As infecções do trato respiratório inferior lideram entre as principais causas de morbidade e mortalidade no mundo. Um dos grandes problemas associados ao tratamento das infecções do sistema respiratório, como as pneumonias, advém da crescente resistência aos mais modernos antibióticos adquirida pelos microrganismos. A terapia fotodinâmica, uma técnica baseada na interação da luz com uma substância fotoativa para causar dano oxidativo a células, tem se destacado como uma interessante alternativa para diversas doenças como diferentes tipos de câncer e infecções. Neste trabalho foi realizada, com experimentos in vitro, uma prova de princípio da possibilidade de inativar, com um protocolo eficiente e seguro, uma das bactérias mais comumente encontradas em quadros de pneumonia, a Streptococcus pneumoniae, com terapia fotodinâmica infravermelha mediada pela indocianina verde. Duas fontes de luz, uma a base de lasers emitindo 780 nm e outra construída com LEDs emitindo 850 nm, foram comparadas para avaliar sua eficiência. Experimentos com a bactéria foram realizados para determinação dos melhores parâmetros de inativação microbiana. Em seguida, ensaios de citotoxicidade foram feitos com macrófagos RAW 264.7 com o intuito de averiguar se as condições microbicidas não apresentavam atividade tóxica para células fagocitárias do sistema imune. Foi possível delinear os parâmetros de concentração de indocianina, tempo de incubação e dose de luz que apresentassem atividade microbicida e que não fossem tóxicas para as células. A interação da terapia fotodinâmica com a ação fagocitária dos macrófagos sobre as bactérias foi avaliada pelo estabelecimento de co-cultura dessas espécies. Concluiu-se que, utilizando-se LEDs de 850 nm fornecendo uma dose de luz de 10 J/cm2 as amostras contendo indocianina verde 5μM, é possível inativar S. pneumoniae de modo eficiente e auxiliar a ação fagocitária de macrófagos. / The lower respiratory tract infections lead among the main causes of morbidity and mortality worldwide. A major problem associated with respiratory tract infections, e.g. pneumonia, stems from from the increasingly resistance to most modern antibiotics developed by microorganisms. Photodynamic therapy, a technique based on the interaction of light and a photoactive substance to cause oxidative damage to cells, has emerged as an attractive alternative for several diseases such as different kinds of cancer and infections. In this work, with in vitro experiments, we accomplished a proof of concept for the possibility of inactivating, with an efficient and secure protocol, one of the most commonly found bacteria in pneumonia cases, Streptococcus pneumoniae, with infrared photodynamic therapy mediated by indocyanine green. Two light sources, one based on 780 nm lasers and the other built with 850 nm LEDs, were compared to evaluate their efficiency. Experiments with bacteria determined the best parameters microbial inactivation. Then, cytotoxicity assays with RAW 264.7 macrophages analyzed if the microbicidal parameters had toxic effects on immune cells. It was possible to delineate the indocyanine concentration parameters, incubation time and dose of light to obtain microbicidal results that weren´t toxic to the cells. Interaction of photodynamic therapy with the phagocytic action of macrophages on the bacteria was assessed by establishing a co-culture with these species. We concluded that, using 850 nm LEDs providing a light dose of 10 J/cm2 to samples containing 5μM indocyanine green, it is possible to inactivate S. pneumoniae and efficiently assist the phagocytic action of macrophages.

A resistência aos antibióticos dos patógenos mais comuns do trato respiratório está aumentando mundialmente. Recentemente, Streptococcus pneumoniae resistente à penicilina tem sido isolado em diversos países, e a freqüência dessas cepas tem elevado de modo alarmante. O aumento da resistência, com conseqüentes implicações terapêuticas, tem levado a uma reavaliação do uso dos antibióticos ß-lactâmicos para o tratamento de infecções pneumocócicas. No presente trabalho, um total de 107 amostras de Streptococcus pneumoniae, obtidas de materiais provenientes de pacientes adultos ambulatoriais e hospitalizados, em dois centros médicos de duas cidades do Rio Grande do Sul (Porto Alegre e Caxias do Sul), os quais apresentavam quadro clínico-radiológico de infecção pulmonar, foram analisadas com o objetivo de estudar-se a resistência do germe à penicilina. As amostras constituídas de escarro (80,4%), lavado brônquico (13,5%) e aspirado traqueal (6,6%) foram coletadas no período compreendido entre Julho de 1998 e Julho de 1999. O material foi semeado em meio de Agar sangue e as colônias suspeitas de Streptococcus pneumoniae foram transferidas para meio de Mueller-Hinton para teste de optoquina e de sensibilidade à penicilina com discos de oxacilina. Um halo de inibição da oxacilina menor do que 20 mm indicava a realização de teste para determinação da concentração inibitória mínima (MIC) com E-test. Um total de nove cepas foi identificado como tendo resistência intermediária à penicilina (MIC 0,1-1,0μg/ml) e nenhuma cepa resistente (resistência elevada: MIC > 2,0 μg/ml) foi identificada. Uma monitorização local das cepas quanto à resistência antimicrobiana é de grande importância para os clínicos no manejo de infecções pneumocócicas. / Antibiotic resistance to the common respiratory tract pathogens is increasing worldwide. Recently, penicillin-resistant pneumococci have been isolated in several countries, and the incidence of these strains has risen alarmingly. The increased of resistance with consequence therapeutic implications has led to a re-evaluation of ß-lactam antibiotics for the treatment of streptococcal infections. In present study a total of 107 isolates of Streptococcus pneumoniae from samples of adult hospitalizated patients ando outpatients were prospectively collected in 2 different medical centers of Rio Grande do Sul - Brazil (Caxias do Sul and Porto Alegre) with clinical and radiologyc signs of respiratory infections, were analyzed with objective to study the penicillin-resistant pneumococci. The samples consisting of sputum (80,4%), bronchial lavage (13,5%) and inhaled traqueal (6,6%) had been colected in the period understood between 98 July and 99 July. The material was sown in agar-blood and the colonies suspicion of Streptococcus pneumoniae had been transferred to Mueller-Hinton for test of optochin and for sensitivity to penicillin test with oxacilin disks. One inhibition of the lesser that 20 mmm for oxacilin indicates the test accomplishment to determine minimum inhibitory concentration (MIC) with E-test. A total of 9 strains were intermediate-resistant to penicillin (MIC 0,1-1,0μg/ml) and neither was resistant to penicillin (MIC > 2,0 μg/ml). Monitoring local of antimicrobial resistant strains is of great importance to clinicians for the management of pneumococcal infections.

O Streptococcus pneumoniae é responsável por altos índices de morbidade e mortalidade pelo mundo. No início do século XX os percentuais de morte causados por essa bactéria atingiram mais de 80%. Com o surgimento da antibioticoterapia, principalmente com o uso da penicilina, o combate ao pneumococo tornou-se mais efetivo. A partir da década de 1980, o combate a essa bactéria começou a ser mais difícil, devido ao aumento da resistência pneumocócica aos antimicrobianos. No Brasil e, de forma geral, no mundo todo são verificados percentuais crescentes de resistência pneumocócica. Tais percentuais variam de um país para outro e, até mesmo, dentro de um mesmo país significativamente. Dessa forma, faz-se necessário o monitoramento desses índices de resistência para se ter conhecimento da efetividade dos antimicrobianos comumente usados contra o pneumococo. A resistência do pneumococo tem origem cromossomal. No caso da eritromicina, os genes de resistência são denominados erm(B) e mef(A/E). Como ocorre com os percentuais de resistência, a prevalência dos genes citados também muda de acordo com a região pesquisada. Assim como a verificação da resistência, é importante a pesquisa da origem da resistência antimicrobiana, no caso dos genes erm(B) e mef(A/E). O presente trabalho verificou a resistência pneumocócica em 64 cepas obtidas de diferentes hospitais de Porto Alegre. Os resultados obtidos foram de 28% de resistência para a penicilina (8% de resistência plena e 20% de resistência intermediária), 15% de resistência plena para a eritromicina, 18% para a tetraciclina (14% intermediárias), 3% plenamente resistentes para o cloranfenicol, 68% de resistência para a associação trimetropima/sulfametoxazol (64% plenamente resistentes), 1% plenamente resistente à ceftriaxona e 0% de resistência à vancomicina. Pôde-se relacionar a presença dos genes com a resistência à eritromicina. O erm(B) mostrou uma predominância maior nessas cepas. Foram 6 cepas com o gene erm(B), 2 com o mef(A/E), uma amostra apresentou os dois genes e apenas uma não apresentou nenhum. Os resultados obtidos nesse trabalho estão relacionados ao período de 2004 e 2005, situando Porto Alegre em relação ao nível de resistência do pneumococo e seus genes de resistência à eritromicina. Cabe salientar que pesquisas subseqüentes devem ser feitas para o monitoramento da resistência do S. pneumoniae e dos determinantes dessa resistência. / The Streptococcus pneumoniae is responsible for high indices of morbility and mortality around the world. In the beginning of century XX the percentages of death caused by these bacteria had reached more than 80%. With the appearance of the antibiotical therapy, mainly with the use of penicillin, the combat to pneumococcus has became more effective. From the decade of 1980, the combat against these bacteria started to be more difficult due to the increase of the pneumococcal resistance to antimicrobials. In Brazil and, of general form, in the entire world has been verified the increase of pneumococcal resistance. Such percentages vary significantly from a country to another one and even inside of the same country. In this way, it’s necessary to have knowledge of the effectiveness of common antibiotics used against pneumococcus through the monitoriment of these resistance’s indices. The resistance of pneumococcus originates from the chromosome. In the case of the erythromycin, the resistance genes are called erm(B) and mef(A/E). As it occurs with the percentages of resistance, the prevalence of these genes also change in accordance with the searched region. As well as the verification of the resistance, the research of the origin of the antimicrobial resistance is important, in the case of the genes erm(B) and mef(A/E). The present work verified the pneumococcal resistance in 64 strains from different hospitals of Porto Alegre. The results were 28% of resistance for penicillin, 14% of resistance for erythromycin, 18% of resistance for tetracyclin, 3% of resistance for chloranphenicol, 68% of resistance for resistance trimetropim/ sulfametoxazole association, 1% of resistance ceftriaxone and 0% of resistance to the vancomycin. The presence of the genes with the erythromycin resistance could be related. The erm(B) showed a high preponderance in these strains. There were 6 erm(B) gene strains, 2 mef(A/E), a sample with the two genes and only one with none gene. It’s important to point out that the values found in this work hasn’t been constant and subsequent research must be made to monitor the S. pneumoniae resistance.

No description available.

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.

Naturally competent bacterial species, which self-induce the recombination mechanism of transformation, are wide spread across the bacterial tree-of-life. However, it remains unclear why competence has evolved in these bacteria. Although it is likely that exact explanations will be different for each species, a common selective factor cannot be excluded. Currently, three dominant hypotheses, which focus on the transformation function, try to explain the benefits of competence. Firstly, competence is thought to increase the rate of adaptation by combining beneficial alleles in single genotypes. Secondly, competence can repair DNA-damage by replacing the damaged DNA fragments with undamaged ones. Thirdly, the DNA uptake during competence is used to recycle environmental DNA fragments for nutrients. One of the naturally competent species is the Gram-positive Streptococcus pneumoniae, which is an opportunistic pathogen generally inhabiting the naso-pharyngeal area of young children. Competence in S. pneumoniae is regulated via density dependent extracellular signaling peptide. Here I use a combination of experiments designed around knockout mutants of the signaling mechanism and next-generation sequencing methods to test the first two hypotheses in S. pneumoniae. First, I extend on the DNA-for-repair hypothesis by showing that competent populations of S. pneumoniae are better protected not only against a DNA-damaging agent, but also against protein synthesis inhibitors. However, the mechanisms underlying this protection differ between types of stress. DNA-damage requires the full process of transformation, while protection against protein synthesis inhibitors only requires the activation of the competent cell state. This shows that benefits of competence cannot be totally explained by the benefits of transformation. Second, I use a long-term evolution experiment, where competent and non-competent strains are kept in the presence and absence of periodic stress, to determine the importance of competence for the generation of genetic variation. I find that competence does not increase the rate of adaptation in S. pneumoniae. The fitness of evolved competent populations was significantly lower than those of non-competent populations evolved over the same period of time. However, the intrinsic costs of competence are mitigated by the addition of short periods of stress exposure. These results confirm the prediction of the fitness associated recombination (FAR) hypothesis that competence is favoured in low-fitness situations. Thirdly, whole genome re-sequencing of the evolved populations allowed me to explore genomic evolution next to fitness changes. The genomic data revealed that competence reduces the mutational load of deleterious mutations rather than generating combinations of beneficial alleles. In addition I show several case of parallel genomic evolution within each treatment and across treatments. This shows that parallel evolution is not restricted by genotypic background (competence) or environment (periodic stress). Finally, these results show that competence has evolved in populations of S. pneumoniae as a mechanism to deal with various forms of stress.

Neonatal sepsis is an important cause of neonatal morbidity and mortality. There are two distinct types of sepsis- early and late onset. Group B streptococcus and Listeria are the most common causes of early onset neonatal sepsis historically. Physicians select antibiotics for neonates with fever based on historically common bacterial pathogens such as GBS, Ecoli, Listeria, and Staphylococcal aureus. However, the landscape of bacterial pathogens causing sepsis and fever in neonates seems to be changing. This could potentially change the first choice of antibiotics for this susceptible population. In this case study, we will present early-onset sepsis in a late preterm infant due to Streptococcus pneumoniae as confirmed by blood culture. The only maternal risk factors present in this case for septicemia were delivery less than 37 weeks. Patient initially had respiratory distress at delivery and required CPAP for 3 days. On day 2 of life, cultures were taken due to acute deterioration. Ampicillin and Gentamycin were given to the patient for empiric coverage initially. On day 2 of antibiotics, cultures were reported positive. Patient’s antibiotics had to be altered at that time to cover the isolated organism. The patient was inadequately treated up until cultures were positive. This case raises the question if Ampicillin and Gentamycin remain the best choice for broad antibiotic coverage in neonates with possible sepsis.

Regulation of penicillin-binding protein Pbp2a in Streptococcus pneumoniae Streptococcus pneumoniae is an extracellular human pathogen that encodes a unique eukaryotic-type Ser/Thr protein kinase StkP in its genome. This enzyme is involved in other cellular processes, such as cell division and cell wall synthesis, through phosphorylation with its substrates. A transmembrane protein MacP has been identified as a substrate of StkP. It is an activator of penicillin-binding protein PBP2a, which is involved in the synthesis of peptidoglycan with its transpeptidase and transglycosylase activities. We found that MacP is phosphorylated by the protein kinase StkP at positions T32 and T56. We confirmed that proteins MacP and PBP2a interact with each other and that phosphoablative and phosphomimetic mutations of the major phosphorylated residues of the MacP protein do not affect the interaction with PBP2a and do not fundamentally affect the function of MacP in vivo. Furthermore, we showed that the ∆macP mutation is synthethically lethal with the ∆pbp1a mutation, confirming that MacP is an activator of the PBP2a protein. MacP is located in the cell septum and interacts with a number of S. pneumoniae cell division proteins. Keywords: Streptococcus pneumoniae, cell division, MacP, PBP2a, phosphorylation