Berlin, Freie Universiẗat, Diss., 1999. / Dateiformat: zip, Dateien im PDF-Format.

Ein Proteintransportsystem genannt Zwei-Partner-Sekretionssystem ist bei gram-negativen Bakterien weit verbreitet. In B. pertussis ist es bereits ausführlich untersucht. Diese Arbeit widmet sich dem Zwei-Partner-Sekretionssystem in Meningokokken. / Characterisation of a protein transoport system in meningococci. This system, called two-partner-secretion-system is allready well examined in B. pertussis.

Neisseria meningitidis ist ein wichtiger Erreger von Meningitis und Sepsis insbesondere bei jungen Menschen, gleichzeitig sind hohe Raten asymptomatischen Trägertums bekannt. Als die Virulenz begünstigende Faktoren wurden unter anderem die Kapsel, Pili, äußere Membranvesikel (OMV) und Lipopolysaccharid (LPS) identifiziert, die es dem Erreger erleichtern, das menschliche Immunsystem zu überwinden. Dabei war bisher die Rolle von Neutrophil Extracellular Traps (NETs) als neu beschriebene Komponente der angeborenen Immunantwort nicht untersucht worden. NETs stellen spinnennetzartige DNA-Strukturen mit globulären Proteindomänen dar, die aus neutrophilen Granulozyten entstehen und als antimikrobiell gelten. Ziel dieser Arbeit war es, die Wirkung von NETs auf Meningokokken zu charakterisieren und mögliche Resistenzmechanismen der Bakterien zu identifizieren. In den vorliegenden Versuchen konnte gezeigt werden, dass Meningokokken an NETs binden und durch diese in ihrer Proliferation gehemmt werden. Eine Lokalisation der Bakterien an die NETs konnte dargestellt werden, LPS und Pili wurden als wichtige Strukturen für die Vermittlung der NET-Bindung identifiziert. OMVs zeigten sich als protektiv gegenüber dem Einfluss der NETs, indem sie die Bindung der Erreger an die NETs blockierten. Wenig empfindlich zeigten sich die Bakterien gegenüber Histonen als den quantitativ bedeutsamsten NET-Proteinen. Meningokokken schützen sich gegenüber dem Einfluss der NETs durch Ausbildung von Kapsel und LPS mit intakter Phosphoethanolamin-Modifikation. Ebenso vermitteln zwei Cathelicidin-Resistenzgene den Bakterien einen Überlebensvorteil. Keine Rolle bei der NET-Resistenz spielten die untersuchten Effluxmechanismen. Neuere Untersuchungen von Lappann et al. indentifizierten Meningokokken und OMVs als potente NET-Induktoren. Damit könnten durch die relativ NET-resistenten Mikroorganismen andere Abwehrmechanismen der Neutrophilen konterkariert werden und eine Immunevasion begünstigt werden. Genauere Untersuchungen diesbezüglich stehen noch aus. / Neisseria meningitidis is an important pathogenic agent of meningitis and sepsis especially in young adults, at the same time high rates of asymptomatic carriage are well-established. Known factors promoting virulence are, among others, the capsule, pili, outer membrane vesicles (OMV) and the lipopolysaccharide (LPS). In host defense against meningococcal disease, the role of Neutrophil Extracellular Traps (NETs), a recently described component of the innate immune response, had not yet been investigated. NETs are web like structures with globular protein domains that arise from neutrophil granulocytes and are considered being antimicrobial. The aim of the present study was to further investigate interactions between N. meningitidis and NETs and to identify possible resistance mechanisms of meningococci. In this thesis I could demonstrate that meningococci bind to NETs and are therewith being restricted in proliferation. A localization of bacteria to NETs was illustrated, and the mediating effect of LPS and pili on binding was identified. OMVs were shown to be protective against the properties of NETs by blocking the binding of pathogens to NETs. Bacteria were minor sensitive to histones which represent the quantitatively most significant group of proteins in NETs. Meningococci are protected against the effect of NETs by the formation of capsule and LPS when correctly modified with phosphoethanolamine. Two genes involved in cathelicidin resistance were shown to be beneficial on the survival of the bacteria. The investigated efflux mechanisms did not affect resistance to NETs though. Recent data by Lappann et al. identified the role of meningococci and OMVs as potent inducers of NET-formation. This might be a strategy of the NET-resilient microorganism to thwart neutrophil phagocytosis or degranulation and to facilitate immune escape, which is yet to be investigated.

This study examined the adherence of Neisseria meningitidis to human buccal epithelial cells (BEC). Past studies have utilized microscopic assays which are tedious and are, at high bacterial concentrations, relatively inaccurate. This study developed a rapid radioactive assay for bacterial adherence which gave results comparable to the microscopic assay. This new assay system was used, then, to examine the kinetics and specificity of meningococcal adherence. Adherence, which increased linearly as a function of multiplicity of infection, reached maximal values within 5 minutes. Adherence was mediated by a bacterial surface protein; pronase-treated meningococcidid not adhere well to BEC. Unlike other adherent Neisseria, adherent meningococci did not exhibit pill. Removal of the capsule with saccharolytic agents greatly increased adherence. These results suggest that Neisseria meningitidis may adhere to human BEC via a non-pilus surface protein which is partially masked by the capsule. The BEC receptor for this protein was examined but remains unidentified.

Proefschrift Universiteit van Amsterdam. / Auteursnaam op omslag: Frits de Vries. Met lit. opg. - Met samenvatting in het Nederlands.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Univ., Diss., 2005--Würzburg.

Infecções por Neisseria meningitidis estão associadas a altos índices de morbimortalidade no mundo. Na Região da Grande São Paulo a Doença Meningocócica (DM) causada por Neisseria meningitidis sorogrupo C começou a se tornar prevalente em 2001 , representando, em 2003, 62,7% de todos os casos de DM sorogrupados sendo que aproximadamente 88,5% dessas cepas eram não sorotipados e não sorosubtipados (C:NST:NSST). Estes dados sugeriam que um fenótipo, C: NST:NSST, tinha emergido na Grande São Paulo e, considerando-se a importância histórica da doença na região, iniciamos o presente estudo com o objetivo de esclarecer a mudança na dinâmica da DM pela determinação das características fenotípicas e genotípicas destas cepas. Para tanto, analisamos por sorotipagem, tipagem das regiões Variáveis da PorA e PorB e do gene 16S RNA ribossomal, 753 cepas de N. meningitidis C isoladas de casos de DM provenientes da Grande São Paulo, no período de 1990 a 2003. Dado a impossibilidade de caracterização do novo fenótipo pelos anticorpos monoclonais disponíveis mundialmente, objetivamos também a produção de hibridomas produtores desses anticorpos para caracterização do fenótipo C:NST:NSST. Foram selecionadas duas linhagens celulares híbridas, produtoras de anticorpos monoclonais que reconhecem as proteínas PorA e PorB deste novo fenótipo. Entre as 255 cepas de N. meningitidis C inicialmente caracterizadas como NST:NSST, 75% (n=191) tomaram-se completamente sorotipadas como 23:P1.14-6. A análise da similaridade do gene 16S RNA ribossomal das cepas analisadas demonstrou um único padrão genético, sugerindo a clonalidade deste novo fenótipo. Os dados obtidos neste trabalho, demonstram a introdução, na Região da Grande São Paulo, de um novo clone de Neisseria meningitidis C apresentando o fenótipo C:23:P1.14-6 e que está sendo responsável pelo aumento dos casos de DM causada por este sorogrupo. / Neisseria meningitidis (Men) is an important cause of morbidity and mortality and is a leading cause of bacterial meningitis and septicemia in children and young adults in Brazil. Meningococcal disease caused by MenC started becoming the most prevalent serogroup in 2001, representing 62.7% of all MD cases serogrouped in 2003 in Greater Sao Paulo and approximately 88.5% of MenC isolates were nonserotypeable and non-serosubtypeable (NST:NSST). This data suggested that a novel invasive isolate (C:NT:NSST) had emerged in GSP, and considering the historical importance of MenC disease in the region, we initiated this study to better understand the dynamics of MD looking at the phenotypic and molecular characteristics of these isolates. To accomplish this goal, we characterized 753 MenC isolates recovered during the period of 1990 to 2003 by serotyping, PorS and PorA VR typing, 16S rRNA gene typing and produced new serotyping monoclonal antibodies (MAbs) to characterize the C:NST:NSST isolates. We were able to select two hybridoma cells that recognizes PorB and PorA proteins. Among the 255 strains initially characterized as NST:NSST, 75% (n=191) of them became completely serotyped as 23:P1 .14-6. Sy 16S RNA ribossomal typing, these strains showed the same pattern suggesting strain clonality. Our data demonstrate the introduction of a new clone of Neisseria rneningitidis C presenting the phenotype C:23:P1.14-6 and that is being responsible for the increase of the cases of DM caused by this serogroup in Great Sao Paulo.

A meningite meningocócica permanece como importante causa de morbidade e mortalidade em todo o mundo. Extratos purificados de LOS de Neisseria meningitidis foram utilizados em um ensaio de ELISA a fim de detectar a especificidade dos anticorpos anti-LOS produzidos por 41 pacientes com doença meningocócica. Durante o período de convalescença, 100% dos pacientes apresentaram aumento da concentração sérica de IgG anti-LOS (L1, L2, L3,7, L6, L8 e L10) em no mínimo três vezes a concentração verificada nos soros coletados durante a fase aguda. O maior aumento foi de oito vezes para IgG anti-L3,7 adicionada de resíduo de ácido siálico. Portadores de Neisseria meningitidis e Neisseria lactâmica na nasofaringe não produziram anticorpos anti-LOS. Através de um ensaio ELISA de Inibição, foi possível verificar a especificidade dos anticorpos anti-LOS, exceto para os anticorpos IgG anti-L10, que apresentaram reação cruzada com outros tipos antigênicos de LOS como L2, L3,7 e L6, revelando-se um importante candidato a antígeno vacinal. / Meningococcal meningitis remains as an important cause of morbidity and mortality all over the world. Purified LOS extracts were used in an ELISA assay to verify the specificity of the antibodies produced due to meningococcal disease. All 41 convalescent sera collected from patients had at lowest a three-folder increase of anti-LOS IgG concentrations when compared with acute sera. A higher increase of eight fold was seen to anti-L3,7 IgG specific antibodies. Safe carries of Neisseria meningitidis and Neisseria lactamica had no detectable specific anti-LOS antibodies. In an Inhibition ELISA assay, except for anti-L10 IgG, antibodies against L1, L2, L3,7, L6 and L8 immunotypes were specific. Anti-L10 antibodies cross-reacted with L2, L3,7 and L6 epitopes.

A meningite meningocócica permanece como importante causa de morbidade e mortalidade em todo o mundo. Extratos purificados de LOS de Neisseria meningitidis foram utilizados em um ensaio de ELISA a fim de detectar a especificidade dos anticorpos anti-LOS produzidos por 41 pacientes com doença meningocócica. Durante o período de convalescença, 100% dos pacientes apresentaram aumento da concentração sérica de IgG anti-LOS (L1, L2, L3,7, L6, L8 e L10) em no mínimo três vezes a concentração verificada nos soros coletados durante a fase aguda. O maior aumento foi de oito vezes para IgG anti-L3,7 adicionada de resíduo de ácido siálico. Portadores de Neisseria meningitidis e Neisseria lactâmica na nasofaringe não produziram anticorpos anti-LOS. Através de um ensaio ELISA de Inibição, foi possível verificar a especificidade dos anticorpos anti-LOS, exceto para os anticorpos IgG anti-L10, que apresentaram reação cruzada com outros tipos antigênicos de LOS como L2, L3,7 e L6, revelando-se um importante candidato a antígeno vacinal. / Meningococcal meningitis remains as an important cause of morbidity and mortality all over the world. Purified LOS extracts were used in an ELISA assay to verify the specificity of the antibodies produced due to meningococcal disease. All 41 convalescent sera collected from patients had at lowest a three-folder increase of anti-LOS IgG concentrations when compared with acute sera. A higher increase of eight fold was seen to anti-L3,7 IgG specific antibodies. Safe carries of Neisseria meningitidis and Neisseria lactamica had no detectable specific anti-LOS antibodies. In an Inhibition ELISA assay, except for anti-L10 IgG, antibodies against L1, L2, L3,7, L6 and L8 immunotypes were specific. Anti-L10 antibodies cross-reacted with L2, L3,7 and L6 epitopes.