidase (TS n=5), Partículas orgânicas associadas à transialidase (PTCTS n=6), e PTCTS com adição de PDTC (n=6). Os animais foram alimentados com dieta enriquecida com colesterol por doze semanas, exceto o CNeg, e receberam seus respectivos tratamentos durante as últimas seis semanas, concomitantemente à dieta hipercolesterolêmica. Tiveram sangue coletado durante as fases basal, pré e pós tratamento, para avaliação bioquímica do perfil lipídico, transaminases hepáticas e quantificação de MPs associadas a antígenos de Mic e LDLox no soro. Ao fim do experimento, foram sacrificados, e tiveram a aorta colhida em conjunto com o monobloco de órgãos para análises morfométrica, histológica, e imunohistoquímica, de forma a verificarmos os efeitos do PTCTS e seus compostos. O composto PTCTS via oral se mostrou eficaz quanto a eliminação de MPs associadas tanto a antígenos de Mic como de LDLox, e apesar de não ter tido os efeitos antiateroscleróticos e antilipemiantes esperados, reduziu não significativamente a área de placa na porção abdominal da aorta, induziu a aorta ao remodelamento positivo, permitindo um fluxo livre mesmo com uma grande área de placa, e evitando assim a obstrução do vaso. Seus componentes, quando administrados isoladamente tiveram efeitos parciais, as PTC sobre o remodelamento do vaso e redução de placa na aorta abdominal, e a TS sobre a redução do colesterol total, comprovando que a associação de ambos possui atividade mais completa e balanceada. A adição do PDTC ao complexo levou ao efeito oposto, ao inibir a atividade de eliminação de MPs apresentada pelo PTCTS, bem como ao induzir remodelamento negativo dos vasos. Nenhum dos componentes apresentou toxicidade nos órgãos estudados. Os dados encontrados neste trabalho experimental reforçam a teoria infecciosa na patogenia da aterosclerose, bem como a de que MPs originadas de tais microorganismos possam estar induzindo a produção de radicais livres e influenciando a oxidação de lípides, de forma que a remoção de tais patógenos do sangue circulante pode ser um novo caminho terapêutico no tratamento da aterosclerose / Atherosclerosis is a chronic systemic process, characterized by lesions, thickening of the arterial wall with accumulation of lipids, inflammatory and fibroproliferative response. Many processes associated with the oxidation of LDL, increase expression of prothrombotic factors, adhesion molecules, proinflammatory cytokines and chemotactic factors are involved in the pathophysiology of atherosclerosis. Previous works have shown the presence of Mycoplasma pneumoniae (Mic) in vulnerable lipid plaques, associated with the release of microparticles (MPs) able to interact with the environment and influence the atherogenic process. Thus, H&S Science and Biotechnology developed the PTCTS complex, which has in its composition natural nanoparticles from medicinal plants (PTC) associated with transialidase enzyme (TS), to combat MPs and Mic, that together have antilipemiante and antiaterosclerotic effects. This study aimed to determine the effects of different components of PTCTS administered orally regarding the efficacy, toxicity and mechanism of action in antiatherosclerotic therapy in hypercholesterolemic rabbits. It was studied White New Zealand rabbits separated randomly into six treatment groups: Negative Control (CNeg n=6), Positive Control (CPos n=6), organic particles (PTC n=6), transialidase (TS n=5) organic particles attached to transialidase (PTCTS n=6) and PTCTS with addition of PDTC (n=6). The animals were fed with a diet enriched with cholesterol for twelve weeks, except the CNeg, and received their treatments during the last six weeks, concurrently with hypercholesterolemic diet. Blood samples were taken during the baseline, pre and post treatment phases for biochemical evaluation of the lipid profile, liver transaminases and quantification of MPs associated with Mic antigens and oxLDL in the serum. At the end of the experiment they were sacrificed and the aorta was harvested together with the monoblock of organs for morphometric analysis, histology and immunohistochemistry, so we could check the effects of PTCTS and their compounds. The compound PTCTS orally is effective at removing MPs associated with both Mic and oxLDL antigens, and despite not having had the expected antiatherosclerotic and antilipemiantes effects, reduced not significantly plaque area in the abdominal portion of aorta, and induced its ascendant portion to positive remodeling, allowing a free flow even with large plate area, avoiding obstruction of the vessel. Its components when administered alone had partial effects, PTC on the remodeling of the vessel and plaque reduction in the abdominal aorta, and TS on the reduction of total cholesterol, proving that the combination of both has more complete and balanced activity. The addition of PDTC to the complex led to the opposite effect to inhibit MPs scavenging activity displayed by PTCTS as well as to induce negative remodeling of vessels. None of the components presented toxicity in the organs studied. The data found in experimental work reinforce the infectious theory on the pathogenesis of atherosclerosis as well as that originating MPs such microorganisms may be inducing the production of free radicals and oxidation of lipids, so that the removal of such pathogens from circulating blood may be a new therapeutic approach in the treatment of atherosclerosis

Fatores de risco clássicos descritos como associados ao desenvolvimento da aterosclerose nem sempre conseguem explicar uma variabilidade da evolução da doença entre um indivíduo e outro. Evidências consistentes mostram que a inflamação exerce papel crucial na patogenia da aterosclerose. Recentemente, a infecção por alguns agentes, principalmente a C. pneumoniae, tem sido progressivamente aventada como possível fator colaborador no processo de aterogênese. O Mycoplasma pneumoniae, bactéria que utiliza colesterol para sua proliferação, foi descrito no interior de placas gordurosas humanas, conjuntamente com a C.pneumoniae. Objetivo - O presente estudo tem como objetivo verificar se antígenos dos agentes infecciosos M. pneumoniae e C. pneumoniae estão presentes na parede das artérias aortas normais dos coelhos, animal que tem sido freqüentemente utilizado no estudo da aterosclerose, e se a introdução de dieta rica em colesterol leva a proliferação desses microorganismos concomitante com o desenvolvimento da placa aterosclerótica e de inflamação. Material e Métodos - Foram estudados 39 coelhos da raça Nova Zelândia, divididos em três grupos conforme a alimentação: Grupo A - dieta normal , Grupo B - dieta com 1% de colesterol por 8 semanas e Grupo C - dieta com 1% de colesterol por 12 semanas. O colesterol total e frações foram dosados bioquimicamente, após sacrifício dos animais. Avaliou-se macroscopicamente a quantidade de placas de gordura na superfície da aorta através da coloração pelo Sudan IV e microscopicamente dois segmentos transversais: um torácico e um abdominal os quais foram submetidos a reações de Imunohistoquímica para antígenos infecciosos. Resultados - Antígenos da C. pneumoniae e M. pneumoniae estiveram presentes em todos os animais estudados, porém em quantidades progressivamente maiores nos grupos A, B e C. Houve forte correlação positiva entre a quantidade de antígenos infecciosos e espessura da camada íntima, inflamação e quantidade de gordura, à histologia tanto no segmento torácico quanto no abdominal. Houve correlação negativa entre perímetro do vaso e tamanho das placas no segmento torácico, cujas placas apresentaram maior porcentagem de fibrose comparadas com o segmento abdominal, indicando que as placas dessa região apresentam mais características evolutivas para placa estável, ou seja, remodelamento negativo do vaso e capa de fibrose. A intensidade da inflamação tanto na adventícia quanto na íntima se correlacionou positivamente à quantidade de ambos os patógenos reforçando a hipótese de que esses patógenos estão influenciando no desencadeamento da inflamação e desenvolvimento de aterosclerose. Os níveis plasmáticos de colesterol total e suas frações também se correlacionaram positivamente com os agentes infecciosos. Em conclusão - A alimentação rica em colesterol levou a aumento da intensidade de antígenos de Mycoplasma pneumoniae e C. pneumoniae presentes na íntima da aorta de coelhos, em correlação com aumento da espessura intimal e inflamação na parede do vaso, favorecendo de forma intensa a hipótese de que esses agentes têm papel importante na aterogênese. A presença desses microorganismos em pequena quantidade nos animais controles reforça a idéia de que esses microorganismos são habitantes usuais de mamíferos e que fatores de risco tais como o colesterol induzem sua proliferação e o desenvolvimento da aterosclerose / Classical risk factors described to be associated with the development of atherosclerosis do not completely explain the variability of the disease occurring among the individuals. Consistent evidences show that inflammation exerts a fundamental role in the pathogenesis and severity of atherosclerosis. Recentently, some infectious agents, specially the Chlamydophila pneumoniae (C.pneumoniae), have progressively been claimed as possible factor acting in the progression of atherosclerosis. Mycoplasma pneumoniae (M.pneumoniae), a bacterium that needs cholesterol for proliferation, has been described in human fat plaques, in conjunction with C.pneumoniae. Objective - The present study has the objective of analyzing if M. pneumoniae and C. pneumoniae antigens are present at the rabbit aorta arterial walls, as the rabbit is an animal model frequently used in studies of atherosclerosis; and if the cholesterol enriched diet may cause proliferation of these microorganisms concomitantly with the development of atherosclerotic plaques and inflammation. Material and Methods - 39 aortas from New Zealand rabbits divided in three groups according to the diet were studied: Group A -normal dieta, Group B - 1% cholesterol enriched diet for 8 weeks and Group C - 1% cholesterol enriched diet for 12 weeks. The serum levels of total and fractions of cholesterol were biochemically measured after animals killed. A macroscopic study of the percentage area occupied by fat at the intimal aorta surface was performed, using the Sudan IV stain. A microscopic study was performed in two transversal segments: a thoracic and an abdominal one, which were submitted to immunohistochemistry for detection of these infectious agents. Results - C. pneumoniae and M. pneumoniae Ags were present in all studied animals, however in progressively higher amounts in groups A, B and C. There was a strong positive correlation between microscopic amounts of infectious antigens and intimal thickness, inflammation and intraplaque fat, both at the thoracic and abdominal segments. There was a negative correlation between vessel perimeter and plaque area at the thoracic segment, where the plaques presented higher percentage of fibrosis compared with the abdominal segment, suggesting that the thoracic region plaques have characteristics for progression to estabilization: negative remodeling of the vessel and fibrous cap. The quantity of Inflammation at both intima and adventitia correlated positively with these infectious agents, re-inforcing the hypothesis that infectious antigens are inducing inflammation and development of atherosclerosis. Serum levels of total cholesterol and their fractions also positively correlated with the amount of infectious antigens. In conclusion - A rich cholesterol diet caused an increase of M. pneumoniae and C.pneumoniae antigens at intima of rabbit aortas, in strong positive correlation with intimal thickness and inflammation of the aorta wall, favoring the hypothesis that these infectious agents have important role in atherogenesis. The presence of small amount of these microorganisms\' antigens at the controls re-inforces the Idea that these microorganisms are usual inhabitants of mammals and that the risk factors such as cholesterol induce their proliferation and acceleration of atherosclerogenesis.

As infecções pneumocócicas são uma importante causa de morbi-mortalidade entre as crianças. Até 2000, era disponível apenas a vacina pneumocócica polissacarídica 23valente, de uso a partir dos 2 anos de idade. Essa vacina era recomendada para crianças com fibrose cística (FC) e para as asmáticas em uso de corticóide oral, dentre outras recomendações. A partir de 2000, licenciou-se a vacina pneumocócica conjugada 7valente, com grande impacto contra infecções causadas pelos sorotipos vacinais. Nos países onde as crianças não são universalmente vacinadas com essa vacina, as recomendações permanecem as mesmas. Atualmente, os adultos asmáticos estão incluídos nas recomendações para vacinação pneumocócica nos EUA. Há poucos estudos sobre o risco de doença pneumocócica em crianças asmáticas per si e naquelas com fibrose cística e sobre a resposta à vacina pneumocócica conjugada. Salienta-se que ainda não há um critério estabelecido para avaliar a resposta sorológica a essa vacina. Recentemente, foi sugerido o critério de 0,35mcg/ml por Elisa para se correlacionar com proteção para doença invasiva pneumocócica. Objetivou-se determinar a concentração dos anticorpos contra os sorotipos vacinais contidos na vacina pneumocócica conjugada 7valente em crianças com asma moderada em uso de corticóide inalatório e em crianças com fibrose cística; avaliando-as pelos critérios de 0,35mcg/ml, 1,3mcg/ml e aumento de 4 vezes o título pós em relação ao pré-vacinal, para cada sorotipo e para a vacina, considerando-se a positividade para 5 sorotipos. Foram avaliadas 18 crianças em cada grupo. A mediana da idade foi de 82,5m nas asmáticas e 69,5m naquelas com FC. Foi colhida amostra para sorologia pré-vacinação e outra após 2 doses da vacina conjugada. As concentrações de anticorpos para os sorotipos vacinais foram quantificadas pelo Elisa. Para 0,35mcg/ml de corte, a grande maioria nos dois grupos já era positiva à inclusão para os sorotipos vacinais e à vacina. Considerando-se o valor de 1,3mcg/ml, entre os que eram negativos, as crianças asmáticas responderam entre 66,7% (9V) e 100% (14), e as com FC, entre 50% (19F) e 100% (6B e 14); e, em relação à resposta vacinal para esse nível, as asmáticas apresentaram 81,8% de resposta, enquanto as com FC, 91,7%. Avaliando-se pelo aumento de 4 vezes o título pós em relação ao pré-vacinal, a melhor resposta aos sorotipos, nos asmáticos, foi de 33,3% (4, 6B, 14 e 18C), e a nos com FC, 61,1% para o 6B; em termos de resposta vacinal, obteve-se 16,7% e 44,4%, para as asmáticas e aquelas com FC, respectivamente. Não houve interferência da vacinação prévia com a vacina pneumocócica polissacarídica. As medianas dos títulos pós em relação aos pré-vacinais, para os sorotipos, nos dois grupos, apresentaram um aumento significante. Apesar de boa parte das crianças apresentarem uma positividade elevada à inclusão, aquelas que eram negativas tenderam a apresentar uma boa resposta à vacina. / Pneumococcal infections are an important morbi-mortality cause among children. Until 2000, it was only available the 23-valent polysaccharide pneumococcal vaccine for children over two years old. This vaccine was recommended for cystic fibrosis (CF) children and to asthmatics children in use of oral corticosteroids, among other recommendations. From 2000, it was licensed the 7-valent conjugated pneumococcal vaccine, with a great impact against the infections caused by the vaccine serotypes. In the countries that dont make a universally use of this vaccine for children, the recommendations remain the same. At the present time, asthmatic adults are included for the pneumococcal vaccine recommendations in the United States. There are few studies about pneumococcal disease risk with cystic fibrosis children and asthmatics, per si, and about the conjugated pneumococcal vaccine response. It points out that there are no a definitive criteria or evaluation established for the serology response for this vaccine. It was suggested, recently, that the level of 0,35mcg/ml, measured by ELISA, is adequate to correlate with the invasive pneumococcal disease protection. The goal of this study was to determine the antibodies concentration of the seven vaccine serotypes from 7-valent conjugated pneumococcal vaccine among children with moderate asthma in use of inhaled corticosteroids and with cystic fibrosis. It was considered the dosage 0,35mcg/ml and 1,3mcg/ml levels and the four-fold increase between pre- and post-immunization concentrations levels, to each serotype and to the vaccine (positivity for five serotypes or more) for positivity. Eighteen children were included in each study group. The age median was 82,5 months for the asthmatics and 69,5 months for the CF children. A blood sample was taken for pre-immunization serology and a second one after the second vaccine dose was given. The antibodies concentrations for the vaccine serotypes were measured by ELISA. Considering the 0,35mcg/ml levels, the majority of children, in both groups, was positive for vaccine serotypes and for the vaccine as well in the beginning. At the 1,3mcg/ml level, among the children with negative serology, asthmatic children responded between 66,7% (9V) and 100% (14), and those with CF, between 50% (19F) and 100% (6B e 14). Related to the vaccine response for this level, the asthmatics had a 81,8% response, while the CF childrens response was 91,7%. Evaluating for the four-fold increase between pre- and post-immunization concentrations, the best response observed for the vaccine serotypes was 33,3% (4, 6B, 14 e 18C) for the asthmatics. In the CF group the best result was 61,1% (6B). In terms of the vaccine response, it was observed that 16,7% and 44,4% were the results for both the asthmatics and CF group, respectively. The polysaccharide vaccine didnt interfere in the results. The medians of the pre- and post-immunization antibodies concentrations for the vaccine serotypes, in both groups, were significantly increased. Despite those children that were already positive for the criteria evaluated, at the first moment of the study, for those children that were negative, the majority had a positive serology towards the vaccination response.

Das hochadaptierte, zellwandlose Bakterium Mycoplasma pneumoniae gehört zu den häufigsten Erregern von ambulant erworbenen Pneumonien. Während den alle 3-7 Jahre auftretenden Epidemien könnten gefährdete Personengruppen bei Verfügbarkeit einer effektiven Vakzinierung vor einer Infektion geschützt werden. Ziel dieser Arbeit war die Erarbeitung von Grundlagen zum Aufbau einer schützenden Immunantwort durch Immunisierung mit rekombinant hergestellten Teilbereichen verschiedener Adhärenzproteine von M. pneumoniae. Da die bereits bekannten Adhäsine des Erregers einerseits unverzichtbare Virulenzfaktoren darstellen und andererseits als dominante Immunogene des Bakteriums beschrieben wurden, war die Feincharakterisierung dieser Proteine im Hinblick auf ihre Eignung als Vakzinekandidat Ausgangspunkt der Untersuchungen. Definierte Teilbereiche des Hauptadhäsins P1 und weiterer adhärenzassoziierter Proteine wurden rekombinant hergestellt und auf ihre Antigenität qualitativ und quantitativ mit Seren experimentell infizierter Tiere und Seren von Patienten mit bestätigter M. pneumoniae-Infektion geprüft. Dabei gingen der C-terminale Bereich des P1-Adhäsins (RP14; AS 1287-1518) und das Protein P30 (RP30; AS 17-274) als hoch antigene Protein(regionen) hervor. Die Oberflächenexposition beider Proteinbereiche wurde experimentell bestätigt. Parallel dazu sollte der Einfluss der monospezifischen Seren gegen die rekombinanten Proteine auf die Adhärenz von M. pneumoniae an verschiedene Zelllinien bewertet werden. Nach Entwicklung eines quantitativen und schnell durchzuführenden in vitro Adhärenzhemmtests wurde gezeigt, dass Antikörper sowohl gegen RP14 als auch gegen RP30 die Adhärenz des Erregers an humane Zellen signifikant reduzieren. Im Hinblick auf die Beeinflussung von mehr als einer an der Adhärenz beteiligten Struktur wurden beide Adhäsinregionen in einem Hybridprotein vereinigt, wodurch die adhärenzhemmende Wirkung des entsprechenden Serums in vitro mehr als additiv verstärkt werden konnte und dem Effekt des polyspezifischen Serums gegen M. pneumoniae entspricht. In Immunisierungs- und Infektionsversuchen mit dem Hybridprotein konnte über eine neu etablierte sehr sensitive real-time PCR basierend auf einer Multicopy-Targetsequenz in M. pneumoniae jedoch nur in einem Teil der Versuchstiere eine signifikante Hemmung der Kolonisierung des Respirationstraktes mit dem Erreger nachgewiesen werden. Eine Steigerung der humoralen Immunantwort durch Zugabe des mukosalen Adjuvans MALP-2 konnte nicht ausreichend belegt werden. Dagegen wurde durch Immunisierung auf intranasalem Wege eine signifikante Erhöhung des sekretorischen IgA-Titers in der BAL auf das Vierfache erreicht. Um einen stabil hohen sekretorischen Antikörperspiegel im Tiermodell zu erreichen, sind weitere Optimierungen des Immunisierungsschemas notwendig. Der Einsatz des Hybridproteins HP14/30, bestehend aus mehreren immunogenen und funktionalen Adhäsinbereichen, hat sich als sehr vielsprechend zur Entwicklung einer effektiven Vakzinierung von Risikopopulationen zur Prophylaxe von Infektionen mit M. pneumoniae erwiesen.

La morphogénèse des ovocoques, dont fait partie le pathogène humain Streptococcus pneumoniae, implique des processus d’élongation et de division associés à la synthèse de la paroi bactérienne. Le composant majeur de cette paroi est le peptidoglycane, un polymère de sucre réticulé par des chaines peptidiques, qui confère la forme de la bactérie et est essentiel à sa survie. La synthèse de peptidoglycane nécessaire à l’élongation et la division bactérienne est effectuée par des complexes protéiques appelés respectivement « élongasome » et « divisome ». Les mécanismes d’assemblage et l’activité de ces complexes dans la cellule bactérienne restent encore non élucidés. Pour imager l’activité des complexes de synthèse du peptidoglycane in vivo à l’échelle du nanomètre, j’ai développé une méthode faisant appel à des dérivés de D-amino acides, à la chimie click et à la microscopie de localisation de molécules uniques (dSTORM ou direct Stochastic reconstruction microscopy). Cette méthode a permis d’obtenir des images à une résolution d’environ 20 nm, révélant des aspects inattendus de la synthèse du peptidoglycane et remettant en question le rôle de certaines protéines dans la morphogenèse du pneumocoque. En combinant ces observations avec les données de la littérature, un modèle simplifié de la morphogénèse des ovocoques est proposé. / The morphogenesis of ovovcocci, which include the human pathogen Streptococcus pneumoniae, involves elongation and division processes associated with cell wall synthesis. The main component of the cell wall is the peptidoglycan, a polymer made of glycan chains cross-linked by peptide chains, which confers the bacterial shape and is essential for cell survival. Peptidoglycan synthesis required for cell elongation and division is performed by large protein complexes called “elongasome” and “divisome”, respectively. The assembly mechanisms and activity of these complexes in the bacterial cell remain mysterious. To image the activity of the peptidoglycan synthesis complexes in vivo at the nanoscale, I developed a method combining D-amino acid derivatives, click chemistry and single-molecule localization microscopy (dSTORM or direct Stochastic reconstruction microscopy). This method allowed obtaining images at a resolution of about 20 nm resolution, revealing unexpected features of peptidoglycan synthesis and challenging the role of some proteins in pneumococcus morphogenesis. By combining these observations with data from the literature, a simplified model of ovococci morphogenesis is proposed.

Lower respiratory tract infectious diseases are a world-wide healthcare burden with bacterial pathogens accounting for a large portion of primary and secondary infections. The human respiratory tract is home to hundreds of species of microbes that comprise the human airway microbiome. These commensals play a crucial role in human health in part by providing colonization resistance against pathogens. In a previous study from the Surette lab it was shown that specific bacterial isolates from the respiratory microbiome inhibits the growth of pathogens aerobically. This included an isolate of Staphylococcus aureus which inhibited the growth of Enterococcus faecium. This activity was further characterized in this thesis and the underlying mechanism was explored through comparative genomics. As well, this observation provided proof-of-concept for a large-scale screen for additional isolates which inhibit pathogen growth. I hypothesized that the respiratory tract microbiota included many other bacteria capable of inhibiting the growth of respiratory tract pathogens in both aerobic and anaerobic environments, and that anaerobic conditions will identify new activities not detected aerobically. To examine and identify potential beneficial bacteria, I have screened ~5000 respiratory tract bacteria from the Surette lab’s airway isolate collection against four pathogens: Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. The respiratory tract commensals were pinned onto the pathogen-lawn and their interaction was expressed as zones of clearing or altered growth phenotypes of the pathogen. The results of the screen showed that anti-pathogen activity was a common feature of respiratory tract commensals. In particular, S. pneumoniae was inhibited by taxonomically diverse members of the microbiota representing three phyla (Proteobacteria, Firmicutes and Actinobacteria). Many of the facultative anaerobes that inhibited S. pneumoniae expressed their activity in anerobic conditions. / Thesis / Master of Science (MSc) / The human respiratory tract harbours commensal and pathogenic bacteria, and the latter cause most of the lower respiratory tract infections. The commensal bacteria help to train the immune system and impede the growth of pathogens through colonization resistance. A previous study by the Surette lab identified bacterial isolates from the respiratory tract that inhibit the growth of select pathogens, among them, a particular strain of Staphylococcus aureus. Based on the results of the earlier study, I hypothesized that the respiratory tract bacteria is a good source of commensals that can inhibit the growth of S. aureus and other respiratory pathogens, such as Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae. To find potential therapeutic bacteria, I screened ~5000 respiratory tract isolates from the Surette lab’s strain collection for the ability to impair growth of target pathogens. Additionally, I further characterized the activity of the previously identified S. aureus strain against various Lactobacillalles strains and used comparative genomics to identify potential biosynthetic genes required for biosynthesis of molecules with antibacterial activity within the genome of S. aureus. The research reported in this thesis demonstrates that many commensal bacteria that live within our airways have the ability to inhibit the growth of bacterial pathogens. This work may provide a new source of antibiotics against respiratory infections and new strategies to reduce susceptibility to infections in vulnerable populations.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2018-01-22T12:47:14Z No. of bitstreams: 1 PATRICIA VOLLÚ SILVA.PDF: 2575547 bytes, checksum: c2489f8abe58d46ea1c2ba8977ba02e1 (MD5) / Made available in DSpace on 2018-01-22T12:47:14Z (GMT). No. of bitstreams: 1 PATRICIA VOLLÚ SILVA.PDF: 2575547 bytes, checksum: c2489f8abe58d46ea1c2ba8977ba02e1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A ilha de patogenicidade pks de 54 kb é responsável pela produção de colibactin, uma molécula genotóxica, que causa rupturas de fita dupla de DNA (DSB) das células hospedeiras, e que parece estar relacionada ao aumento do risco de desenvolvimento de câncer colorretal. Colibactin foi inicialmente descrita em Escherichia coli, mas também pode ser encontrada em outras enterobactérias, como Klebsiella pneumoniae. A biossíntese de colibactin requer a atividade enzimática da fosfopantoteinil transferase (PPTase) ClbA, que também pode contribuir para a produção dos sideróforos enterobactin e yersiniabactin em E. coli. O objetivo deste trabalho foi avaliar a virulência em amostras de E. coli e de K. pneumoniae, presença da ilha de patogenicidade pks e determinar o papel de colibactin e do sideróforo enterobactin em infecções por K. pneumoniae. Para tal, uma coleção de 218 amostras de E. coli isoladas de pacientes com infecção do trato urinário atendidas no Instituto Nacional do Câncer, Brasil e 258 amostras de K. pneumoniae isoladas de sangue de pacientes internados no Centre Hospitalier Universitaire de Toulouse, França foram avaliadas. A tipificação filogenética e a presença do gene de virulência fyuA em E. coli e do gene clbN em ambas bactérias foram avaliadas por PCR. O fenótipo hipermucoviscoso (HMV) de K. pneumoniae foi determinado pelo teste string. Foi ainda investigado o papel de colibactin e enterobactin em um modelo de pneumonia em camundongos C57BL/6JRJ e em infecções de células HeLa, utilizando a cepa K. pneumoniae SB 4496 e mutantes isogênicos deficientes em clbA, clbN ou entD. Os resultados demonstraram que entre as amostras de E. coli pesquisadas, o grupo filogenético B2 foi o mais prevalente (44,9%), seguido pelos grupos filogenéticos A (13,8%), B1 (22,0%) e D (19,3%). O gene fyuA, relacionado a produção do sideróforo yersiniabactin, foi detectado em todos os grupos filogenéticos, no entanto, a detecção do referido gene foi mais frequente no grupo B2 (P = 0,0228), sendo detectado em 74,5% das amostras deste grupo. O gene clbN, relacionado a produção de colibactin, foi detectado em 14,7% das amostras de E. coli. Vale ressaltar que todas as amostras clbN positivas pertenciam ao grupo filogenético B2, as quais também portavam o gene fyuA. Adicionalmente, três amostras de E. coli apresentaram efeito citopático em células HeLa, independente da produção de colibactin e da presença dos genes hlyC/A, hlyF, cdt e cnf1. O gene clbN foi detectado em 5,8% das amostras de K. pneumoniae. Também foi observado que a detecção de clbN foi estatisticamente significantemente (P < 0,0001) entre as amostras caracterizadas como HMV, este gene foi observado em 35% das amostras HMV analisadas. Além disso, a síntese de colibactin não pôde ser mantida pela PPTase EntD e a produção de sideróforos pela K. pneumoniae SB 4496 foi continuada por outro sistema independente de PPTases EntD e ClbA. Os resultados obtidos neste trabalho parecem indicar que a produção de colibactin não afeta a sobrevivência de K. pneumoniae hipervirulenta (hvKP) nos tecidos pulmonares de camundongos C57BL/6JRJ, nas condições estudadas. No entanto, é importante salientar que a toxina colibactin produzida por hvKP é capaz de induzir genotoxicidade em células HeLa. Ambos os genes clbA e clbN foram necessários para a manutenção da megalocitose e DBS induzida por colibactin em K. pneumoniae / The 54-kb pks pathogenicity island produces a genotoxic molecule named colibactin, which causes double-strand DNA breaks (DSB) in the host cells and enhanced colorectal cancer development. Colibactin was initially described in Escherichia coli, but can be found in other enterobacteria, including Klebsiella pneumoniaE. colibactin biosynthesis requires the enzymatic activity of phosphopantetheinyl transferase (PPTase) ClbA, which may also support the enterobactin and yersiniabactin siderophores synthesis in E. coli. The goal of this work was to evaluate the virulence of E. coli and K. pneumoniae isolates, presence of pks pathogenicity island and to determine the role of colibactin and enterobactin siderophore in K. pneumoniae infections. For this purpose, it was evaluated a collection of 218 E. coli isolates obtained from patients with urinary tract infection assisted at Instituto Nacional do Câncer, Brazil, and 258 K. pneumoniae isolates collected from blood samples from patients admitted to the Centre Hospitalier Universitaire in Toulouse, France. Phylogenetic typing and the detection of fyuA virulence gene in E. coli and clbN gene in both bacteria were assessed by PCR. K. pneumoniae hypermucoviscous (HMV) phenotype was determined by the string test. The role of colibactin and enterobactin in a C57BL/6JRJ mice pneumonia model and in HeLa cells infection was investigated using K. pneumoniae SB 4496 strain and isogenic mutants deficient in clbA, clbN or entD. Among the E. coli isolates, the phylogenetic group B2 was more prevalent (44.9%) followed by phylogroups A (13.8%), B1 (22.0%) and D (19.3%). The fyuA gene, involved in the yersiniabactin production, was detected in all phylogenetic groups studied; however, its presence was more frequently detected among the phylogroup B2 (P = 0.0228), with a high percentage of 74.5%. The clbN gene, associated to colibactin production, was detected in 14.7% of E. coli isolates. It is noteworthy that all clbN positive isolates belong to the phylogroup B2 and carry the fyuA gene. In addition, three E. coli isolates studied showed cytopathic effect in HeLa cells independently of colibactin production and the presence of hlyC/A, hlyF, cdt and cnf1 genes. Regarding K. pneumoniae, clbN gene was detected in 5.8% of the isolates. It was also observed that this detection was statistically significant (P < 0.0001) among the isolates of the HMV phenotype group, being this gene observed in 35% of the HMV isolates analyzed. In addition, it was observed that colibactin synthesis could not be maintained by the PPTase EntD. Indeed, siderophores production by K. pneumoniae SB 4496 was successful continued by another system that does not require the activity of PPTases EntD and ClbA. Results obtained in this work seem to indicate that production of colibactin does not affect the survival of hypervirulent K. pneumoniae (hvKP) in C57BL/6JRJ lung mice. Despite that, it is interesting that colibactin produced by hvKP is capable of inducing genotoxicity in HeLa cells. Both clbA and clbN genes were required for the maintenance of megalocytosis and DBS induced by colibactin in K. pneumoniae

Toll-like receptors (TLRs), named after toll proteins identified in Drosophila melanogaster, are the pattern recognition receptors in the innate immune system that detect microbes. TLRs are mono, membrane-spanning, as well as non-catalytic receptors, which are mainly expressed in sentinel cells, such as the dendritic cells, neutrophils and macrophages. While humans have ten TLRs (TLR 1 to 10), the mouse has another three (TLRs 11, 12, 13). TLRs are made up of glycoproteins, which have luminal ligand-binding sites consisting of leucine-rich repeat (LRR) for detection of pathogens leading to activation of immune cells. TLR1, 2, 4, and 6 are responsible for recognition of lipids (such as triacetylated lipopeptide), peptidoglycan, and lipopolysaccharide (LPS). However, the TLR3, 7, 8, and 9 mainly recognize nucleic acids, such as double-stranded RNA (dsRNA) and CpG DNA, while the TLR13 detects ribosomal RNA sequences. So far, there are no data on the localization and immunological functions of TLR10. I studied the expression, localization and role of TLR10 in S. pneumoniae infection. First, I examined the expression of TLR10 in lungs of pig, cattle, dog, rat, and chickens. The light and electron microscopic data show TLR10 expression in vascular endothelium and smooth muscles in lungs of control and inflamed animals. Further, we found altered basal level of expression and localization of TLR10 in bovine neutrophils treated with E. coli lipopolysaccharide. These data show the expression of TLR10 in the lungs of tested animal species, and its alteration by LPS in bovine neutrophils. The next study was designed to investigate the regulation of TLR10 expression and to address its role in neutrophil chemotaxis. E. coli LPS activated human neutrophils showed temporal and spatial change in TLR10 expression. Confocal microscopy showed cytosolic and nuclear distribution of TLR10 in normal and activated neutrophils. TLR10 in E. coli LPS-activated neutrophils colocalized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, suggested its endocytosis. Live cell imaging of LPS activated neutrophils showed TLR10 translocation to the leading edge. Neutrophils upon TLR10 knockdown were unable for fMLP-induced migration. TLR10 knockdown reduced the number of membrane pseudopods in activated neutrophils without altering the expression of key proteins of actin nucleation process, ARP-3 and Diap1. These data show TLR4-mediated pathway for regulation of TLR10 expression, and that TLR10 may have a role in neutrophil chemotaxis. Next, I examined the role of TLR10 in innate immune response to S. pneumoniae infection in U937 human macrophage cell line. S. pneumoniae are major causative agents of pneumonia, meningitis and bacteremia. A significant increase in TLR10 mRNA expression was found in S. pneumoniae (107 cfu for 6hr) challenged macrophages. TLR10 knockdown significantly reduced production of IL-1β, IL-8, IL-17 and TNF-α and no significant change in IL-10 expression, and also significantly diminished nuclear translocation of NF-κB but without affecting the phagocytosis of S. pneumoniae. Altogether, I report the that TLR10 is expressed in the normal and inflamed lungs in cattle, pigs, dogs, rats, chickens and humans. The expression of TLR10 is altered in activated neutrophils, and it plays a role in neutrophils chemotaxis and production of pro-inflammatory cytokines in macrophages infected with S. pneumoniae.

Escherichia coli and Klebsiella pneumoniae cause a wide range of infections. Multidrug-resistance strains carrying extended-spectrum β-lactamases (ESBLs) has become a growing problem worldwide. The CTX-M type ESBLs has emerged distinctly, especially in Escherichia coli and Klebsiella pneumoniae. CTX-M type has been associated with many outbreaks of infections both in the hospitals and community. CTX-M-15 is now identified as the most predominantly distributed CTX-M enzyme. Clonal outbreaks of CTX-M-15 producing Enterobacteriaceae have been described in many countries including the United Kingdom, and Escherichia coli is the most commonly involved species. A total of 100 isolates were received in 2010 from London St George’s hospital, England, 50 Escherichia coli, 17 Klebsiella spp, 9 Enterobacter spp, 13 Proteus spp, 6 Lactose fermenting coliforms, 2 Pantoea spp, one Serratia marcescens, one Morganella morganii, and one Hafnia alvei. The antimicrobial susceptibility results showed that 5 Escherichia coli and one Klebsiella pneumoniae isolates were found to be resistance to cefotaxime, ceftazidime, ceftriaxone, cefotaxime, ciprofloxacin, and gentamicin, making them multi-drug resistant bacteria. None of the isolates showed resistance to imipenem, ertapenem, or morepenem, thus making carbapenems the drug of choice for the treatment of these infections due to multi-resistant isolates. The overall frequency of CTX-M-15 type ESBL-producers detected in this study was 6 (6%) most of them 5/6 (83%) were from Escherichia coli and one was (17%) Klebsiella pneumoniae isolates. The 6 CTX-M-positive isolates were typed by PFGE, only two strains of Escherichia coli showed more than 85% similarity, owing to clonal homology for both strains. The rest strains showed less than 85% similarity. S1 nuclease plasmid profiles were obtained for ESBL-producers isolates. A total of one to three plasmids per isolate, ranging from approximately 78.0 to 152.0 kb, were observed. The plasmids from most isolates were assigned to be IncFA and IncFB replicons. Analysis of phylogenetic groups showed group A and group B2. The method of phylogenetic classification of exteraintestinal pathogenic Escherichia coli depends on examine and combination of two preserved genes (chuaA and yjaA) and the DNA fragment TSP. Primer walking and PCR experiments were used for the genetic environment studies which showed 5 different genetic constructions for the described blaCTX-M-15 genes. Conjugation studies were used to detect the transferability of the plasmids harbouring the reported blaCTX-M-15 genes. Three isolates were found transferable by conjugation. In conclusion, this study reports the presence of hospital highly resistant blaCTX-M-15 in St George’s hospital. The spread of blaCTX-M-15 is probably due to horizontal gene transfer harbouring ISEcp1 and the conjugative properties of plasmids carrying blaCTX-M-15.