Les épidémies de méningite sévissent depuis plusieurs décades dans la Ceinture Africaine de la méningite. En 2010, une vaccination de masse avec le vaccin conjugué MenAfriVac® a été entreprise dans la région afin d’éliminer les épidémies dues au méningocoque (Nm) A, principal sérogroupe en cause. L’objectif de cette thèse est de décrire l’épidémiologie des méningites au Niger avant et après l’introduction du vaccin MenAfriVac®; contribuer à une meilleure compréhension des dynamiques des épidémies en comparant les caractéristiques des foyers épidémiques selon le sérogroupe responsable; évaluer l’efficacité et l’efficience des stratégies de surveillance et de réponse vaccinale. Pour cela, nous avons utilisé pour la première fois des donnéesagrégées au niveau géographique de l’aire de santé concernant des cas suspects et des cas confirmés au laboratoire de 2002 à 2015. Nous avonsconsidéré la situation historique d’avant l’introduction du MenAfriVac®, la situation simulant l’élimination du NmA et la situation réelle de réémergence du NmC. La première partie de cette thèse a montré un changement de l’épidémiologie des méningites au Niger, avec l’élimination du NmA à partir de 2011 et la réémergence explosive du NmC en 2015. Les groupes d’âge et les aires du pays les plus affectés depuis 2002 sont également connus. Le pic épidémique était plus précocement détecté au niveau de l’aire de santé. Les incidences annuelles les plus élevées étaient retrouvées dans le sud-est du pays. Nous avons aussi trouvé que les épidémies à NmX présentaient un pic plus élevé que celles à NmA au niveau de l’aire de santé,pendant que le pic du NmW paraissait plus faible. Le sérogroupe C présentait une dynamique épidémique similaire à celle du NmA. Les incidences annuelles montraient des différences moins prononcées, probablement dues auxlongues durées des épidémies à NmA. En simulant l’élimination du NmA, l’efficacité de la réponse était plus importante pour une surveillance au niveau de l’aire de santé et une vaccination du district, alors que l’efficience était optimisée avec la vaccination au niveau de l’aire de santé. Pendant l’épidémie à NmC de 2015, l’efficacité et l’efficience étaient plus élevées pour la stratégie de surveillance au niveau de l’aire de santé et la vaccination du district que pour les autres stratégies. Les stratégies de prévention et de riposte aux épidémies de méningites nécessitent d’être adaptées selon les caractéristiques des sérogroupes de méningocoque prédominant. La surveillance de niveau subdistrict devrait être plus appropriée à la réponse aux épidémies suite à l’élimination des méningites de sérogroupe A et pour l’épidémie à NmC de 2015. / Meningitis epidemics strike since several decades in the African Meningitis Belt. In 2010, a mass vaccination with the conjugate vaccine MenAfriVac® was conducted in the region in order to eliminate epidemics due to meningococcal (Nm) A, the main serogroup in cause. The objective of this thesis is to describe the epidemiology of meningitis in Niger before and after MenAfriVac® introduction; to contribute to a better understanding of epidemics’ dynamics by comparing the characteristics of localized epidemics by serogroup responsible; to evaluate the effectiveness and the efficiency of surveillance and response strategies.For this, we used for the first time data aggregated at the geographical level of health area concerning suspected cases and laboratory confirmed cases from 2002 to 2015.We considered the historical situation of before MenAfriVac® introduction, the situation simulating NmA elimination and the real situation of NmC re-emergence. The first part of this thesis showed a change of meningitis epidemiologyin Niger, with the elimination of NmA from 2011 and the explosive re-emergence of NmC in 2015.The age groups and the country’s area the most affected since 2002 were also known. The epidemic peak was more early detected at the health area level. The highest annual incidences were found in the south eastern part of the country, bordering Nigeria. We also found that epidemics due to NmX tended to have higher peak force at health center level than those due to serogroup A, while the peak force of NmW appeared to be lower. The serogroup C appeared to have epidemic dynamic similar to NmA. Annual incidences showed less pronounced differences, possibly due to the trend for longer duration of NmA epidemics.Whensimulating NmA elimination, effectiveness of the response was highest for health area level surveillance and district level vaccination, while efficiency was optimized with health area level vaccination. For 2015 NmC epidemic, effectiveness and efficiency were higher for health area level surveillance and district level vaccination compared to other strategies. Strategies for prevention and response to meningitis epidemics need to be adapted according to the characteristics of predominant meningococcal serogroups.Subdistrict level surveillance may be most appropriate for meningitis epidemic response following elimination of serogroup A meningitis and for the 2015 NmC epidemic.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-12-10T12:32:54Z No. of bitstreams: 1 iaralice-souza.pdf: 1338553 bytes, checksum: 59fa26df56bce23aa1b5861c460f716f (MD5) / Made available in DSpace on 2012-12-10T12:32:54Z (GMT). No. of bitstreams: 1 iaralice-souza.pdf: 1338553 bytes, checksum: 59fa26df56bce23aa1b5861c460f716f (MD5) Previous issue date: 2001 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / Neisseria meningitidisdo grupo C é uma bactéria encapsulada causadora dediversas doenças, está associada à altas taxas de mortalidade e portanto é de grande importância para a saúde pública. Bio-Manguinhos está desenvolvendo uma vacina conjugada formada pela ligação covalente do polissacarídeo capsular àanatoxina tetânica e esta vacina, atualmente, está sendo avaliada em estudos clínicosde Fase II em crianças de 1 a 9 anos. A quantificação de componentes livres como polissacarídeos e proteínas faz parte do controle de processo de vacinas conjugadas e tem o objetivo de evitar o aparecimento de reações adversas exacerbadas e/ou redução da imunogenicidade do componente vacinal. A Organização Mundial de Saúde preconiza níveis máximos de proteína livre no conjugado vacinal de 5%, mas não estabelece um limite máximo para o polissacarídeo livre para a vacina conjugada contra o grupo C. Desta forma, o objetivo deste estudo foi desenvolver e validar métodos de controle de qualidade adequados para separar e quantificar estes componentes livres presentes na vacina meningocócica C conjugada brasileira, utilizando a técnica de eletroforese capilar (EC). Para a separação da proteína livre foram comparados os modos de eletroforese capilar de zona livre (CZE) e cromatografia eletrocinética micelar (MEKC). Diferentes condições de migração da amostravariando-se parâmetros como pH, temperatura, tensão, concentração do tampão, ciclodextrinas e de surfactante dodecil sulfato de sódio (SDS) foram estudadas. Os resultados demonstraram que a melhor separação do conjugado foi obtida por MEKC utilizando tampão tetraborato de sódio (TBNa) 150 mM, 25 mM de SDS, 60°C, 30 kV e pH 9,3. Entretanto, nos modos de EC estudados não foi possível obter a separação completa dos componentes, sendo necessária a utilização de outro processo. Por outro lado, por CZE foi possível observar a separação da proteína ativada da nativa, demonstrando a necessidade de otimização da reação de ativação da proteína, a fim de aumentar orendimento da reação de conjugação. A separação completa do açúcar livre presente no conjugado foi obtida empregando CZE utilizando tampão TBNa 50 mM, 40°C, 30 kV e pH 10. Com as condições escolhidas foi possível determinar o conteúdo de polissacarídeo livre nos lotes de conjugado e validar o método proposto, que se mostrou linear na faixa de 0,047 a 0,164 mg/mL, apresentou efeito matriz, 0,0154 mg/mL de limite de detecção e 0,0454mg/mL de limite de quantificação. Após as etapas de validação, foram quantificados alguns lotes de conjugado e observou-se um alto teor de açúcar livre nos lotes com longo período de estocagem a 4°C. Desse modo, fez-se a avaliação de um lote recentemente produzido e obteve-se o valor de 19,08% de polissacarídeo livre. A fim de estimar o tempo de estocagem máximo do conjugado foram realizadas análises com 30, 60 e 90 dias de estocagem a 4°C. Os valores encontrados até 60 dias não foram significativamente diferentes dosdeterminados no tempo zero. No entanto, com 90 dias de estocagem ocorreu uma modificação do perfil do conjugado que impossibilitou a sua quantificação. A metodologia desenvolvida e validada será introduzida no controle de qualidade do lote de conjugado que será submetido aos estudos clínicos de Fase III e na rotina da vacina conjugada estudada. Além disto, o conhecimento adquirido poderá ser empregado no controle de qualidade de outras vacinas conjugadas contra bactérias encapsuladas de interesse epidemiológico no país. / Neisseria meningitidisgroup C is an encapsulated bacterium that causes several diseases and is associated with high mortality rates becoming a serious public health problem. Bio-Manguinhos is developing a conjugate vaccine constituted by covalent attachment of capsular polysaccharide to tetanus toxoid, which iscurrently being evaluated in Phase II clinical studies in children between 1-9 years. Free components quantification is a vaccine process control assay and intended to prevent exacerbated adverse reactions occurrence and/or vaccine immunogenicity reduction. The World Health Organization recommends 5% of free protein maximum level in the conjugate vaccine, but does not set a limit for the free polysaccharide contents. Thus, the aim of this study was to develop and validate quality control methods appropriate to separate and quantify free components present in the conjugate vaccine against N. meningitidisgroup C, using capillary electrophoresis (CE) technique. For free protein separation, free capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were compared and different sample migration conditions were studied by varying parameters such as pH, temperature, voltage, buffer concentration, cyclodextrin and surfactant sodium dodecyl sulfate (SDS). The results showed that the best separation was obtained by MEKC using sodium tetraborate buffer (TBNA) 150 mM, 25 mM SDS, 60°C, 30 kV and pH 9.3. H owever, the CE did not induce a complete separation of the components suggesting that other techniques should be necessary. On the other hand, native and activated protein separation was possible using CZE, demonstrating the necessity of optimize protein activation reaction in order to increase the conjugation reaction yield. The total free sugar conjugate was completely separated from the conjugate by CZE using 50 mM TBNA buffer, 40°C, 30 kV and pH 10. In these conditions it was possible to determine the free polysaccharide content and validate the proposed method, which was linear in 0.047 to 0.164 mg/mL range, showed a matrix effect, 0.0154 mg/mL of detection limit and 0.0454 mg/mL ofquantification limit.After the validation steps, some conjugate batches were quantified and high levels of free sugar were observed in batches storaged at 4°C for long periods. On the other hand a conjugate batch recently produced was evaluated and showed19.08% of free polysaccharide. In order to estimate the maximum storage time a conjugate batch was analyzed30, 60 and 90 days after the production steps. The values found up to 60 days were not significantly different from that one determined at the initial time. However, with 90 days of storage there was a change in the conjugate profile that impaired its quantification. The methodology developed and validated will be used to evaluate the conjugate batch that will be submitted to Phase III clinical studies and in the routine quality controlof the conjugate vaccine. Moreover, the acquiredknowledge could be used in quality control of other conjugate vaccines against encapsulated bacteria of epidemiological importancein the country.

In the search for a vaccine providing comprehensive protection against meningococcal disease, one vaccine currently under development contains the immunogenic proteins PorA and FetA in meningococcal outer membrane vesicles (OMVs). To achieve high levels of coverage against disease-causing isolates, the antigenic variability of these proteins could be overcome using knowledge of meningococcal epidemiology and population structure. In this study, the possible implications of variable expression levels of PorA and FetA on vaccine efficacy were investigated. Producing OMVs containing consistent amounts of FetA is difficult due to iron-repressed expression; therefore, meningococcal strains were constructed which constitutively expressed FetA at increased levels for OMV vaccine production and analysis. In mice, OMVs from modified strains induced antibodies against both PorA and FetA. These antibodies acted synergistically in a serum bactericidal assay; however, antibodies against FetA were weakly bactericidal alone. The potential to increase levels of PorA- and FetA-specific bactericidal antibodies with a prime-boost strategy, using OMV and protein inoculums, was also tested. While successful for a weakly-immunogenic PorA variant, a similar strategy did not increase bactericidal activity against FetA. Although antibodies against FetA can be induced following OMV immunisation, sufficient antigen expression in target bacteria is also required for bactericidal killing; therefore, the variability and regulation of porA and fetA transcription was investigated in a range of isolates. Despite differences in regulation among clonal complexes, variable expression is unlikely to be an issue for vaccine coverage. In particular, regulation of fetA by iron is reduced in many isolates due to a deletion in the sequence bound by the regulatory protein, Fur. Therefore, a vaccine targeting PorA and FetA may provide high levels of protection against meningococcal disease; however, an alternative formulation or immunisation strategy is required to improve coverage against FetA.

RTX (Repeat in ToXin) superfamily consists of many proteins divided into several groups according to their different functions and characteristics: toxins, metalloproteases, lipases, proteins of the S-layer, bacteriocins and proteins with unknown function. However, all of them can be characterized by the following features: i) they contain tandemly repeated (6-50) nonapeptide glycine-rich calcium-binding consensus sequences GGXGXDX[L/I/V/W/Y/F]X (where X is any amino acid residue) in the C-terminal part of the protein. The presence of these repeats is a sine qua non condition for RTX protein family membership; ii) secretion from the cell occurs without a periplasmic intermediate by a mechanism which involves recognition of a signal sequence at the C-terminus of the protein by membrane-associated proteins that export the toxin across a channel spanning the entire bacterial envelope directly to the outside of the cell (Type I Secretion System); iii) the genes for protein synthesis, activation and secretion are mostly grouped together on the chromosome and form rtx operons. RTX toxins are the largest protein group of the RTX family. To this group belong mostly the proteins with molecular weight ranging from 100 to 200 kDa, with posttranslational fatty acid acylation mediated by a specific activating...

Les espèces Neisseria gonorrhoeae et Neisseria meningitidis, sont des bactéries pathogènes obligatoires de l'Homme, qui ont acquis différents mécanismes de défense pour détecter et combattre le stress oxydant généré par les mécanismes de défense de l'hôte lors de l'infection. La protéine PilB périplasmique, ferait partie de ces mécanismes et serait de ce fait associée à leur pathogénicité. PilB est composée de trois domaines : un domaine N-terminal (Nter) à activité disulfure oxydoréductase, et les domaines central et C-terminal à activité Méthionine Sulfoxyde Réductase (Msr) respectivement de classe A et B. L'étude des domaines isolés de PilB avait montré que le domaine Nter réduit sélectivement le domaine MsrB. Par ailleurs, le domaine Nter présente un repliement de type DsbE. Les DsbE sont des disulfure oxydoréductases périplasmiques impliquées dans la maturation des cytochromes c. En particulier, la DsbE1 de N. meningitidis a été identifiée par le Dr Adeline Gand lors de son doctorat.Lors de ma thèse, l'étude des protéines PilB de N. meningitidis et de Fusobacterium nucleatum m'a permis de montrer que : 1) la sélectivité de réduction du domaine Nter pour le domaine MsrB n'est pas conservée, 2) la sélectivité de réduction des domaines Nter observée sur les domaines isolés n'est pas retrouvée sur les PilB entiers ; et 3) dans tous les PilB, la réduction du domaine MsrB par le domaine Nter peut se faire selon un mécanisme intramoléculaire. De plus, nous avons étudié in vivo l'effet de la délétion du gène pilB sur la survie d'une souche de N. meningitidis en présence d'agents oxydants. D'autre part, le domaine N-terminal de la protéine DsbD (nDsbD) de N. meningitidis a été identifié comme étant le réducteur périplasmique de PilB et de la DsbE1 de N. meningitidis. Enfin, la caractérisation de l'activité apocytochrome c réductase de la DsbE1 de N. meningitidis a été complétée par des approches in vitro et in vivo chez N. meningitidis / The Neisseria gonorrhoeae and Neisseria meningitidis species are human obligatory pathogenic bacteria, which acquired various defense mechanisms to detect and fight oxidative stress generated by mechanisms of host defense during infection. The periplasmic PilB protein, specific to these bacteria, would be part of such mechanisms and would be associated with their pathogenicity. PilB is composed of three domains: an N-terminal domain (Nter) with disulfide oxidoreductase activity, and central and C-terminal domains with Methionine sulfoxide reductase activity (Msr) of A and B class respectively. The study of isolated domains of PilB showed that the Nter domain selectively reduced MsrB domain. Moreover, this Nter domain presents a DsbE-fold. The DsbE are periplasmic disulfide oxidoreductases involved in the maturation of cytochrome c. In particular, Dr. Adeline Gand identified the DsbE1 from N. meningitidis during his PhD. During my PhD, the study of PilB proteins from N. meningitidis and Fusobacterium nucleatum allowed me to show that: 1) the selective reduction of Nter domain for the MsrB domain is not conserved, 2) the selective reduction of Nter domains observed on the isolated domains is not found in entire PilB, and 3) in all PilB, the MsrB domain reduction by Nter domain could be an intramolecular mechanism. Moreover, we studied the in vivo effect of the pilB gene deletion on the survival of a strain of N. meningitidis in the presence of oxidants. And, the N-terminal domain of DsbD protein (nDsbD) from N. meningitidis was identified as the reducing partner of periplasmic PilB and DsbE1 of N. meningitidis. Finally, the characterization of apocytochrome c reductase activity of DsbE1 N. meningitidis was complemented by in vitro and in vivo approaches in N. meningitidis

Neisseria meningitidis ist mit jahrlich etwa 700.000 Erkrankungsfallen weltweit und einer Mortalitat von circa 7% einer der häufigsten Ausloser der bakteriellen Hirnhautentzündung. Der entscheidende Schritt zur Auslosung einer Meningitis ist die Uberwindung der Blut-Hirn-Schranke. Diese im menschlichen Korper einmalig dichte Barriere wird maßgeblich durch Tight-Junctions spezialisierter Endothelzellen der Hirnkapillaren aufrecht erhalten. Ob N. Meningitidis diese Barriere auf einem parazellulären oder transzellulärem Weg uberwindet, ist nicht vollstandig geklart. In dieser Arbeit wurde der Einfluss von N. meningitidis auf die Tight-Junction Proteine Occludin und ZO-1 unter Nutzung des HBMEC Zellkulurmodelles untersucht. Neben einer verminderten Genexpression von Occludin zeigte sich dabei eine Abspaltung eines 50 kDa Fragmentes von Occludin. Gleichzeitig konnte eine Umverteilung von Occludin von den Zellgrenzen in das Zytoplasma beobachtet werden. ZO-1 hingegen wurde weder in seiner Exprimierung, noch in seiner intrazellularen Verteilung beeinflusst. Mittels eines in dieser Arbeit etablierten Assays zur Bestimmung der Permeabilitat eines HBMEC-Monolayer als vereinfachtes in-vitro Modell der Blut-Hirn-Schranke konnte bestatigt werden, dass durch die Beeinflussung von Tight-Junction Proteinen die parazellulare Permeabilitat steigt. In weiteren Analysen konnten diese Prozesse auf eine gesteigerte Aktivitat von Matrixmetalloproteinase 8 zurückgefuhrt werden. Die Ergebnisse dieser Arbeit zeigen einen neuen Mechanismus auf, durch den N. meningitidis im Stande ist, die-Hinr-Schranke auf einem parazellulärem Weg zu überwinden. / Neisseria meningitidis is a leading cause of bacterial meningitis with about 700.000 cases per year and a mortality of approximately 7%. A crucial step in the pathogenesis of meningococcal meningitis is the crossing of the blood-brain barrier (BBB). This barrier mainly consists of tight junctions of the microvascular endothelial cells. It remains unclear whether N. meningitidis passes the BBB by a paracellular or transcellular route. In this work the influence of N. meningitidis on the tight junction proteins occludin and ZO-1 was investigated by using the HBMEC cell-culture model. Using QRT-PCR a reduced expression of occludin was seen, western-blot analyses showed a cleavage of occludin into a fragment of 50 kDA. Immonulourescence further showed a complete dissociation of occludin from the cell membrane, whereas ZO-1 was not influenced. By establishing a permeability-assay an increased permeability of the HBMEC monolayer after infection with N. meningitidis was demonstrated. Further work showed, that these effects are dependent of an increased activity of matrix metalloproteinase-8. These results reveal a new mechanism that could enable N. meningitidis to cross the BBB by a paracellular route.

Neisseria meningitidis is a facultative human pathogen that occasionally shows strong resistance against serum complement exposure. Previously described factors that mediate meningococcal serum resistance are for example the capsule, LPS sialylation, and expression of the factor H binding protein. I aimed for identification of novel serum resistance factors, thereby following two approaches, i) the analysis of the impact of global regulators of gene expression on serum resistance; and ii) a comparative analysis of closely related strains differing in serum resistance. (i) Of six meningococcal global regulators of gene expression studied, only mutation of the zinc uptake regulator Zur reduced complement deposition on meningococci. Little was known about meningococcal Zur and regulatory processes in response to zinc. I therefore elucidated the yet unidentified meningococcal Zur regulon comparing the transcriptional response of the N. meningitidis strain MC58 under zinc-rich and zinc-deficient conditions using a common reference design of microarray analysis. The meningococcal Zur regulon comprises 17 genes, of which 15 genes were repressed and two genes were activated at high zinc condition. Amongst the Zur-repressed genes were genes involved in zinc uptake, tRNA modification, and ribosomal assembly. A 23 bp meningococcal consensus Zur binding motif (Zur box) with a conserved central palindrome was established (TGTTATDNHATAACA) and detected in the promoter region of all regulated transcriptional units (genes/operons). In vitro binding of meningococcal Zur to the Zur box of three selected genes was shown for the first time using EMSAs. Binding of meningococcal Zur to DNA depended specifically on zinc, and mutations in the palindromic sequence constrained Zur binding to the DNA motif. ii) Three closely related strains of ST-41/44 cc from invasive disease and carriage which differed in their resistance to serum complement exposure were analysed to identify novel mediators of serum resistance. I compared the strains’ gene content by microarray analysis which revealed six genes being present in both carrier isolates, but absent in the invasive isolate. Four of them are part of two Islands of horizontally transferred DNA, i.e. IHT-B and –C. The working group furthermore applied a comprehensive screening assay, a transcriptome and a proteome analysis leading to identification of three target proteins. I contributed to establish the role of these three proteins in serum resistance: The adhesin Opc mediates serum resistance by binding of vitronectin, a negative regulator of the complement system; the hypothetical protein NMB0865 slightly contributes to serum resistance by a yet unknown mechanism; and NspA, recently identified to bind the negative complement regulator factor H, led to considerable reduced complement-mediated killing. / Neisseria meningitidis ist ein fakultatives Humanpathogen, welches mitunter sehr resistant gegenüber Serumkomplement-Exposition ist. Bereits beschriebene Faktoren, welche die Serumresistenz von Meningokokken fördern, sind beispielsweise die Kapsel, LPS-Sialylierung und Expression des fH-bindenden Proteins. Das Ziel dieser Arbeit war die Identifikation neuartiger Serumresistenzfaktoren, wobei ich zwei Ansätzen verfolgte: i) Die Analyse des Einflusses von globalen Regulatoren der Genexpression auf die Serumresistenz; und ii) eine vergleichenden Analyse von eng verwandten Stämmen, die sich hinsichtlich ihrer Serumresistanz unterschieden. i) Von sechs untersuchten globalen Regulatoren der Genexpression, war die Komplementdeposition auf Meningokokken nur nach Mutation des Regulators der Zinkaufnahme, Zur, reduziert. Über Zur selbst und die regulatorischen Prozesse in Reaktion auf Zink war in Meningokokken wenig bekannt. Ich habe daher das bisher nicht bestimmte Zur-Regulon von Meningokokken aufgeklärt, wofür ich mittels Mikroarrays die transkriptionelle Antwort des N. meningitidis-Stammes MC58 unter Zink-Überfluss und Zink-Mangel zu vergleichen. Das Zur-Regulon von Meningokokken umfasst 17 Gene, von denen unter Zinküberfluss 15 reprimiert und zwei aktiviert wurden. Unter den Zur-reprimierten Genen fanden sich Gene, die in die Aufnahme von Zink, die Modikation von tRNAs und den Zusammenbau des Ribosoms involviert sind. Ein 23 bp langes Binde-Konsensusmotiv für Meningokokken-Zur (Zur-Box) mit einem konservierten zentralen Palindrom wurde ermittelt (TGTTATDNHATAACA) und in der Promotorregion aller regulierten Transkriptionseinheiten (Gene/Operons) detektiert. In vitro-Bindung des N. meningitidis Zur an die Zur-Box dreier ausgewählter Genen konnte mittels EMSAs erstmals gezeigt werden. Die Bindung von Zur an DNA war spezifisch abhängig von Zink, und Mutationen in der palindromischen Sequenz hemmten die Zur-Bindung an das DNA-Motiv. ii) Drei eng verwandte Stämme des ST-41/44-Komplexes aus invasiver Erkrankung und Trägertum, die sich in ihrer Resistenz gegenüber Serumkomplement-Exposition unterschieden, wurden analysiert um neuartige Mediatoren der Serumresistenz zu identifizieren. Der Gengehalt der Stämme wurde mittels Mikroarray-Analyse verglichen. Dies offenbarte sechs Gene, die in den beiden Trägerstämmen vorhanden, aber in dem invasiven Isolat abwesend waren. Vier dieser Gene liegen innerhalb zweier Inseln horizontal transferierter DNA, d.h. IHT-B und –C. Weiterhin führte die Arbeitsgruppe eine Transkriptom- und Proteom-Analyse der drei Stämme sowie einen umfangsreichen Screening-Assay durch. Diese Ansätze führten zur Identifikation dreier Kandidaten-Proteine für die weitere Analyse. Ich wirkte daran mit, die Rolle dieser Proteine für die Serumresistenz von Meningokokken zu ermitteln: Das Adhäsin Opc vermittelt Serumresistenz durch Bindung von Vitronektin, einem negativen Regulator des Komplementsystems; das hypothetische Protein NMB0865 trägt über einen bisher unbekannten Mechanismus geringfügig zur Serumresistanz bei; und NspA, für welches vor Kurzem erkannt wurde, dass es den negativen Komplementregulator Faktor H bindet, führte zu beträchtlich reduzierter Abtötung durch Komplement.

Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions. Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed. Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments. / Neisseria meningitidis ist ein kommensal lebendes, fakultativ pathogenes Bakterium, welches unter nicht vollständig verstandenen Umständen lebensbedrohliche Krankheitsbilder bei Menschen verursacht. Aktuelle Studien haben gezeigt, dass die stringente Antwort einen Einfluss auf die bakterielle Virulenz haben kann. Aus diesem Grund untersucht diese Arbeit die Regulation der stringenten Antwort, insbesondere die Rolle von RelA, sowie den Einfluss der stringenten Antwort auf die Ex-vivo-Fitness der Meningokokken. Die Ergebnisse wurden für den Trägerstamm α522 und den hyperinvasiven Stamm MC58 erhoben und miteinander verglichen. Wachstumsexperimente zeigten, dass sich beide Wildtyp-Stämme in ihren Ex-vivo-Phänotypen nicht unterscheiden. Jedoch wurden mittels quantitativer Echtzeit-PCR (qRT-PCR) Unterschiede zwischen beiden Stämmen in der Genexpression von relA gefunden. Zudem war die α522 relA Mutante im Gegensatz zu der MC58 relA Mutante nicht in der Lage, in menschlichem Vollblut zu überleben. Allerdings zeigten in Saliva und Liquor beide Stämme den gleichen Phänotyp. Außerdem war der Trägerstamm auf eine kurze, nicht-codierende AT-reiche Region (ATRrelA) in der Promotorregion von relA angewiesen, um im menschlichen Blut überleben zu können. Darüber hinaus zeigten Zellkulturexperimente mit humanen Epithelzellen, dass die Deletion relA die Invasionsrate in beiden Stämmen signifikant verringerte, obwohl die Adhäsionsrate durch die Deletion unbeeinflusst blieb. Um besser verstehen zu können, weshalb die Deletion von relA unter bestimmten Bedingungen letal ist, wurden mit In-silico- und experimentellen Analysen nach Unterschieden in den Aminosäurebiosynthesewegen beider Stämme gesucht. Es zeigte sich, dass Stamm MC58 in der Lage ist alle 20 Aminosäuren zu synthetisieren, während Stamm α522 eine Auxotrophie für Cystein und Glutamin aufweist. Ferner zeigten die In-vitro-Wachstumsversuche, dass RelA bei Aminosäuremangel essentiell für beide Stämme ist. Darüber hinaus zeigte eine MC58 Mutante mit einer ATRrelA –Kopie in der relA Promotorregion ein im Vergleich zum Wildtyp-Stamm verbessertes Wachstum in mit L-Cystein und/oder L-Glutamin angereichertem Minimalmedium. Gegensätzlich dazu zeigte der Stamm α522 keine Unterschiede im Wachstum zwischen dem Wildtyp und einer ATRrelA Deletions-Mutante. Dies deutet darauf hin, dass ATRrelA an dem komplexen regulatorischen Zusammenspiel der stringenten Antwort und dem L-Cystein- beziehungsweise dem L-Glutamin-Metabolismus beteiligt ist. Es lässt sich vermuten, dass RelA zu der Virulenz von Meningokokken in einer stamm- und umgebungsspezifischen Weise beiträgt. Abschließend hebt diese Arbeit die Rolle von kleinen regulatorischen Elementen für die bakterielle Virulenz hervor und postuliert, dass die Virulenz der Meningokokken auf ihrer Fähigkeit basiert, sich der durch den Wirt gegebenen Umgebung anzupassen.