The work focuses on the significance of pneumolysin in contribution to virulence of Streptococcus pneumoniae serotype 1 isolates. Methods include bioinformatics as well as in vitro assays. A SNP within nucleotide sequence of the second virulence factor, hyaluronidase, is a subject for screen of meningitis isolates.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen and commensal organism of the nasopharynx. A major virulence factor of the pneumococcus is the cholesterol dependent, pore forming cytolysin pneumolysin. This toxin acts extracellularly, but the mechanism of release has not been well elucidated. Despite being a catalase negative organism, the pneumococcus produces up to millimolar concentrations of hydrogen peroxide through the activity of pyruvate oxidase. In all strains analyzed, deletion of the pyruvate oxidase gene yielded a significant reduction in the amount of PLY observed in the supernatant via western blot. A single strain, WU2 was also observed to have a significant (p<.05) reduction in the amount of PLY observed in the supernatant when treated with extracellular catalase. Furthermore, a significant correlation between hydrogen peroxide production and PLY release was observed in a panel of 15 clinical isolates.

In der vorliegenden Arbeit wurde die Auswirkung der Behandlung muriner Mikrogliazellen mit Agonisten von TLR2, TLR4 und TLR9 untersucht. Zu diesem Zweck wurden murine Mikrogliazellkulturen angelegt. Als TLR - Agonisten dienten Pam3Cys und HKAL (TLR2), Pneumolysin und LPS (TLR4) sowie CPG (TLR9). Die Stimulation muriner Mikrogliazellen mit den verschiedenen TLR - Agonisten führte zur Freisetzung von NO und TNF-α. Durch den Einsatz unterschiedlicher Konzentrationen der TLR - Agonisten konnten Dosis - Wirkungs - Kurven für die Freisetzung von NO und TNF-α erstellt werden. Anhand der EC50 wurde die Potenz der TLR - Liganden beurteilt. Für die Freisetzung von NO wies LPS die höchste stimulatorische Potenz auf, gefolgt von Pneumolysin, CpG und Pam3Cys. Für die TNF-α - Freisetzung besaß ebenfalls LPS die höchste stimulatorische Potenz, auch hier folgten Pneumolysin und CpG. Das verwendete Pam3Cys löste sich nicht optimal, vermutlich aus diesem Grund wurde durch die Pam3Cys - Gabe keine maximale Stimulation erreicht. Darum konnte die EC50 für die TNF-α - Freisetzung fur Pam3Cys nicht ermittelt werden. Die EC50 für die TNF-a - Freisetzung war jeweils höher als die entsprechende EC50 für die Freisetzung von NO. Die Behandlung mit HKAL führte zur starken NO - und TNF-a - Freisetzung. Ein direkter Vergleich der Potenz von HKAL mit der der anderen Liganden ist nicht möglich, da die Konzentration von HKAL in Zellzahl pro ml gemessen wird. Die Konzentrationen von Pam3Cys, Pneumolysin und LPS werden jedoch in µg/l gemessen.  Die Stimulation von Mikrogliazellen über verschiedene TLR hatte eine relativ gleich starke Sezernierung von NO und TNF-a zur Folge. Die Costimulation der Mikrogliazellen mit Konzentrationen von zwei unterschiedlichen TLR - Agonisten, welche allein jeweils zur maximalen NO - Produktion geführt hatten, resultierte nicht in einer weiteren Erhöhung der NO - Freisetzung.  Niedrig dosiert zeigte die Pneumolysinbehandlung einen immunstimulatorischen Effekt. Das Maximum an Stimulation, gemessen an der Zunahme der NO - Produktion, wurde bei einer Pneumolysin - Konzentration von 0,3 µg/ml (6nM) beobachtet. Eindeutige zytotoxische Effekte anhand der signifikant geringeren NO - Freisetzung waren bei Konzentrationen von 3 µg/ml bzw. 10 µg/ml (60 nM bzw. 200 nM) nachweisbar. Durch die Isolectin-B4 - Färbung wurden bei diesen Konzentrationen pneumolysinbedingte Zellschäden dargestellt. Bei den anderen Substanzen wurde in hohen Konzentrationen keine Zytotoxität beobachtet Die Behandlung TLR4 - defizienter Mikrogliazellen mit den spezifischen TLR4 - Agonisten Pneumolysin und LPS führte zu einer signifikant geringeren Freisetzung von NO im Vergleich zu Wildtypzellen. Schlussfolgernd kann gesagt werden: Die Stimulation von Mikrogliazellen über unterschiedliche TLR resultiert in einer relativ einheitlichen Freisetzung von NO und TNF-α. Die gleichzeitige Stimulation mit zwei jeweils niedrigdosierten TLR - Agonisten führt zu einem additiven oder supraadditiven Effekt. Nicht nur bakterielle Substanzen, sondern auch endogene Stoffe sind Agonisten an TLR - Rezeptoren. Der additive Effekt durch die simultane Stimulation mehrerer TLR erhöht nicht nur die Sensitivität von Mikroglia während Infektionen, sondern kann ebenfalls Wechselwirkungen zwischen exogenen und endogenen Agonisten von TLR zur Folge haben. Dies kann ein Grund für die Exazerbation oder Induktion autoimmuner Krankheiten durch Infektionen sein.

Streptococcus pneumoniae é responsável por milhões de mortes anuais no mundo todo. As vacinas atuais são baseadas em polissacarídeos capsulares e apresentam cobertura limitada. Assim, diversas proteínas tem sido estudadas como alternativas vacinais, dentre as quais se destacam PspA e PLY. Os objetivos deste estudo foram caracterizar produção de anticorpos e a resposta celular induzida pela imunização com vacinas híbridas PspA-PLY em modelo de pneumonia. Os anticorpos apresentaram elevada capacidade de ligação a pneumococos expressando PspAs de família 2, bem como um aumento na deposição de complemento nestes isolados. A imunização com as proteínas híbridas protegeu os camundongos do desafio sistêmico com pneumococo contendo PspA de família 2. Foi desenvolvido um modelo de pneumonia lobar em camundongos, onde a imunização com PspA-PLY induziu um aumento de IL-6 e TNF nos pulmões após o desafio, levando a uma redução no número de bactérias nos pulmões dos animais. Os dados sugerem que a fusão de PspA à PLY é capaz de ampliar a cobertura protetora a isolados expressando PspAs distintas, e que essa mesma vacina á capaz de proteger os animais contra pneumonia, pela indução precoce de citocinas como IL-6 e TNF-a nos pulmões. / Streptococcus pneumoniae is responsible for millions of deaths annually around the globe. The current vaccines are based on capsular polysaccharides, and have limited coverage. Thus, several proteins have been investigated as alternative vaccines, including PspA and PLY. The present study aimed at characterizing the antibody production and cellular immune response induced by mouse immunization with PspA-PLY hybrid vaccines in a pneumonia model. The induced antibodies demonstrated a strong recognition of pneumococci expressing family 2 PspA molecules, as well as an enhancement in complement deposition in their surface. Immunization with the fusion protein protected mice from systemic challenge with a pneumococcal isolate bearing a family 2 PspA. A model of lobar pneumonia was developed in BALB/c mice, where the immunization with the PspA-PLY fusion induced an increase in IL-6 and TNF-a production in the lungs after pneumococcal challenge, leading to a reduction on bacteria load in the lungs of immunized mice. The data suggests that fusion of PspA to PLY is capable of increasing vaccine coverage aginst pneumococcal infection with isolates bearing family 2 PspAs, and confer protection in mice against pneumonia, by the early production of IL-6 and TNF-a in the lungs.

Streptococcus pneumoniae é responsável por milhões de mortes anuais no mundo todo. As vacinas atuais são baseadas em polissacarídeos capsulares e apresentam cobertura limitada. Assim, diversas proteínas tem sido estudadas como alternativas vacinais, dentre as quais se destacam PspA e PLY. Os objetivos deste estudo foram caracterizar produção de anticorpos e a resposta celular induzida pela imunização com vacinas híbridas PspA-PLY em modelo de pneumonia. Os anticorpos apresentaram elevada capacidade de ligação a pneumococos expressando PspAs de família 2, bem como um aumento na deposição de complemento nestes isolados. A imunização com as proteínas híbridas protegeu os camundongos do desafio sistêmico com pneumococo contendo PspA de família 2. Foi desenvolvido um modelo de pneumonia lobar em camundongos, onde a imunização com PspA-PLY induziu um aumento de IL-6 e TNF nos pulmões após o desafio, levando a uma redução no número de bactérias nos pulmões dos animais. Os dados sugerem que a fusão de PspA à PLY é capaz de ampliar a cobertura protetora a isolados expressando PspAs distintas, e que essa mesma vacina á capaz de proteger os animais contra pneumonia, pela indução precoce de citocinas como IL-6 e TNF-a nos pulmões. / Streptococcus pneumoniae is responsible for millions of deaths annually around the globe. The current vaccines are based on capsular polysaccharides, and have limited coverage. Thus, several proteins have been investigated as alternative vaccines, including PspA and PLY. The present study aimed at characterizing the antibody production and cellular immune response induced by mouse immunization with PspA-PLY hybrid vaccines in a pneumonia model. The induced antibodies demonstrated a strong recognition of pneumococci expressing family 2 PspA molecules, as well as an enhancement in complement deposition in their surface. Immunization with the fusion protein protected mice from systemic challenge with a pneumococcal isolate bearing a family 2 PspA. A model of lobar pneumonia was developed in BALB/c mice, where the immunization with the PspA-PLY fusion induced an increase in IL-6 and TNF-a production in the lungs after pneumococcal challenge, leading to a reduction on bacteria load in the lungs of immunized mice. The data suggests that fusion of PspA to PLY is capable of increasing vaccine coverage aginst pneumococcal infection with isolates bearing family 2 PspAs, and confer protection in mice against pneumonia, by the early production of IL-6 and TNF-a in the lungs.

Abstract New rapid methods for sensitive and specific detection of pneumococci are not only needed to improve the diagnosis of pneumococcal disease but are also essential for vaccine and carriage studies. The purpose of this study was to develop sensitive PCR methods for the detection and quantification of S. pneumoniae and to study the applicability of these methods to detecting pneumococci in clinical samples. A previously described PCR method was first developed further by introducing a Europium-labelled hybridisation probe for the detection of amplification products. The hybridisation method was easy to use and improved the specificity of the PCR assay. The developed PCR assay was established as a sensitive method for detecting pneumococcal DNA when the presence of pneumococcal DNA in over 2500 middle ear fluid (MEF) samples of children with acute otitis media (AOM) was studied by using the method. Pneumococcal findings increased by 76% when using PCR detection in addition to culture, compared to using culture alone. However, the PCR-positive, culture-negative AOM events represented a less severe type of disease compared to the culture-positive events. A positive PCR finding seems to indicate the presence of viable, although often non-culturable pneumococci within the middle ear cleft. To be able to rapidly detect and quantify the initial numbers of pneumococcal genome copies in clinical samples, a real-time PCR method for the detection and quantification of pneumococcal DNA was developed. In real-time PCR, amplification and detection of amplification products occur simultaneously, which makes it possible to monitor the phase of the reaction at a particular stage or continuously. The method developed here was applied to the analysis of MEF samples and to investigating the nasopharyngeal carriage of pneumococcus. The sensitivities of bacterial culture and real-time PCR in detecting pneumococci were also compared. The real-time PCR assay was found to be rapid and sensitive and to provide information about the differences between the numbers of bacteria in samples. However, the quantitative results were shown to be dependent on the DNA extraction method applied. The real-time PCR method developed appears to be a good aid in research where an accurate and sensitive pneumococcal diagnosis is needed.

Abstract The purpose of this study was to develop sensitive and specific laboratory diagnostic methods for the demonstration of pneumococcal surface antigens or pneumococcus-specific antibodies in clinical samples. The work took account of epidemiological aspects of both pneumococcal disease and nasopharyngeal carriage of pneumococcus. We first compared the sensitivity of pneumococcal culture and antigen detection methods in nasopharyngeal samples in a developing country setting and then investigated the possibility of improving the sensitivity of the antigen detection by introducing an enrichment step in the procedure. — Further investigations were designed to determine the validity of pneumolysin-specific immune complex bound antibody assay as a tool for diagnosing pneumococcal ALRI in a developing country setting. Finally, we developed an enzyme immunoassay for the detection of pneumococcal capsular polysaccharide antigens, using type-specific antibodies produced in-house in rabbits through immunisation with an in-house-produced pneumococcal whole cell vaccine. The method was tested in nasopharyngeal and middle ear fluid samples. The first results indicated that antigen detection might be more sensitive than culture in demonstrating pneumococci in URT, particularly in children with prior antimicrobial therapy. Antigen detection is a feasible method for studies on pneumococci in developing countries. For type-specific demonstration of S. pneumoniae, detection of pneumococcal antigen after an enrichment step proved a sensitive method that can be applied for epidemiologic study purposes, e.g., in vaccine trials, in areas without ready access to a good microbiology laboratory. Determination of IC-bound pneumolysin IgG antibodies appears to be a useful method for species-specific diagnosis of pneumococcal infections. The results indicating pneumococcal aetiology in ALRI patients in this study compare well with the best results obtained by the use of lung aspirates. Increasing the number of serial samples improves the sensitivity of the assay, but even two samples provide more positive findings than other methods currently in routine use. Criteria of positivity need to be confirmed in subsequent larger studies with both healthy controls and patients with confirmed pneumococcal disease. It is also important to control the findings in patients with pneumonia of non-pneumococcal origin. The novel enzyme immunoassay was shown to work well with enrichment culture samples, with an almost 100% sensitivity compared with the culture. Middle ear fluid samples were too diluted for the enzyme immunoassay method used, and only 74% sensitivity compared with culture was achieved. Provided that adequate samples can be obtained, the method will be a useful complement to the current laboratory methods used to diagnose pneumococcal disease. With the existence of a broad spectrum of microbiological and immunological methods, it is imperative to seek international consensus for standard methods to demonstrate pneumococcus. Otherwise it is very difficult to compare results from different clinical studies. A WHO Working Group recently proposed a standard method for detecting upper respiratory carriage of pneumococcus, but a lot of work remains to be done in other areas of research on pneumococcal infections.

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive, encapsulated bacterium capable of causing significant morbidity and mortality throughout the world. A hallmark of S. pneumoniae infection is infiltration of neutrophils (PMNs) that assist in controlling the spread infection but may also contribute to pathology. Paradoxically, studies have shown that limiting PMN infiltration into the lumen of the lung during infection actually betters clinical outcome in experimental S. pneumoniae infection. The final step in PMN luminal trafficking is a Hepoxilin A3 (HXA3)-dependent migration across the pulmonary epithelium. HXA3 is a PMN chemoattractant that forms gradients along the polarized epithelial face, drawing PMNs from the basolateral to the apical surface during proinflammatory responses. HXA3 requires assistance of an integral- membrane protein transporter to escape the cell and form the gradient. The pulmonary HXA3 transporter is currently unidentified. In this work, we identify the pulmonary HXA3 transporter as the ATP-Binding Cassette Transporter (ABC transporter) Multi-drug Resistance Associated Protein 2 (ABCC2, MRP2). We demonstrate that MRP1 and MRP2 are divergent ABC- transporters that control transepithelial PMN migration through efflux of a distinct anti-inflammatory substance and the pro-inflammatory HXA3 in the context of Streptococcus pneumoniae infection. Enrichment of MRP2 on the plasma membrane requires detection of the bacterial virulence factors pneumolysin (PLY) and hydrogen peroxide. PLY and hydrogen peroxide not only coordinate MRP2 apical membrane enrichment but also influence HXA3-dependent PMN transepithelial migration. They influence migration through stimulation of epithelial intracellular calcium increases that are crucial for HXA3 production as well as MRP2 translocation to the plasma membrane. PLY and hydrogen peroxide are not sufficient in their signaling alone, however, and require at least one additional bacterial signal to induce HXA3/MRP2 proinflammatory activities.

Streptococcus pneumoniae é causa de doenças como pneumonia, otite, meningite e sepse. As vacinas hoje disponíveis têm cobertura limitada porque são baseadas no polissacarídeo capsular, que varia com os mais de 90 sorotipos da bactéria, além de levarem à substituição de sorotipos na população por outros não presentes nas formulações. Visando reduzir o custo e aumentar a cobertura, têm-se estudado vacinas baseadas em proteínas que ofereceriam proteção independente de sorotipo. Este trabalho teve por objetivo a obtenção, avaliação da estabilidade e da resposta imune em camundongos de híbridos de duas proteínas de S. pneumoniae: a proteína de superfície do pneumococo (PspA) e a pneumolisina geneticamente detoxificada (PdT), sem ou com espaçadores moleculares, rígido ou flexível, entre as moléculas. Os genes dos híbridos com espaçadores foram clonados através da técnica de overlap extension PCR. O gene das proteínas foi expresso em E. coli e as proteínas obtidas foram reconhecidas por anticorpos produzidos contra a célula inteira de pneumococo através de Western Blot. A purificação dos híbridos foi feita utilizando homogeneizador de alta pressão, precipitação de impurezas com detergente catiônico CTAB e diferentes etapas cromatográficas. A estabilidade foi analisada periodicamente através de SDS-PAGE e Western Blot. O híbrido sem espaçador mostrou-se instável, por isso a presença de atividade proteolítica foi investigada através de diversos ensaios para detecção dessas enzimas, mostrando que a instabilidade não decorreu de hidrólise por proteases. Os fragmentos resultantes da quebra tiveram a porção N-terminal sequenciada para identificar o sítio de clivagem, que estava localizado na junção das duas proteínas. Esse sítio foi retirado das construções seguintes e espaçadores moleculares foram incluídos entre os dois antígenos. A molécula com espaçador flexível foi obtida na forma solúvel durante o cultivo, porém durante a purificação ocorreu precipitação irreversível. O clone para produção do híbrido com espaçador rígido permitiu a obtenção da proteína, que foi purificada e teve a estabilidade avaliada periodicamente à 4&deg; C e -20&deg; C por Western Blot empregando anticorpos contra célula inteira de pneumococo, indicando que a introdução do espaçador rígido aumentou a estabilidade do híbrido em relação à molécula sem espaçador. Além disso, diferentes concentrações de estabilizantes foram avaliadas, mostrando que em presença de trealose 1M ou glicerol 50&#37; o híbrido com linker rígido permaneceu estável por pelo menos 4 meses a 4&deg; C. A molécula com espaçador rígido produziu níveis de anticorpos em camundongos comparáveis aos níveis obtidos com a molécula sem espaçador, que foram capazes de proteger 100&#37; dos animais em ensaio de desafio letal intranasal e inibir a atividade hemolítica da pneumolisina. O aumento de estabilidade do híbrido alcançado com a inserção do linker rígido juntamente com a retirada do sítio de clivagem e com a presença de estabilizantes permitem que esta molécula seja utilizada numa vacina pneumocócica proteica. Como estudos vêm demonstrando que seria necessário mais de uma proteína para formular esta nova vacina, a produção deste híbrido traz a grande vantagem de obtenção de dois antígenos em um único processo de produção, o que pode resultar em uma diminuição do custo da dose. / Streptococcus pneumoniae is cause of diseases like pneumonia, otitis, menngitis and sepsis. The vaccines available nowadays has limited coverage because they are based on the capsular polysaccharyde, which varies among the more than 90 bacteria serotypes and they lead to serotype substitution on the population for others not present on the vaccine formulations. In order to reduce the cost and increase the coverage, vaccines based on pneumococcal proteins that would offer serotype independent protection have been studied. The objective of this thesis was the obtainment, stability evaluation and immune response evaluation in mice of hybrids composed of two proteins of S. pneumoniae: pneumococcal surface protein A (PspA) and genetically detoxified pneumolysin (PdT), with or without molecular linkers, rigid and flexible, between the molecules. The genes with molecular linkers were cloned using the overlap extension PCR technique. The genes were expressed in E. coli and the proteins were recognized by anti pneumococcal whole cell in Western Blot. The hybrids were purified using high pressure homogeneizer, precipitation of impurities using cationic detergent CTAB and different chromatography steps. The stability was periodically analyzed through SDS-PAGE and Western Blot. The hybrid without linker was unstable and the presence of proteolytic activity was investigated through several methods for protease activity detection, showing that instability was not due to protease hydrolysis. The N-terminal portion of the degraded protein fragments was sequenced in order to identify the cleavage site, which was localized exactly between the two proteins. This site was removed from the other hybrids and molecular linkers were included between the two antigens. The molecule with flexible linker was obtained on the soluble form during expression, but during purification it precipitated irreversibly. The molecule with rigid linker were expressed, purified and had its stability analyzed periodically at 4&deg; C and -20&deg; C by Western Blot using anti pneumococcal whole cell, indicating that the insertion of the rigid linker increased the hybrid stability when compared with the hybrid without linker. Besides that, different stabilizers in different concentrations were evaluated and it was found that the presence of trehalose 1 M or 50&#37; glycerol stabilized the hybrid with rigid linker for at least 4 months at 4 &deg; C. The molecule with rigid linker produced levels of antibodies in mice comparable to the hybrid without linker. These antibodies were able to protect 100&#37; of animals from lethal intranasal challenge and inhibit the haemolytic activity of pneumolysin. The stability increase due to the rigid linker together with the removal of the cleavage site and the stabilizers presence allow this hybrid molecule to be used on a novel pneumococcal vaccine. Since studies have shown that it would be necessary more than one protein to formulate this new vaccine, the production of this hybrid brings the great advantage of producing two antigens in one single process, which can decrease the dosage cost.

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