Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

Streptococcus pneumoniae (pneumococo) é um importante patógeno humano, responsável por graves infecções das vias respiratórias. O principal fator de virulência desse microrganismo é a cápsula polissacarídica (PS), antígeno das vacinas atuais, que são elaboradas com os PS purificados de cepas de pneumococo prevalentes na população. O objetivo desse trabalho foi desenvolver um processo de purificação do PS do sorotipo 14, responsável por 39% das doenças em crianças de 0-6 anos no Brasil. A metodologia de purificação envolveu separação celular por microfiltração tangencial e concentração do microfiltrado com membrana de ultrafiltração tangencial de 50 kDa. O produto dessa etapa foi diafiltrado com dodecil sulfato de sódio em membrana de ultrafiltração tangencial de 30 kDa, seguido de precipitação com ácido tricloroacético a 5%, precipitação por etanol (20% e 60%) e cromatografia de troca aniônica. A pureza do PS foi avaliada pelo conteúdo de proteínas e ácidos nucleicos remanescentes e o tamanho por cromatografia de exclusão molecular. O PS foi obtido com pureza e tamanho requeridos pelos órgãos regulatórios e o rendimento final do processo foi de 65%. / Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for severe respiratory tract infections. The main virulence factor of this microorganism is the capsular polysaccharide (PS), which is the antigen of all current vaccines, which are prepared with purified PS of pneumococcal strains prevalent in the population. The objective of this work was to develop a new purification process for PS of serotype 14, responsible for 39% of diseases in children of 0-6 years old in Brazil. The purification method involved cell separation by tangential microfiltration and concentration of cell-free culture broth containing PS by tangential ultrafiltration (50 kDa). The product of this step was diafiltrated with sodium dodecyl sulfate by tangential ultrafiltration (30 kDa), following by 5% trichloroacetic acid precipitation, 20% and 60% ethanol precipitation and anion exchange chromatography. The PS purity was evaluated by the content of residual proteins and nucleic acids, and the molecular mass by size exclusion chromatography. The purity and molecular mass requirements were achieved and the process global yield was 65%.

This diploma thesis deals with the issue of the attitude of parents towards selected infectious diseases which are preventable by vaccines in the Czech Republic. The primary focus is on parents' opinions on vaccination against invasive diseases caused by meningococci, haemophili and pneumococci. The theoretical part discusses the issue of vaccination, its importance and organizational structure; it further describes the selected pathogens, the diseases they cause and the epidemiological situation. It also considers the factors that may lead many parents to distrust and be critical of vaccination. The main aim of the practical part is to find out by means of an online questionnaire which social, geo-demographic, economic and other factors influence the choice of parents to have their children vaccinated against the given bacteria. Statistical methods of χ2 -independence test and binary logistic regression have been used to evaluate the data obtained from the questionnaire with the help of the SPSS program. The results show that parents with lower education and fewer children are more likely to get their child vaccinated; furthermore, it is the parents in the vicinity of where the outbreak of the given disease occurred or those who obtain sufficient information from a pediatrician and consider this...

De nombreux pathogènes sont soupçonnés d'interagir entre eux lorsqu’ils circulent dans les populations humaines. L'effet de ces interactions entre pathogènes peut être synergique ou antagoniste. Les avancées technologiques récentes en microbiologie ont favorisé la multiplication des études sur les interactions entre pathogènes chez l’hôte humain, notamment au sein des voies respiratoires. En effet, ces interactions peuvent jouer un rôle dans la dynamique de transmission des pathogènes concernés et avoir un impact conséquent sur la santé publique. Néanmoins, hormis certains systèmes plus souvent étudiés comme l’interaction entre la grippe et le pneumocoque pour laquelle des hypothèses mécanistiques spécifiques ont été suggérées, les connaissances concernant les mécanismes biologiques à l’origine de ces interactions restent limitées. De ce fait, un nouveau champ d'étude se développe soulevant de nombreuses questions. Du point de vue de la modélisation épidémiologique, il est nécessaire de répondre aux questions suivantes : comment formaliser les mécanismes sous-jacents à ces interactions dans des modèles mathématiques ? Quelles sont les conséquences de ces interactions sur la dynamique des infections dans les populations ? Dans quelles conditions et avec quelles méthodes les détecter à partir de données écologiques d’incidence, telles que celles rapportées par les systèmes de surveillance ? L’objectif du présent travail de thèse est de répondre à ces questions à partir de méthodes de modélisation statistique et mathématique, en travaillant spécifiquement sur les interactions entre grippe et pneumocoque. Bien que peu utilisée dans ce contexte, la modélisation mathématique permet une approche globale de ces phénomènes car elle formalise le lien entre l’échelle individuelle où ils se produisent et l’échelle de la population à laquelle ils peuvent être détectés. Dans un premier temps, j’ai conçu et développé un nouveau modèle agent afin de simuler la propagation simultanée de deux pathogènes en interaction dans une population humaine. Ce modèle a permis d’étudier au niveau populationnel les conséquences des phénomènes complexes liés à ces interactions, formalisées au niveau individuel. Notamment, j’ai montré que différentes hypothèses sur les mécanismes d’interaction entre la grippe et le pneumocoque conduisaient à des dynamiques d’incidence spécifiques. Dans un second temps, j'ai simulé à partir du modèle agent précédent des données similaires à des données de surveillance, pour lesquelles les mécanismes d’interaction et leur intensité étaient contrôlés. Les méthodes statistiques et mathématiques classiquement employées dans la littérature ont été appliquées à ces données. Les résultats ont montré leur capacité à identifier des associations entre pathogènes à partir d'un certain seuil, variable selon les méthodes et les mécanismes d'interaction. Enfin, dans le cadre d’une collaboration avec le CNRP et Santé Publique France, nous avons proposé une nouvelle méthode d'analyse des interactions entre pathogènes à partir de séries temporelles, basée sur l’étude de leur saisonnalité. Cette méthode a permis d'identifier une association faible dans les données françaises de syndromes grippaux et infections invasives à pneumocoque sur la période 2000-2014. Ainsi, les résultats et développements issus de ce travail de thèse permettent, par une approche de modélisation mathématique, une meilleure compréhension de l’impact des interactions au sein de l’hôte sur la dynamique d’incidence au niveau populationnel. Du fait du grand nombre de facteurs impliqués (pathogènes, hôte, environnement), la co-circulation de pathogènes en interaction dans une population est un système complexe que la modélisation est particulièrement apte à appréhender. Une meilleure compréhension de ces phénomènes est un enjeu capital pour le futur, notamment quant au développement de mesures de santé publique globales visant à réduire le fardeau des maladies infectieuses. / Several pathogens have been suggested to interact with each other while circulating within human populations. These between-pathogen interactions may be synergistic, when one pathogen favours another, or antagonistic when one pathogen is detrimental to the other. Recent technological developments in the field of microbiology have created new opportunities for studying between-pathogen interactions within the human host, and particularly in the respiratory tract. These interactions may have dramatic consequences on the transmission dynamics of the implicated pathogens, and consequent public health impacts. However, despite some mechanistic hypotheses having been formulated, especially for the well-studied influenza-pneumococcus system, the underlying biological mechanisms are still poorly understood. This developing field raises numerous questions. From an epidemiological modelling perspective, how should these interaction mechanisms be formalized into models? How do these interactions impact the transmission dynamics and burden of the involved pathogens? Under which conditions, and with which methods can interactions be detected from ecological incidence data, classically reported from surveillance systems? The aim of this thesis is to address these questions using statistical and mathematical modelling tools, with a specific focus on the interaction between influenza and pneumococcus. While mathematical models have scarcely been used to address between-pathogen interactions, they are powerful tools that allow for a global approach by precisely formalizing the interactions at the individual scale and linking them to the population scale at which data are collected and phenomena observed. First, I conceived and developed a new agent-based model which simulates the co-circulation of two interacting pathogens in a human population. This model specifically formalizes between-pathogen interactions at the individual level, resulting in global dynamics at the population level. Notably, I demonstrated that different hypotheses regarding interaction mechanisms between influenza and pneumococcus lead to specific incidence dynamics and interaction burdens. Second, in order to construct in silico data mimicking surveillance data, I simulated a large number of interaction scenarios from the previous agent-based model. These simulated datasets were analysed using a variety of statistical and mathematical methods classically applied in between pathogen association studies. Results showed that all methods consistently detected between-pathogen associations as long as the simulated interaction strength remained above a threshold, which varied according to the method and the simulated interaction mechanism. Lastly, collaborating with the National Center for Pneumococcal Reference and Santé Publique France, we developed a new method to analyse between-pathogen interactions from incidence time series, based on the analysis of their seasonality patterns. By applying this method to French data of influenza-like illnesses and invasive pneumococcal diseases over the 2000-2014 period, we identified a small association, consistent with previous studies. The mathematical models developed and results presented in this thesis provide new understanding of the impact of between-pathogen interactions at the population level and the efficiency of available methods to assess them. Because the co-circulation of pathogens in populations is a complex system involving a large number of factors related to the pathogens, the host, and the environment, the development of mathematical models will be critical in the future. A better comprehension of these phenomena is of major importance as it may lead to new opportunities to reduce the public health burden of infectious diseases.

<p>Influenza is a highly contagious respiratory disease. Yearly epidemics and pandemics account for high morbidity and mortality worldwide. Lung immunopathology is a major factor causing death following influenza. In addition, secondary bacterial superinfections that occur after influenza further complicate the lung immunopathology and contribute to higher morbidity and mortality. The research presented in this thesis addressed important, understudied questions in the complicated field of tissue immunopathogenesis and host defense to influenza and pneumococcal infections. Firstly, in a model of acute respiratory influenza infection, we found that the classically proinflammatory cytokine TNF plays a dual and biphasic role at different times post-infection. While it does have pro-immune roles in the beginning stages, TNF acts as a negative type 1 immune regulator at later points of infection. TNF controls the level of immune activation and has a key role in preventing lung immunopathology and aberrant tissue remodeling. Secondly, to further investigate mechanisms of lung pathology, we elucidated the role of bacterial replication and over activated host immune responses during bacterial superinfection following influenza. In our model of pulmonary <em>Streptococcus pneumoniae</em> infection after influenza, we found that dual infected animals experience rapid weight loss and succumb to infection. Bacterial outgrowth, dysregulated cytokine and chemokine expression, and severe lung neutrophilia and immunopathology are linked to the poor clinical outcome. Combined treatment with both an antibiotic azithromycin and corticosteroid dexamethasone best improves clinical outcome, bacterial clearance, cellular and cytokine responses, and immunopathology. Thirdly, in our continuing interest for improved therapies during pulmonary infections, we tested the transgenic expression of type I IFN as a treatment during <em>S. pneumoniae</em> infection. We found that IFN-a controls bacterial outgrowth and improves clinical outcome. Together, our findings provide novel insights into the mechanisms of lung immunopathology and treatment protocols for pulmonary influenza and pneumococcal infections.</p> / Doctor of Philosophy (Medical Science)

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 é o principal causador da pneumonia bacteriana. As vacinas atualmente disponíveis contêm polissacarídeo capsular conjugado ou não com proteínas carreadoras. No entanto, elas apresentam elevado custo ou proteção reduzida nos grupos de risco (crianças abaixo de 5 anos de idade e idosos). Proteínas de superfície de S. pneumoniae, como a PsaA e PspA, são consideradas fortes candidatas vacinais. Com o objetivo de se desenvolver uma vacina de ampla cobertura e baixo custo contra pneumococos, os genes psaA e pspA foram clonados em vetores de expressão em E. coli, pAE e pET e as proteínas expressas foram purificadas por cromatografias de afinidade e de troca aniônica. O rendimento de proteína recombinante obtido com a construção baseada em pET foi 3 vezes maior que o obtido com pAE. Condições de cultivo foram estabelecidas utilizando meio definido com indução por IPTG e/ou por lactose. As cepas recombinantes estão adequadas para serem usadas em estudos para escalonamento da produção em biorreatores. / Streptococcus pneumoniae is the main causative agent of bacterial pneumonia. The current vaccines available contain capsular polysaccharide conjugated or not with carrier proteins. However these are either too expensive or do not protect the high-risk groups. Surface proteins of S. pneumoniae, such as PsaA and PspA, are considered strong vaccine candidates. With the aim of developing a broad-coverage and low-cost vaccine against pneumococcus, the psaA and pspA genes were cloned in E. coli expression vectors, pAE and pET and the expressed proteins were purified through affinity and anion exchange chromatography. The yield of the recombinant protein obtained with the construction based in pET was 3-fold higher than that obtained with pAE. Culture conditions were established using defined media with IPTG and/or lactose induction. The recombinant strains are now ready to undergo studies for scale-up of production in bioreactors.

Meningitis is an infection of the meninges, a layer of tissue surrounding the brain. In cases of pneumococcal meningitis (where the bacterium Streptococcus pneumoniae is the causat- ive agent) this causes severe inflammation, requiring intensive care and rapid antibiotic treatment. The contribution of variation in host and pathogen genetics to pneumococcal meningitis is unknown. In this thesis I develop and apply statistical genetics techniques to identify genomic variation associated with the various stages of pneumococcal meningitis, including colonisation, invasion and severity. I start by describing the development of a method to perform genome-wide association studies (GWAS) in bacteria, which can find variation in bacterial genomes associated with bacterial traits such as antibiotic resistance and virulence. I then applied this method to longitudinal samples from asymptomatic carriage, and found lineages and specific variants associated with altered duration of carriage. To assess meningitis versus carriage samples I applied similar analysis techniques, and found that the bacterial genome is crucial in determining invasive potential. As well as bacterial serotype, which I found to be the main effect, I discovered many independent sequence variants associated with disease. Separately, I analysed within host-diversity during the invasive phase of disease and found it to be of less relevance to disease progression. Finally, I analysed host genotype data from four independent studies using GWAS and heritability estimates to determine the contribution of human sequence variation to pneumococcal meningitis. Host sequence accounted for some variation in susceptibility to and severity of meningitis. The work concludes with a combined analysis of pairs of bacterial and human sequences from meningitis cases, and finds variation correlated between the two.

Streptococcus pneumoniae é o principal causador da pneumonia bacteriana. As vacinas atualmente disponíveis contêm polissacarídeo capsular conjugado ou não com proteínas carreadoras. No entanto, elas apresentam elevado custo ou proteção reduzida nos grupos de risco (crianças abaixo de 5 anos de idade e idosos). Proteínas de superfície de S. pneumoniae, como a PsaA e PspA, são consideradas fortes candidatas vacinais. Com o objetivo de se desenvolver uma vacina de ampla cobertura e baixo custo contra pneumococos, os genes psaA e pspA foram clonados em vetores de expressão em E. coli, pAE e pET e as proteínas expressas foram purificadas por cromatografias de afinidade e de troca aniônica. O rendimento de proteína recombinante obtido com a construção baseada em pET foi 3 vezes maior que o obtido com pAE. Condições de cultivo foram estabelecidas utilizando meio definido com indução por IPTG e/ou por lactose. As cepas recombinantes estão adequadas para serem usadas em estudos para escalonamento da produção em biorreatores. / Streptococcus pneumoniae is the main causative agent of bacterial pneumonia. The current vaccines available contain capsular polysaccharide conjugated or not with carrier proteins. However these are either too expensive or do not protect the high-risk groups. Surface proteins of S. pneumoniae, such as PsaA and PspA, are considered strong vaccine candidates. With the aim of developing a broad-coverage and low-cost vaccine against pneumococcus, the psaA and pspA genes were cloned in E. coli expression vectors, pAE and pET and the expressed proteins were purified through affinity and anion exchange chromatography. The yield of the recombinant protein obtained with the construction based in pET was 3-fold higher than that obtained with pAE. Culture conditions were established using defined media with IPTG and/or lactose induction. The recombinant strains are now ready to undergo studies for scale-up of production in bioreactors.

ortalidad en niños menores de 5 años, a pesar de contar con medidas preventivas como la vacunación.  Objetivo: Evaluar el impacto de la vacuna antineumocócica conjugada sobre la incidencia de neumonía en niños menores de 5 años, a nivel nacional y departamental, así como las hospitalizaciones y mortalidad a nivel nacional, desde 2001-2019 en el Perú. Además, realizar un análisis entre departamentos con coberturas altas de vacunación y aquellos que alcanzan coberturas bajas.  Metodología: Diseño: Series de tiempo definidas por la introducción de la vacuna antineumocócica heptavalente (PCV7) en el periodo de tiempo entre 2009 y 2011 a nivel nacional y departamental. Posteriormente, se realizó un análisis multivariado contrastando la incidencia de casos entre los departamentos con alta y baja cobertura de vacunación. Procedimiento de obtención de datos: Los datos agrupados sobre la incidencia, hospitalización y mortalidad por neumonía fue obtenida de la CDC (Centro de Enfermedades Contagiosas); la cobertura de vacunación fue obtenida como base de datos del Ministerio de Salud (MINSA) Análisis específicos: se realizó un análisis multivariado contrastando la incidencia de casos de neumonía entre los departamentos con alta y baja cobertura de vacunación.  Resultados: Para las hospitalizaciones a nivel nacional, la tendencia de cambio post vacunación fue negativa y significativa (p <0.001). La incidencia y mortalidad tuvieron cambios no significativos. A nivel regional, Callao, Lima, Moquegua, Cusco, Huancavelica, Pasco, Loreto, San Martín y Ucayali tuvieron tendencia de cambio post vacunación negativa y significativa (p <0.001). Conclusiones: La vacunación ha demostrado ser efectiva para disminuir hospitalizaciones por neumonía en algunos departamentos y a nivel nacional. Sin embargo, existen factores individuales que pueden alterar la efectividad de la intervención, propias de la estrategia aplicada y del tipo de estudio utilizado. / Introduction: Pneumonia is an acute respiratory infection, the most common bacterial cause is Streptococcus pneumoniae and represents one of the major causes of mortality in children under 5 years of age, despite preventive measures. Objective: Assess the impact of the pneumococcal vaccine on the incidence of pneumonia in children under 5 years of age, at the national and departmental level, as well as hospitalizations and mortality at the national range, over 2001-2019 in Peru. In addition, perform an analysis between departments that achieve high vaccination coverage and those with low coverage.  Methodology: Study design: Time series interrupted by the inclusion of the heptavalent pneumococcal vaccine (PCV7) in the period between 2009 and 2011 at the national and departmental level. Multivariate analysis, contrasting the incidence of cases between departments with high and low vaccination coverage. Data collection procedure: Pooled data on pneumonia incidence, hospitalization, and mortality obtained from the CDC (Center for Communicable Diseases); vaccination coverage obtained as a database from the Ministry of Health (MINSA) Specific analyzes: multivariate contrasting the incidence of pneumonia cases between departments with high and low vaccination coverage. Results: Hospitalizations at the national level had a negative trend of change after vaccination (p <0.001). At regional level, Callao, Lima, Moquegua, Cusco, Huancavelica, Pasco, Loreto, San Martín and Ucayali had a negative and significant change trend post vaccination (p <0.001). Conclusions: Vaccination has proven to be effective in some departments and at the national level. However, there are individual factors and limitations of the study that may affect the outcome. / Tesis