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Streptococcus pneumoniae est la cause principale de pneumonies, otites, méningites et septicémies. La transformation génétique naturelle constitue l’élément clé de son adaptation aux changements environnementaux. Elle s’effectue par intégration d’ADN d’origine externe dans le chromosome de la bactérie, et a lieu pendant un état physiologique particulier appelé compétence.Mon travail de thèse a consisté à étudier les acteurs principaux de la régulation de la compétence (ComD, ComE) et les protéines impliquées dans la prise en charge, le traitement de l’ADN transformant et la recombinaison (DprA, RecA). J’ai notamment résolu la structure du facteur de transcription ComE par cristallographie aux rayons X, et réalisé une étude fonctionnelle de sa fixation sur un de ses promoteurs. Les résultats obtenus ont permis de proposer un mécanisme selon lequel la dimérisation induite par la phosphorylation de ComE, couplée à sa fixation sur la séquence promotrice d’ADN, provoquerait une courbure de l’ADN. Cette courbure permettrait la fixation de l’ARN polymérase, activant ainsi la transcription des gènes nécessaires à la mise en place de la compétence. / Streptococcus pneumoniae is the leading cause of community-acquired infections worldwide. The natural genetic transformation is the key to its adaptation to environmental changes. It takes place with the integration in its chromosome of exogenous DNA, during a physiological state called competence.During my thesis I have focused on the main actors of competence regulation (ComD, ComE) and on proteins involved in exogenous DNA processing and recombination (DprA, RecA). In particular, I have solved the structure of the transcriptional activator ComE by X-ray crystallography, and carried out a functional study of its binding to its promoter. The results obtained allowed us to propose a mechanism regarding the transcriptional activation by ComE of the genes necessary for the set up of the competence : the phosphorylation-induced dimerization, coupled to the binding of ComE to its DNA promoter, would curve the DNA and allow the binding of the RNA polymerase.

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Otitis media (OM), one of the commonest of childhood diseases, causes much suffering. OM exists in a variety of forms, two of which are acute otitis media (AOM) and otitis media with effusion (OME). The clinical courses of these conditions differ, AOM usually presenting with earache, fever and/or aural discharge, and the OME usually with hearing impairment. The tympanic membrane (TM) mirrors the events in the middle ear cavity, and pars flaccida (PF) is the initial site of inflammatory changes in the TM. PF is rich in mast cells (MCs), which by releasing various mediators, may trigger TM inflammation. The aims of the present studies were to investigate early inflammatory changes in the TM in rat models of OM; after mast cell degranulation, in response to AOM, and OME, after myringotomy in AOM and in normal ears. Furthermore, we developed a new rat AOM model, that excludes surgical trauma and resembles the natural route of infection in man. AOM and OME elicited the first inflammatory response in PF of the TM. The response to OME was discrete, but a slight increase in macrophages was found. During the first 48 hours of AOM, the inflammatory response was intense, following a bimodal pattern. This reaction is similar to that found after MC degranulation. In AOM, macrophages were the predominant cell in PF, while in pars tensa (PT), polymorphonuclear cells (mainly neutrophils) predominated. When myringotomy was performed in AOM ears, the healing time was shorter than that of myringotomy in normal ears. The highly inflamed lamina propria seemed to promote healing. During early AOM, as well as following myringotomy, fibrin extravasates into PF and PT. This fibrin deposition may be involved in regulating the inflammatory response. Repeated nasal challenge with the otitis media pathogen Streptococcus pneumoniae provoked AOM and concomitant TM stimulation reduced the number of AOM cases. This new rat AOM model has the advantage of avoiding trauma in the middle ear cavity, while eliciting an intense inflammatory response in the middle ear cavity (MEC).

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Protein-carbohydrate interactions are pivotal to many biological processes, from plant cell wall degradation to host-pathogen interactions. Many of these processes require the deployment of carbohydrate-active enzymes in order to achieve their intended effects. One such class of enzymes, glycoside hydrolases, break down carbohydrate substrates by hydrolyzing the glycosidic bond within polysaccharides or between carbohydrates and non-carbohydrate moieties. The catalytic efficiency of glycoside hydrolases is often enhanced by carbohydrate-binding modules (CBMs) which are part of the modular structure of these enzymes. Understanding the carbohydrate binding function of these modules is often key to studying the catalytic properties of the enzyme. This thesis investigates the molecular determinants of carbohydrate recognition by CBMs that share similar amino acid sequences and overall three-dimensional structures and thus fall within the same CBM family. Specifically this research focused on two families; plant cell wall binding family 6 CBMs and the alpha-glucan binding family 41 CBMs. Through X-ray crystallography, isothermal titration calorimetry and other biochemical experiments, the structural and biophysical properties of CBMs were analyzed. Studying members of CBM family 6 allowed us to establish the overall picture of how similar CBMs interact with a diverse range of polysaccharide ligands. This was found to be due to changes in the topology of the binding site brought about by changes in amino acid side chains in very distinct regions of the binding pocket such that it adopted a three-dimensional shape that is complementary to the shape of the carbohydrate ligand. Members of CBM family 41 were shown to have nearly identical modes of starch recognition as found in starch-binding CBMs from other families. However family 41 CBMs are distinct as they are found mainly in pullulanases (starch debranching enzymes) and have developed binding pockets which are able to accommodate alpha-1,6-linkages, unlike other starch-binding CBM families. These are the first studies comparing multiple CBMs from within a given CBM family at the molecular level whose results allow us to examine the distinct modes of carbohydrate recognition within a CBM family. Analysis of the family 41 CBMs revealed that these CBMs are mainly found in pullulanases from pathogenic bacteria. Members from Streptococcal species were shown to specifically interact with glycogen stores within mouse lung tissue, leading us to investigate the role of alpha-glucan degradation by the pullulanase SpuA in the pathogenesis of Streptococcus pneumoniae. SpuA targets the alpha-1,6-branches in glycogen granules, forming alpha-1,4-glucan products of varying lengths. The overall three-dimensional structure of SpuA in complex with maltotetraose was determined by X-ray crystallography and showed that its active site architecture is optimal for interacting with branched substrates. Additionally, the N-terminal CBM41 module participates in binding substrate within the active site, a novel feature for CBMs. This is the first study of alpha-glucan degradation by a streptococcal virulence factor and aids in explaining why it is crucial for full virulence of the organism.

[Truncated abstract] Pneumococcal exposure is high and life-long in developing countries including Papua New Guinea (PNG), with children under 2 years of age being at most risk for early upper respiratory tract pneumococcal carriage and infection. Deaths from pneumococcal diseases such as pneumonia and meningitis are common and likely the result of an absence of vaccination programmes. The need for effective and affordable pneumococcal vaccines has led to the testing of protein antigens including pneumolysin (Ply) and pneumococcal surface protein A (PspA) as novel vaccine antigens. Little is known on the immune responses to these proteins in humans, particularly in high-risk populations where such vaccines will be of most benefit. In this study, we examined the roles of naturally acquired antibody and cellular immune responses in mothers and newborns to Ply and PspA family 1 (PspA1) and family 2 (PspA2) in protection against or risk for early carriage in a high-risk PNG population. Antibodies to Ply, PspA1 and PspA2 were measured in plasmas of 241 mothers and 115 newborns (cords) from PNG, and 50 Australian mothers using an enzyme-linked immunosorbent assay (ELISA). Pernasal swabs were collected from PNG mothers at the time of delivery, one month post-partum, and weekly within the first month of life from their newborns to determine pneumococcal carriage. Cellular immune responses to Ply, PspA1 and PspA2, the TLR2/TLR4 ligands, LTA and LPS and to PHA were measured in cord blood mononuclear cells (CBMC) of 84 PNG versus 33 Australian newborns. Innate and T-cell cytokine responses in the PNG newborns were then analysed to determine their effect on infant pneumococcal carriage. ... No protective effect against infant pneumococcal carriage was observed with maternal and cord IgG levels for all antigens. Maternal carriage at time of delivery increased the risk for infant pneumococcal carriage in the first month of life (HR: 1.93, 95% CI 1.36 – 2.73, p = 0.001) with 70% of infants being colonised. Papua New Guinean newborns produced higher innate IL-10 and IFN-¿ (p = 0.003) and TNF-a (p < 0.001) to Ply compared to Australian newborns with no significant differences observed for IL-6 or IL-12. IFN-¿ responses to LPS and LTA (p = 0.005 and p < 0.001) were higher in PNG than Australian newborns, while IL-6, IL-10 (p < 0.001) and TNF-a (p = 0.002) to LPS with LTA-induced IL-6 and IL-10 (p < 0.001) were higher in Australian newborns. T-cell IL-5, IL-10, IL-13, IFN-¿, IL-6 and TNF-a response levels to PspA and PHA stimulation were significantly high in PNG newborns. No differences were observed for cytokine responses to Ply and PspA between PNG infant pneumococci carriers and non-carriers. Papua New Guinean infants are colonised by pneumococci very early in life and this may be influenced by high maternal carriage rates. PspA- and Ply-IgG levels are high in PNG mothers and undergo cross placental transfer but do not appear to be protective against early pneumococcal carriage. In PNG newborns, PspA elicits T-cell responses, while Ply drives more innate cellular responses, neither were demonstrated to have a protective effect against early carriage though further work is required to better define these and their relation to immune development in early childhood.

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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.