Entre membros da Família Enterobacteriaceae a produção de β-lactamases de espectro estendido (ESBL – Extended-Spectrum β-lactamase) se constitui em um importante mecanismo de resistência a antibióticos β-lactâmicos. O objetivo deste estudo foi determinar a prevalência de ESBL em diferentes gêneros da Família Enterobacteriaceae em um hospital universitário no sul do Brasil. Além disso, avaliou-se o teste de triagem proposto pelo CLSI, o teste que utiliza discos combinados e técnica de PCR para detecção de ESBL na Família Enterobacteriaceae. De forma complementar, foi feita pesquisa de KPC em amostras com resistência (plena ou intermediária) ao ertapenem. Foram analisados 731 isolados da Família Enterobacteriaceae, obtidos a partir de amostras clínicas de pacientes hospitalizados. A prevalência de isolados produtores de ESBL na Família Enterobacteriaceae foi de 26,8% (196/731). Destacam-se Providencia spp. com uma prevalência de 91,7% (11/12), seguida de Klebsiella pneumoniae com 56,7% (59/104) e Enterobacter spp. com 40,7% (48/118). Entre os antibióticos utilizados no Teste Confirmatório Fenotípico, a cefepima foi o substrato que detectou o maior percentual dos isolados (90,6% - 183/202) como produtores de ESBL. Utilizando a técnica de PCR foi possível detectar blaTEM em 89,6% (208/232), blaSHV em 59% (137/232) e blaCTX-M em 37,9% (88/232) dos isolados. Comparando o perfil de suscetibilidade global das amostras ESBL com as demais amostras consideradas como não produtoras de ESBL, notou-se um grande decréscimo na sensibilidade de todos antimicrobianos testados (p < 0,05) nas ESBL positivas. Apenas os carbapenêmicos (Imipenem e Meropenem) apresentaram total eficácia in vitro. Outros antibióticos com maior percentual de sensibilidade para isolados produtores de ESBL foram Amicacina, Doxaciclina e Piperacilina/Tazobactam com 35,1%, 29,9% e 28,0% de sensibilidade, respectivamente. Entre os microrganismos com teste de triagem para ESBL positivo, 39 apresentaram resistência (plena ou intermediária) ao ertapenem, mas em nenhuma destas amostras foi detectado o gene blaKPC por técnica de PCR.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2015-05-26T17:18:09Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese doutorado Alexsandra Scavuzzi.pdf: 8878953 bytes, checksum: 50dba197129c93a6d62fed598bc1f2d1 (MD5) / Made available in DSpace on 2015-05-26T17:18:09Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese doutorado Alexsandra Scavuzzi.pdf: 8878953 bytes, checksum: 50dba197129c93a6d62fed598bc1f2d1 (MD5) Previous issue date: 2015-02-27 / Foi investigada a ocorrência de genes de resistência a quinolonas (qnrA, qnrB e qnrS), a presença de mutações em gyrA, gyrB e parC, a expressão de bombas de efluxo (acrB e acrF) e mutações no gene ramR, como também foi avaliado alterações ultraestruturais provocadas pela polimixina B, meropenem e pela associação entre polimixina B e meropenem em isolados de Klebsiella pneumoniae portadores de blaKPC-2 provenientes de infecção e colonização, em pacientes de hospitais de Recife-PE, Brasil. Trinta isolados de K. pneumoniae foram selecionados para este estudo, destes, seis isolados foram selecionados para seqüenciamento de DNA e dois foram selecionados para detecção de alterações ultraestruturais. A detecção dos genes qnr, acrB e acrF foram realizadas por PCR seguidas por sequenciamento de DNA. As mutações em ramR e nas QRDRs de gyrA, gyrB e parC foram detectadas por sequenciamento, a expressão das bombas de efluxo foi determinada por RT-PCR e as alterações ultraestruturais foram detectadas por microscopia eletrônica de transmissão e varredura. Dos isolados analisados, 73,3% (n=22) apresentaram o gene qnrB, sendo detectadas por seqüenciamento as variantes qnrB1 e qnrB12. Esse é o primeiro relato publicado da presença de genes qnrB1 e qnrB12 com blaKPC-2 em K. pneumoniae, como também o primeiro relato mundial da presença do gene qnrB12 em K. pneumoniae. Foram observadas mutações em gyrA S83 em dois isolados e mutação em ramR em um isolado. Todos os isolados apresentaram genes para as bombas de efluxo acrB e acrF. A RT-PCR realizada mostrou que as bombas estavam sendo expressas. Quando submetidos à concentração clinicamente relevante para meropenem, a análsie de microscopia eletrônica mostrou que os isolados apresentaram alterações morfológicas, retração do material citoplasmático, incapacidade de divisão celular, rompimento da parede celular e extravasamento do conteúdo citoplasmático. Quando submetidos à concentração clinicamente relevante de polimixina B os isolados apresentaram perda de membrana celular, retração do material citoplasmático, extravasamento do conteúdo citoplasmático e presença de compartimento membranares. Quando submetidos à concentração clinicamente relevante de meropenem associado com polimixina B, os isolados apresentaram uma maior intensidade das alterações ultraestruturais visualizadas. Portanto, foi observada uma maior alteração celular quando os isolados eram submetidos à associação de meropenem e polimixina. Os resultados obtidos são preocupantes porque foram detectados isolados de K. pneumoniae portadores do gene blaKPC-2, qnrB, mutação em gyrA, expressão de bombas de efluxo acrB e acrF e mutação em ramR, infectando e colonizando pacientes, podendo ser importantes reservatórios para disseminação de diversos mecanismos de resistência no ambiente hospitalar.

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Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-10-17T12:39:08Z No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DissertaçãoParcial (Cap.I,II,III,IV e VII) - Polyane Lima - PPGIBA.pdf: 832056 bytes, checksum: a70651fc03df55df1e12db3ec2f97adb (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-10-17T12:39:21Z (GMT) No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DissertaçãoParcial (Cap.I,II,III,IV e VII) - Polyane Lima - PPGIBA.pdf: 832056 bytes, checksum: a70651fc03df55df1e12db3ec2f97adb (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Made available in DSpace on 2018-10-17T12:39:21Z (GMT). No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DissertaçãoParcial (Cap.I,II,III,IV e VII) - Polyane Lima - PPGIBA.pdf: 832056 bytes, checksum: a70651fc03df55df1e12db3ec2f97adb (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2018-06-21 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Klebsiella pneumonia is a pathogenic and opportunistic Gram-negative bacterium, which can cause pneumonia, consequently lung injury and mortality according to the exacerbation of inflammatory response. Therefore, many efforts have been made to discovery alternative therapies to control the events involved in inflammation, increasing resolution and reducing the risk of tissue injury. Recently studies described such as potential anti-inflammatory the isomer of palmitoleic acid 16:1 n-9 in humans circulating monocytes and in macrophages derived from monocytes in humans. Thus, we investigated the immunomodulatory effect of 16:1 n-9 on experimental infection with K. pneumoniae. In this study, we performed in vitro LPS stimulation and 16:1 n-9 treatment using AMJ2-C11 lineage macrophages and bone marrow derived macrophages (BMDM). Although both macrophages presented different biology, we observed for BMDM the decrease production of inflammatory mediators such as NO, IL-6, KC, MCP-1 after the treatment with 16:1 n-9. In efferocytosis process of neutrophils infected with K. pneumoniae, the treatment with 16:1 n-9 increased phagocytosis by MDMO and IL-1β production, and decreased IL-10. In vivo experiments, mice infected with lethal dose of K. pneumoniae and treated with 16: 1 n-9 (100 μg/mL) had increased survival. The immunomodulatory effects of 16:1 n-9 during the experimental infection by K. pneumoniae were characterized for decrease in neutrophil recruitment in the acute phase of infection, and increased mononuclear cells in the chronic phase, indicating action on resolution of inflammation. The effect on the production of inflammatory, proteic and lipid mediators has also been described, and the data contribute to the resolution effect, however it is not efficient in eliminating the infectious agent. In addition, treatment with 16:1 n-9 had a great influence on the metabolism of eicosanoids. Thereby, we suggest that 16:1 n-9 treatment in pneumonia is potential in resolving an inflammatory process, without immunosuppressive effects, and can be used only as an adjuvant of other pharmacological therapies. / Klebsiella pneumoniae é uma bactéria gram-negativa, oportunistas, podendo causar pneumonia, consequentemente lesão pulmonar intensa e mortalidade correlacionadas com exacerbação da resposta inflamatória. Estudos buscam terapias alternativas para controlar os eventos envolvidos na inflamação, aumentando a resolução e reduzindo o risco de lesão tecidual. Recentemente, um isômero do Ácido palmitoleico, 16:1 n-9 foi descrito em monócitos circulantes e em macrófagos derivados de monócitos, com potencial antiinflamatório. Dessa forma, investigamos o efeito imunomodulador de 16:1 n-9 na infecção experimental por K. pneumoniae. Neste estudo, realizamos experimentos in vitro, com estímulo de LPS e tratamento com 16:1 n-9 em macrófagos de linhagem AMJ2-C11 e macrófagos derivados de medula óssea (MDMO) e observamos que apesar da biologia dos macrófagos serem diferentes, o tratamento diminui a produção de mediadores inflamatórios, como NO, IL-6, KC, MCP-1 nos MDMO. No processo de eferocitose de neutrófilos infectados com K. pneumoniae, o tratamento com 16:1 n-9, aumentou a fagocitose por MDMO e potencializou a produção de IL-1β e diminuiu IL-10. Nos nossos experimentos in vivo, camundongos infectados com dose-letal de K. pneumoniae e tratados com 16:1 n-9 (100 μg/mL) tiveram sobrevida aumentada. Quando analisamos os efeitos imunomoduladores do 16:1 n-9 durante a infecção experimental por K. pneumoniae, observamos a diminuição no recrutamento de neutrófilos na fase aguda da infecção, e aumento das células mononucleares na fase tardia da infecção, que nos indicaram ação na resolução da inflamação. O efeito na produção dos mediadores inflamatórios, proteicos e lipídicos também foram descritos, e os dados colaboram com o efeito de resolução, apesar de não ser eficiente na eliminação do agente infeccioso. Por outro lado, o tratamento com 16:1 n-9 teve grande influência no metabolismo de eicosanoides. Assim, sugerimos que o tratamento com 16:1 n- 9 na pneumonia apresenta-se com capacidade de resolução em processo inflamatório, não sendo imunossupressor, mas podendo ser utilizado apenas como um adjuvante de outras terapias farmacológicas.

A aterosclerose é um processo complexo, multifatorial que ainda não está totalmente esclarecido. Foi proposto que a resposta imune mediada por processos infecciosos e/ou inflamatórios influencia na patogênese de lesões ateroscleróticas. Os receptores TolI-likes (TLRs) estão envolvidos na resposta inata e em outros eventos fisiológicos através da interação com seus ligantes endógenos e exógenos e talvez envolvidos no processo aterogênico. Tem por objetivo analisar a expressão dos receptores Toll-like 2 e 4 (TLR2 e TLR4) associando o processo de sinalização com a presença de agentes infecciosos tais como a Chlamydia pneumoniae (CP) e Mycoplasma pneumoniae (MP), em pacientes com infarto do miocárdio (MI) e em aneurismas aórticos. Foram obtidos fragmentos de aortas ascendentes de pacientes submetidos à cirurgia de revascularização do miocárdio (G1, n=13) e de fragmentos de pacientes submetidos à cirurgia de correção de aneurisma aórtico (G2, n=14). Amostras congeladas e parafinadas foram analisadas por Imunohistoquímica (lHO) e Hibridização in situ (HIS) para detecção e localização da presença dos patógenos e TLRs. Realizou-se uma semiquantificação em microscópio (O, ausente; 1, discreto e focal; 2, moderado e focal e 3, intenso e difuso). Observou-se o grau de inflamação e de acúmulo de gordura. Outrossim, realizou-se PCR em tempo real (SYBR Green) para pesquisa de DNA de CP e MP, como também análise da expressão de mRNA de TLR2 e de TLR4. Na lHQ, constatou-se presença de MP, CP, TLR2 e TLR4 (G1 e G2), maior quantidade de MP (p=0,012) e de TLR4 (p=0,017) no G2. Houve correlação de CP com MP (r=0,810 e p=0,003) e de TLR2 com TLR4 (r=0,569 e p=0,034). Na HIS, constatou-se presença de MP, CP, TLR2 e TLR4 (G1 e G2), não houve diferenças significativas comparando-se os grupos (G1 x G2), porém houve correlação, no G1, de CP com TLR4 (r=0,730 e p=0,040) e de infiltrado inflamatório com células adiposas (r=0,700 e p=0,036). No G2, houve várias correlações: MP com CP (r=0,620 e p=0,016), MP com TLR4 (r=0,662 e p=0,010), CP com TLR2 (r=O,733 e p=0,003), CP com TLR4 (r=0,589 e p=0,027) e de TLR2 com TLR4 (r=0,714 e p=0,004). A PCR em tempo real mostrou presença de CP, pela segunda extração de DNA realizada (G2). Não houve diferença de expressão dos TLRs entre os grupos. A expressão de TLR2 foi maior do que de TLR4 no G1 (p=0,006). O grau de inflamação e o acúmulo de gordura foram maiores no G2 do que no G1(p=0,001). Estes dados sugerem uma relação da co-infecção CP e MP, na gravidade do processo inflamatório presente em placas ateroscleróticas e em pacientes com infarto do miocádio, como também, participação dos receptores Toll-like 2 e 4. / The atherosclerosis is a complex and multifactorial process that is not still completely elucidated. It has been proposed that immune-mediate response to inflammatory and/or infectious processes is implicated in the pathogenesis of the atherosclerotic lesions. Toll-like receptors (TLRs) are involved in the innate response and other physiological events through binding to endogenous and exogenous ligands and it may be involved in the atherogenic process To investigate the Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) expression in atheroma plaques and its association with the presence of infectious agents such as Chlamydia pneumoniae (CP) and Mycoplasma pneumoniae (MP) in patients with myocardial infarction (MI) and aortic aneurysms. Fragments of ascending aorta were obtained from MI patients submitted to surgeries of revascularization of the myocardium (G1, n=13) and correction of aortic aneurism (G2, n=14). Frozen and paraffined samples slices were analyzed by Immunohistochemistry (lHQ) and in situ Hybridization for detection and localization of TLR2 and TLR4 expression and CP and MP antigens. There was semiquantification in microscope (0, absent; 1, discreet and focal; 2, moderate and focal; and 3, intense and diffuse). Histopathology was also carried out to investigate the inflammation degree and fat accumulation in these tissues. Real time PCR using SYBR Green System detection was used to stydy DNA CP and MP, also to analyze expression of mRNA TLR2 and TLR4. Using lHQ, it was verified presence of MP, CP, TLR2 and TLR4 (G1 and G2), larger amount of MP (p=0.012) and TLR4 (p=0.017) in G2. In G1 group, MP was positively correlated with CP (r=0.810, p=0.003), in G2, TLR2 with TLR4 (r=0.569, p=0.034). Using HIS, it was verified presence of MP, CP, TLR2 and TLR4 (G1 and G2), there were not significant differences between groups (G1 x G2), however, It was shown correlation between in G1, CP with TLR4 (r=0.730, p=0.040) and also inflammation with fat accumulation (r=0.700, p=0.036). In G2, there were several correlations: presence of MP with CP (r=0.620, p=0.016), MP with TLR4 (r=0.662, p=0.010), CP with TLR2 (r=0.733 p=0,003), CP with TLR4 (r=0.589, p=0.027) and TLR2 with TLR4 (r=0.714, p=0.004). Real time PCR showed presence of CP DNA using second purification accomplished (G2). There was not difference of expression TLRs among the groups. The expression of TLR2 was higher than TLR4 in G1 (p=0.006). Increased degree of inflammation and fat accumulation was also find in G2 than in G1 (p=0.001). These results are suggesting that the gravity of the inflammatory process in atherosclerotic plaques strongly are related to the presence of MP and CP co infection and expression of TLR2 and TLR4, as well in MI patients under myocardial revascularization.

Abstract Chlamydia pneumoniae is an obligate intracellular gram-negative bacterium, which causes respiratory infections in humans and may participate in the development of chronic diseases like atherosclerosis, chronic obstructive lung disease, adult-onset asthma and late-onset Alzheimer’s disease. It can infect various cell types, e.g. vascular endothelial cells, smooth muscle cells and monocyte-derived macrophages in vitro. It has been speculated that circulating macrophages disseminate the infection in the body, and that genetically susceptible individuals become chronically infected. Quantification of C. pneumoniae growth inside cultured cells is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally this has been done by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary and multiple inclusions are also seen. Therefore we developed a new method based on the real-time PCR quantification of chlamydial genomes adjusted to the number of human genomes and used it to quantify the exact amounts of C. pneumoniae in infected cells. The susceptibility of monocyte-macrophages from healthy individuals to C. pneumoniae infection in vitro was studied first. Intracellular growth of C. pneumoniae was used as an indicator of susceptibility to infection, and it was compared to serum levels of CRP, soluble CD14, human HSP-IgG, human HSP-IgA, C. pneumoniae IgG and IgA antibodies. The growth of C. pneumoniae in infected macrophages was highly variable, ranging from 0 to 638 chlamydial genomes per human genome. C. pneumoniae growth associated positively with serum C. pneumoniae IgA (titer ≥10) and hHSP-IgG and negatively with soluble CD14 concentration. The association between chlamydial IgA antibodies, hHSP-IgG and C. pneumoniae growth was statistically significant only among men. Age did not correlate with the growth. Therefore we hypothesize that persons whose macrophages cannot restrict the growth of C. pneumoniae are more prone to chronic infection by this agent. In the next study, we evaluated the effects of innate immunity genes CD14 -260 C>T, TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu and IL-6 -174 G>C polymorphisms on C. pneumoniae growth in human macrophages in vitro. The growth of C. pneumoniae was highest in CD14 -260 C>T TT genotype cells and the difference to CC or CT genotype was statistically significant. The G-allele of the IL6 -174 G>C polymorphism had a positive influence on chlamydial growth; the difference was statistically significant only between CC and GC genotypes. TLR2 Arg753Gln, TLR4 Asp299Gly, LBP Phe436Leu polymorphisms showed no effect on chlamydial growth.

Abstract Chlamydia pneumoniae is a common, widespread pathogen that causes acute and chronic infections. Serological diagnosis of C. pneumoniae infection is primarily based on the microimmunofluorescence (MIF) method, but only a fourfold IgG antibody increase between paired sera and the presence of IgM antibodies have generally been accepted as markers of acute infection. At the present, no commonly accepted, reliable serological or other methods for the diagnosis of chronic C. pneumoniae infection exist. We evaluated C. pneumoniae specific serological tests in different populations, followed the kinetics of C. pneumoniae antibodies in multiple sera obtained from the same individuals, compared anti-human IgA FITC conjugates in MIF test and evaluated C. pneumoniae specific antibody tests before and after coronary events in case-control pairs matched for the time point of serum sampling, place of residence, and treatment. We showed that reinfection or reactivation is needed for the persistence of elevated IgG and IgA antibody levels. In chronic infections and upon reactivation, chronic processes may be better diagnosable based on IgA persistence than IgG levels because of the rapid disappearance of IgA levels after seroconversions. The cycle of reinfection and reactivation seems to be faster than previously thought in crowded conditions, such as in military service, since we recorded several antibody changes between the arrival and departure sera of military recruits during 6-month service. The presence of antibodies does not provide protection from reinfection. Commercial anti-human IgA conjugates act differently in MIF tests, and there is marked variation in their ability to detect IgA antibodies. The EIA test used here overestimated the prevalence and persistence of IgA antibodies when compared to MIF. The best compability between MIF and EIA antibody levels was seen in the participants with high titers. Only high IgA MIF titers to C. pneumoniae at the baseline predicted future coronary events. In the present study, seroconversions both in the participants who developed a coronary event and in the controls were detected by MIF and EIA, but mostly in different persons. Seroconversion suggesting reinfection or reactivation of persistent infection may have a role in accelerating chronic processes, because the participants with MIF seroconversion between consecutive sera had a slightly higher risk for coronary events than the controls. EIA seroconversions were more common in the controls than in the cases before the coronary event. The difference in the kinetics of EIA and MIF antibodies warrants future research and supports the use of the MIF method as a golden standard in the measurement of C. pneumoniae IgG and IgA antibody levels and seroconversions. In their diagnostic practice, laboratories should use, compare, and validate more C. pneumoniae IgA antibody tests in addition to IgG tests. Unspecific findings in C. pneumoniae EIA tests require re-estimation and a new way to interpret the results. Chlamydia experts should speak for MIF and rethink the meaning of IgA antibodies and recommendations in the diagnosis of C. pneumoniae infections.

Genome of S. pneumoniae contains only one copy of the gene coding eukaryotic type protein kinase StkP and corresponding phosphatase PhpP. These two enzymes form a functional signaling pair regulating cell division, which could be used in the future for the design of new bacteriostatic compounds. Not only kinase and phosphatase are important components of the system, but also other members of this pathway - specific substrates of these enzymes. The identification of the Ser/Thr phosphoproteom with a focus on the membrane fraction provided information not only about already known substrates such as LocZ, Jag and DivIVA but also about an unknown protein P15 with a molecular weight about 15 kDa. In this thesis the protein was identified as rhodanase (spr0595) by MS MALDI TOF. However, its subsequent deletion did not confirm it as a StkP/PhpP substrate. Therefore we investigated another substrate, protein FtsA, which has already been identified as a substrate of this kinase in a previous study (Beilharz et al., 2012). FtsA is an essential cell division protein that anchors FtsZ filaments into the membrane. Phosphorylation of this protein was detected on the Thr residue at position 404. Using phosphoablative substitution we found out, that Thr404 is indeed phosphorylated by protein kinase StkP, however, FtsA...

The Streptococcus pneumoniae-derived toxin, pneumolysin, has been reported to augment neutrophil-mediated inflammatory responses in murine models of experimental infection of the airways, and to favour invasive pneumococcal disease. The laboratory research presented in this thesis has been designed to investigate the possible proinflammatory interactions of pneumolysin with human neutrophils in vitro, as well as the underlying mechanisms of these. Addition of pneumolysin (0.0167 - 41.75 ng/ml) to neutrophils caused dose-related enhancement of the following proinflammatory activities of these cells: superoxide generation, elastase release, expression of the β2-integrin CR3, phospholipase A2 activity and production of leukotriene B4 and prostaglandin E2, oxidative inactivation of α-1-proteinase inhibitor, and synthesis and release of interleukin-8. Pneumolysin-mediated enhancement of these neutrophil activities was observed in the absence of detectable cytotoxicity and was most striking when the toxin was added together with the bacterial chemoattractant N-formyI-L-methionyl-L-leucyl-L-pnenylalanine (FMLP, 1 µM). Treatment of neutrophils with pneumolysin also resulted in uncontrolled influx of Ca2+ into the cells in the setting of membrane depolarisation and efflux of K+, which appeared to be a consequence of the pore forming actions of the toxin. Importantly, the proinflammatory interactions of pneumolysin with neutrophils were completely attenuated by exclusion of Ca2+ from the cell-suspending medium. These observations identify novel proinflammatory properties of pneumolysin which result from pore formation in the plasma membrane, influx of Ca2+ and augmentation of Ca2+ -activitable neutrophil functions. / Thesis (DPhil)--University of Pretoria, 2005. / Immunology / unrestricted

La recombinaison homologue (RH) est une réaction universelle qui assure la maintenance des génomes. Elle participe aussi à leur variabilité. De fait, dans les trois domaines du vivant elle est au centre de nombreux processus biologiques tels que la réparation des dommages à l'ADN ou encore le brassage génétique au cours de la méiose. Chez les bactéries, la RH est également impliquée dans les processus de transferts horizontaux qui favorisent les échanges génétiques entre les espèces. La transformation génétique est l'un de ces processus largement répandus parmi les bactéries. Celle-ci a spécifiquement lieu lorsque les cellules entrent dans un état physiologique particulier nommé la compétence. Comparativement aux autres types de transferts horizontaux, la transformation génétique à la caractéristique d'être entièrement contrôlée par la cellule receveuse qui dirige l'intégration d'ADN exogène capturé dans son milieu extérieur. Le but de ma thèse a été d'améliorer notre compréhension moléculaire de la voie de RH spécifique de la transformation génétique de Streptococcus pneumoniae qui permettrait à ce pathogène non seulement d'échapper aux vaccins mais également d'acquérir de nouvelles résistances aux antibiotiques. Bien qu'elle présente des variations d'un organisme à l'autre, la RH peut fondamentalement être décomposée en trois phases successives catalysées par la recombinase RecA chez les bactéries. La phase présynaptique consiste en la polymérisation de la recombinase sur l'ADN simple brin (sb) générant un nucléofilament. L'étape synaptique comprend l'appariement du nucléofilament avec celui des deux brins de la séquence homologue qui lui est complémentaire. Elle aboutit à l'apparition d'une D-loop, intermédiaire de recombinaison résultant de la jonction entre les deux molécules ADN engagées. Enfin, la phase postsynaptique constitue l'étape finale durant laquelle les jonctions entre les molécules ADN sont maturées puis clivées de manière à maintenir l'intégrité double brin du génome. L'action optimale de la recombinase au cours de ces trois étapes nécessite l'assistance de partenaires protéiques spécifiques au processus au sein duquel la réaction de RH intervient. Dans le cadre de la transformation génétique, la RMP (recombination mediator protein) DprA et la protéine de liaison à l'ADNsb SsbB sont deux partenaires de RecA spécifiquement produits par les cellules en état de compétence. Nous avons révélé in vitro que leurs actions conjuguées permettent d'améliorer l'efficacité de la RH catalysée par RecA en facilitant l'étape présynaptique de la réaction. De plus, nous avons montré que contrairement à SsbB, la protéine SsbA constitutive et essentielle ne stimule pas la RH spécifique de la transformation génétique de S. pneumoniae. Ce résultat suggère que les deux paralogues n'interviennent pas au cours des mêmes processus biologiques. La protéine RadA, ubiquitaire et très conservée parmi les bactéries, est un autre partenaire de RecA constitutivement produit par les cellules mais spécifiquement induit lors de la transformation génétique de S. pneumoniae. Par une combinaison d'approches in vivo, in vitro et structurale, nous avons mis en évidence d'une part, l'appartenance de RadA à la famille SF4 d'hélicases de type DnaB, et d'autre part son rôle clef dans la phase postsynaptique de la RH. Ce travail a permis de proposer un modèle inédit du mécanisme de migration de branches permettant la maturation des intermédiaires de recombinaison. Via son interaction avec RecA, nous proposons que RadA accède aux deux brins de l'ADN receveur de part et d'autre de la D-loop. Ainsi chargé symétriquement, RadA transloquerait de manière divergente le long des deux ADNsb dans le sens 5'-3' permettant ainsi l'ouverture du duplex receveur. L'incorporation de l'ADNsb envahissant serait ainsi facilitée par l'action de RadA tant au cours de la transformation génétique que des processus de maintenance faisant intervenir la RH / Homologous recombination (HR) is a central biological process in living organisms across all three domains of life. Crucial both for genomic maintenance and genetic plasticity, HR acts either to repair harmful DNA breaks or to produce new DNA combinations on chromosomes. In bacteria, HR is also used to exchange genetic material between different strains or species through horizontal gene transfers processes. Genetic transformation is one of those processes, widely distributed among species. Genetic transformation, occurring during a distinct physiological state called competence, is entirely directed by the recipient cell which uses exogenous DNA as a source of transferred genetic material. The goal of my PhD was to provide a molecular understanding of the specific HR pathway operating during the genetic transformation process of Streptococcus pneumoniae which enables this human pathogen to escape vaccine by capsular serotype switching as well as to acquire new antibiotics resistance genes. Although HR mechanisms vary among different organisms and cell types, the same three basic steps are conserved. During the pre-synaptic phase, the highly conserved recombinase named RecA in bacteria polymerises along ssDNA. The synaptic step proceeds through invasion of this nucleofilament into complementary duplex DNA, promoting the formation of joint molecules called D-loops. Finally, the post-synaptic phase corresponds to the maturation and clivage of thoses branched structures to preserve genomic integrity. To complete this strand exchange reaction, RecA requires the assistance of several specific partners depending on the context of the HR event. During genetic transformation, the recombination mediator protein DprA and the ssDNA binding protein SsbB are such partners specifically produced in the competence state. We have shown in vitro that their interplay with RecA improves HR efficiency by facilitating the pre-synaptic step of the reaction. Moreover, we have shown that, contrary to SsbB, the essential SsbA protein does not stimulate this specific HR reaction, suggesting that the two paralogous proteins could be implicated in different processes. The ubiquitous bacterial RadA in an other RecA partner constitutively produced by the cells and specifically induced during genetic transformation of S. pneumoniae. Using a combination of in vivo, in vitro and structural approaches, we have shown that RadA is a DnaB-like helicase implicated in the post-synaptic phase of HR. This structural and functional analysis of RadA leads to an unprecedented model of DNA branch migration acting to maturate D-loops structures. Through its interaction with RecA, RadA gains access to both strands of the recipient duplex DNA on both sides of the D-loop. Once symmetricaly loaded RadA could translocate divergently thereby unwinding the complementary duplex DNA thus facilitating the incorporation of ssDNA during genetic transformation as well as in genomic maintenance processes.