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Descrição de um novo clone de Neisseria meningitidis Sorogrupo C, Grande São Paulo, 1990 a 2003 / Emergence of new clone of Neisseria meningitidis Serogroup C in Grande São Paulo, 1990 a 2003Ana Paula Silva de Lemos 23 September 2005 (has links)
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.
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Haem utilisation by pathogenic neisseriaeTurner, Paul Christopher January 2000 (has links)
No description available.
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Complications and sequelae of meningococcal disease in Québec, 1990-1994Erickson, Lonny January 1998 (has links)
Objectif : To determine the frequency and the nature of complications and sequelae of serogroup B and serogroup C meningococcal disease, during a recrudescence caused by a virulent clone of serogroup C, serotype 2a Neisseria meningitidis. To evaluate the quality of life of survivors. Methods. The study population included all cases of culture-proven serogroup B and C meningococcal disease reported in the province of Quebec, Canada, between 1 January 1990 and 31 December 1994. Complications and sequelae were assessed by review of medical files, postal questionnaires, and telephone interviews. Results. There were 167 cases of serogroup B and 304 cases of serogroup C infection. The largest number of cases was observed in the under 1 year age group for serogroup B and in the 10-19 year age group for serogroup C. Fatality rates were 7% for serogroup B and 14% for serogroup C. %). Only 3% of survivors of serogroup B cases had physical sequelae. 15% of survivors of serogroup C infection had one or more significant physical sequelae (skin scars 12%, amputations 5%, significant sensorineural hearing loss 2%, renal failure 1%, other sequelae 4%. Among cases without identified physical sequelae who completed the questionnaire, 19% reported a reduction in their quality of life attributable to the disease. Conclusions. These results confirm the gravity of disease caused by serogroup C, serotype 2a Neisseria meningitidis and support vaccination for control of outbreaks and epidemics of disease caused by this particular strain.
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Construction of a novel epitope expression vector based on the B-subunit of the diphtheria toxinJohnson, Nicholas January 1993 (has links)
No description available.
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Bedeutung des Klasse A Scavenger Rezeptors für die Zytokinsekretion von humanen dendritischen Zellen nach Kontakt mit dem humanen Pathogen Neisseria meningitidis / Influence of the class A scavenger receptor for the cytokine secretion by human dendritic cells after contact with the human pathogen Neisseria meningitidisVillwock, Andrea January 2008 (has links) (PDF)
Meningokokken gehören zu den wichtigsten Erregern bakterieller Sepsis und Meningitis. Der Schweregrad des Krankheitsverlaufs bei Meningokokkenerkrankungen korreliert mit der Konzentration an proinflammatorischen Zytokinen im Serum. Dendritische Zellen (DZ) bilden die erste Abwehr am humanen Epithel des Nasopharynx, welches die Eingangspforte von Neisseria meningitidis darstellt und sind eine wichtige Quelle proinflammatorischer Zytokine. In dieser Arbeit konnte gezeigt werden, dass bekapselte Meningokokken-Stämme bei DZ signifikant weniger proinflammatorische Zytokine als isogene Kapsel-defiziente Stämme oder obligat unbekapselte Stämme induzieren. Dieser Effekt ist unabhängig von der chemischen Zusammensetzung der Kapsel, da aufgereinigtes Kapselpolysaccharid der Serogruppe B nicht den reduzierenden Effekt der Zytokininduktion beeinflusste. Darüber hinaus spielt die Kapsel-O-Acetylierung bei Serogruppe C, W-135 und Y nur eine untergeordnete Rolle bei der Erkennung von Meningokokken durch DZ. Microarray Versuche zum Transkriptionsprofil von DZ, die mit dem konstitutiv unbekapselten Trägerisolat alpha 14, dem bekapselten MC58 oder dem isogenen unbekapselten Stamm MC58siaD durchgeführt wurden, zeigten nach 4 h ein identisches Profil von proinflammatorischen Zytokinen. Nur der phagozytierte unbekapselten Stamm MC58siaD zeigte eine differentielle Regulation von weiteren Zytokinen. Jedoch glich sich das Profil nach 18 h Infektion durch alle drei Stämme an. Der Scavenger Rezeptor der Klasse A (SR-A) wurde als Hauptrezeptor identifiziert, der die Erkennung und Phagozytose von Meningokokken durch DZ initialisiert. Eine Assoziation phagozytierter Meningokokken mit SR-A konnte mittels Elektronenmikroskopie bestätigt werden. Nach Infektion von THP-1 Makrophagen mit bekapselten Serogruppe B und C Stämmen, den isogenen Kapsel-defizienten Stämmen und dem obligat unbekapselten Stamm alpha 14 wurde auf Transkriptionsebene keine differentielle Regulation der SR-A nachgewiesen. Lediglich eine minimale Hochregulation des SR-A auf der zellulären Oberfläche konnte nach einer Stunde Infektion verzeichnet werden. Nach Infektion von DZ oder THP-1 Makrophagen mit MC58siaD kommt es zur Dephosphorylierung des SR-A. Unter Verwendung von globalen Phagozytose-Inhibitoren konnte gezeigt werden, dass für die maximale Induktion der proinflammatorischen Zytokine TNF-alpha, IL-6 und IL-1 Phagozytose von N. meningitidis benötigen wird, dies ist jedoch für die IL 8 Produktion nicht notwendig. Außerdem konnte gezeigt werden, dass mit dem spezifischen SR-A Inhibitor poly G eine Reduktion von TNF-alpha, IL-6, IL-1 und IL-8 zu verzeichnen war. Folglich ist die Phagozytose über SR-A nötig, um TNF-alpha, IL-6 und IL-1 zu induzieren, jedoch nur die Erkennung aber nicht die Phagozytose via SR-A die IL-8 Produktion initiiert. Die Aufnahme von Neisseria meningitidis über den SR-A durch DZ ist damit nicht nur für die Phagozytose und Abtötung verantwortlich sondern auch für die Zytokininduktion wichtig ist. Es gibt jedoch auch Meningokokken Stämme, die nicht vom SR-A erkannt werden. Mit alpha 14 konnte erstmals ein Meningokokken-Stamm identifiziert werden, der nicht an SR-A bindet. Die Induktion von Zytokinfreisetzung durch alpha 14 erfolgt dementsprechend unabhängig von SR-A und nach Kontakt von alpha 14 mit humanen DZ ist keine Veränderung der Phosphorylierungsstatus dieses Rezeptors zu beobachten. Die erhobenen Daten legen eine zentrale Rolle von SR-A in der Induktion von Immunität gegen N. meningitidis nahe. / Meningococci belong to the most important pathogens which cause bacterial sepsis and meningitis. The severity and outcome of meningococcal disease correlates with the concentration of proinflammatory cytokines in the serum. Dendritic cells (DC) build a first line of defence within the nasopharyngeal epithelium, which is the port-of-entry for Neisseria meningitidis and are an important source of proinflammatory cytokines. Infection of DC with encapsulated meningococcal strains induces significantly lower levels of proinflammatory cytokines than isogenic capsule-deficient mutants or constitutively unencapsulated strains. It could be shown that this effect does not depend on the chemical composition of the meningococcal capsule, because purified capsular polysaccharide of serogroup B is unable to modulate cytokine secretion. O-acetylation of serogroup C, W-135 and Y capsules plays a minor role in the recognition of meningococci by DC. Microarray studies on the expression of cytokine genes by DC infected with constitutively unencapsulated carriage isolate alpha 14, encapsulated serogroup B strain MC58 or the isogenic unencapsulated strain MC58siaD showed an identical pattern for proinflammatory cytokines after 4 h of infection. Only the unencapsulated strain MC58siaD which is easily phagocytosed induced additional cytokines. However, the cytokine profile after 18 h of infection was equal for all three strains. Scavenger receptor A (SR-A) was identified as the main receptor mediating recognition and phagocytosis of meningococci by DC. Association of phagocytosed meningococci with SR-A was visualized by electron microscopy. Infection of THP-1 macrophages with encapsulated serogroup B und C strains, their isogentic capsule-deficient muntants or the obligatory unencapsulated strain alpha 14 did not induce differential regulation of SR-A on the transcriptional level and only minor up-regulation of SR-A on cellular surface after one hour of infection. After infection of DC or THP-1 macrophages with MC58siaD dephosphorylation of SR-A can be detected. Using global phagocytosis inhibitors, it could be determined that the induction of the proinflammatory cytokines TNF-alpha, IL-6 and IL-1 require phagocytosis of N. meningitidis, whereas induction of IL-8 does not. In addition, the specific SR-A-inhibitor poly G also leads to a reduction of TNF-alpha, IL-6, IL-1 and IL-8 secretion. Taken together these data indicate that phagocytosis via SR-A is necessary for TNF-alpha, IL-6 and IL-1 secretion, whereas recognition but not phagocytosis via SR-A enhances IL-8 secretion. Therefore, the uptake of Neisseria meningitidis via SR-A is not only important for phagocytosis and killing but also involved in cytokine induction. However, not all meningococcal strains are recognized via SRA. The cytokine induction by DZ infected with alpha 14 is completely independent of SR-A and after contact of human DC with alpha 14 no changes in SR-A phosphorylation can be detected. These data suggest that SR-A plays a central role in the induction of immunity against N. meningitidis.
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Interaktion pathogener Neisserien mit zellulären Rezeptoren: Molekulare Untersuchungen zu neisseriellen Adhäsinen und ihrer Wechselwirkung mit humanen CEACAMs / Interaction of pathogenic Neisseria with cellular receptors: Molecular analysis of neisserial adhesins and their interaction with human CEACAMsKüspert, Katharina January 2007 (has links) (PDF)
Neisseria meningitidis und Neisseria gonorrhoeae sind humanpathogene Vertreter der Gattung Neisseria. Diese Erreger verfügen über eine Reihe von Virulenzfaktoren, um erfolgreich den menschlichen Körper zu besiedeln. Dabei spielen die neisseriellen OpaCEA-Proteine eine wichtige Rolle. Diese Adhäsine binden an bestimmte Rezeptoren der humanen CEACAM-Familie, die auf unterschiedlichen Zelltypen im menschlichen Körper exprimiert werden. Die Interaktion der OpaCEA-Proteine mit der stark konservierten aminoterminalen Domäne von bestimmten CEACAMs führt zu einer Aufnahme der Bakterien in die Zellen. Dort können sie, vor der humoralen Immunantwort geschützt, persistieren oder sich durch Transzytose in tiefer gelegene Gewebe weiter ausbreiten und letztendlich eine disseminierte Krankheit auslösen. Da CEACAMs eine entscheidende Rolle bei der Infektion mit pathogenen Neisserien spielen, wurde in der vorliegenden Arbeit die Interaktion dieser zellulären Rezeptoren mit pathogenen Neisserien und die daraus resultierenden molekularen Ereignisse auf bakterieller bzw. zellulärer Seite näher untersucht. Zunächst wurde eine neuartige Methode entwickelt, die im Gegensatz zu herkömmlichen Strategien eine schnelle und quantitative Erfassung von Neisserien-CEACAM-Interaktionen ermöglicht. Bei dieser Methode wurden GFP-fusionierte, lösliche aminoterminale CEACAM-Domänen eingesetzt, die spezifisch an OpaCEA-exprimierende Bakterien binden. Einzelne Bakterien einer Population, die mit den fluoreszierenden Rezeptordomänen assoziierten, konnten aufgrund ihrer erhöhten Fluoreszenz im Durchflußzytometer sehr schnell und quantitativ bestimmt werden. Diese Rezeptordomänen wurden außerdem als molekulares Werkzeug zur Erstellung eines OpaCEA-induzierten Transkriptionsprofils von Opa-positiven Meningokokken verwendet. Transkriptomanalysen mittels Mikroarrays zeigten, dass die Interaktion OpaCEA-exprimierender Meningokokken mit der löslichen, aminoterminalen Domäne von CEACAM1 eine Herrunterregulation von 56 Genen sowie eine Hochregulation von sieben Genen in Neisseria meningitidis MC58 zur Folge hatte. Dabei konnte ein hochreguliertes Gen identifiziert werden, dessen Genprodukt aufgrund seiner Homologie zu einem bakteriellen -Hämolysin möglicherweise virulenz-assoziiert ist. Die Erstellung dieses Transkriptionsprofils beruhte auf der Interaktion zwischen der aminoterminalen Domäne von CEACAM1 und seinen bis heute einzig bekannten neisseriellen Liganden, den OpaCEA-Proteinen. Bemerkenswerterweise konnte im Rahmen der vorliegenden Arbeit ein Opa-negativer Meningokokkenstamm isoliert werden, der ebenfalls an CEACAM1 bindet und von CEACAM1-exprimierenden Zellen internalisiert wird. Da dieser Meningokokkenstamm keine Opa-Proteine exprimierte, muß er über ein weiteres Adhäsin verfügen, das mit CEACAM1 assoziiert. Interessanterweise konnte gezeigt werden, dass für die CEACAM1-vermittelte Aufnahme dieser Opa-negativen Meningokokken mehrere extrazelluläre Domänen des Rezeptors notwendig sind. Im Gegensatz zur Aufnahme OpaCEA-exprimierender Bakterien war die aminoterminale Domäne essentiell, aber nicht ausreichend für diesen phagozytischen Vorgang, der unabhängig vom Aktinzytoskelett erfolgte. Auch bei Bindungsstudien mit löslichen CEACAM1 Konstrukten gab es Differenzen zwischen den opaquen und nicht-opaquen Bakterienstämmen. So zeigte sich, dass im Gegensatz zu OpaCEA-exprimierenden Meningokokken, die mit monomeren Formen des löslichen Rezeptors assoziieren konnten, Opa-negativen Meningokokken nur mit multimerisierten Formen des Rezeptors interagierten. CEACAM1 stellt den einzigen Rezeptor aus der CEACAM-Familie dar, mit dem Opa-negative Meningokokken interagieren können. Demgegenüber assoziieren OpaCEA-exprimierende Neisserien mit mehreren Mitgliedern der CEACAM-Familie, unter anderem mit CEACAM3. In der vorliegenden Arbeit konnte gezeigt werden, dass die c-Jun N-terminale Kinase an Signaltransduktionswegen, die durch Interaktion von OpaCEA-exprimierenden Neisserien mit CEACAM3 ausgelöst wurden, beteiligt ist. Erstaunlicherweise konnte durch Inhibition der c-Jun N-terminalen Kinase CEACAM3-vermittelte Aufnahme der opaquen Bakterien in die Zelle reduziert werden. Da die Aktivierung der c-Jun N-terminalen Kinase unabhängig von der Phosphorylierung der ITAM-ähnlichen Sequenz erfolgte, scheint dieses Molekül an einem neuartigen Signalweg beteiligt zu sein, der komplementär zu bereits bekannten CEACAM3-vermittelten Signalprozessen abläuft. Die in der vorliegenden Arbeit zusammengefassten Befunde liefern neue Einblicke in die Wechselwirkung zwischen pathogenen Neisserien und ihren Wirtszellen und können als Ausgangspunkt für interessante weiterführende Analysen dienen. / The human specific pathogens Neisseria meningitidis and Neisseria gonorrhoeae have developed several mechanisms to colonize their human host. Colonization can be mediated by binding of neisserial OpaCEA proteins to members of the human CEACAM-family, which are expressed in various human tissues and cell types. Interaction of OpaCEA proteins with the aminoterminal domain of these receptors results in the uptake of Neisseria into the cells, allowing the bacteria to escape host defences and to persist within the host. Besides, bacteria can transcytose across the mucosal barrier and cause systemic dissemination. As CEACAMs play an important role during infection with pathogenic Neisseria the aim of this doctoral thesis was to investigate Neisseria-CEACAM interactions and resulting molecular events on bacterial and cellular levels. Therefore, a novel method was developed which allows rapid and quantitative analysis of Neisseria-CEACAM association. Soluble GFP-tagged aminoterminal domains of CEACAMs are utilized for recognition of CEACAM-binding bacteria. Bacteria binding this receptor domains are therefore labelled with a fluorescent protein. Analysis via flow cytometry allows a rapid and specific detection of receptor binding events on a single bacteria level . The soluble receptor domains were also used as a molecular tool for microarray analysis to assess OpaCEA induced changes on transcriptional level in Opa-positive Neisseria meningitides MC58. Interaction of OpaCEA-expressing meningococci with soluble aminoterminal domains of CEACAM1 resulted in downregulation of 56 bacterial genes and upregulation of seven other genes. Interestingly, one of these upregulated genes might be involved in virulence due to its homology to an -hemolysin of other bacteria. OpaCEA proteins are the only neisserial proteins known that associate with CEACAM1. However, in this study a meningococcal strain was isolated, which does not express Opa-proteins but nevertheless specifically interacts with CEACAM1. Therefore, these Opa-negative meningococci must express an additional adhesin, which mediates CEACAM1-interaction. Internalisation of Opa-negative meningococci via CEACAM1 requires several extracellular domains of this receptor. In contrast to OpaCEA-expressing bacteria, the aminoterminal domain was necessary but not sufficient for internalisation. Differences between CEACAM1-binding of Opa-negative and Opa-positive meningococci were also observed by binding-assays with soluble CEACAM1 receptor. While Opa-positive meningococci associated with monomeric forms of soluble CEACAM1, Opa-negative meningococci bound exclusively to multimeric forms of soluble CEACAM1. CEACAM-interactions with Opa-negative meningococci were only observed for CEACAM1. OpaCEA-expressing Neisseria can interact with several members of the CEACAM family, amongst others CEACAM3. Interestingly, interaction of OpaCEA-expressing Neisseria with CEACAM3 causes the activation of c-Jun N-terminal kinase. Inhibition of this kinase resulted in a reduced CEACAM3-mediated uptake of Neisseria into the cells. Remarkably, activation of c-Jun N-terminal kinase was independent of phosphorylation of the ITAM-like sequence of CEACAM3. Therefore, c-Jun N-terminal kinase seems to be involved in a novel signaling pathway, complementary to known CEACAM3-mediated signaling processes. The results presented in this study give new insights regarding the interaction of pathogenic Neisseria and host cells and pave the way for further interesting studies.
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Vergleichende Proteomanalyse von Biofilmen und planktonischen Zellen bei dem humanen Infektionserreger Neisseria meningitidis / Comparative proteomic analysis of biofilms and planktonic cells of the human pathogen Neisseria meningitidisvan Alen, Tessa January 2010 (has links) (PDF)
Neisseria meningitidis ist ein humaner Infektionserreger, der Meningitis und Sepsis hervorruft. Das asymptomatische Trägertum im Nasenrachenraum ist entscheidend für die Übertragung des Bakteriums und dessen Interaktion mit dem menschlichen Wirt. Frühere Beobachtungen legen die Annahme nahe, dass Meningo¬kokken im Tonsillengewebe in einem biofilmähnlichen Stadium vorliegen. Daher werden in vitro Biofilme als Modell für das Trägertum verwendet. Expressionsunterschiede zwischen Biofilmen und planktonisch gewachsenen pathogenen Neisserien wurden in wenigen Transkriptomanalysen untersucht, während bisher keine Proteomanalysen durchgeführt wurden. Kartierungen des Proteoms und des Immunoproteoms von Meningokokken liegen allerdings vor. In dieser Studie wurde das Biofilmproteom des unbekapselten N. meningitidis Stammes WUE3671 im Vergleich zum Proteom der planktonisch gewachsenen Bakterien untersucht. Dazu wurde ein auf Silikonschläuchen basierendes Biofilmmodell mit kontinuierlichem Fluss etabliert. Es erfolgte eine Anreicherung bakterieller Biomasse über 48 h, wobei die kolonie-bildenden Einheiten bei 24 h ein Plateau erreichten. Licht- und Elektronen¬mikroskopie belegten die deutliche Zunahme der Biomasse über 48 h und zeigten zudem eine Struktur-ierung des 48 h Biofilms in eine apikale Region mit überwiegend vitalen Meningokokken und eine basale Region mit einer verstärkten Anzahl von Bakterien mit avitalem Erscheinungs-bild. Das Proteom von N. meningitidis Biofilmen, die 24 beziehungsweise 48 h gewachsen waren, wurde mit dem einer exponentiell gewachsenen planktonischen Kultur mit 2D-Gelelektro¬phorese verglichen. Unterschiedlich exprimierte Proteine wurden mit Massen-spektrometrie identifiziert und die Ergebnisse mit Spectral Counting und, wenn möglich, mit spezifischen Antikörpern abgesichert. Die Expression von ungefähr 2 % aller Proteinspots im Biofilm unterschied sich von der in planktonischen Zellen wenigstens um das 2-fache. Es wurden Veränderungen beobachtet, die mit einem Nährstoff- und Sauerstoffmangel sowie einer Zunahme von reaktiven Sauerstoffspezies (reactive oxygen species, ROS) in Verbindung gebracht werden können. Die Expression der Proteine SodC und MntC war im Biofilm deutlich erhöht, was mutmaßlich auf ROS im Biofilm zurückzuführen ist. In dieser Arbeit konnte gezeigt werden, dass MntC in der Tat essentiell für Biofilmwachstum, nicht aber für planktonisches Wachstum ist. Die Daten zu SodC und MntC legen die Hypothese nahe, dass Meningokokken im Biofilm trainiert werden mit Mediatoren des Immunsystems, wie ROS, umzugehen. Zudem wird NMB0573, ein Lrp-Homolog, als wesentlicher globaler Regulator für metabolische Anpassungen im Biofilm postuliert. Es konnte über die Proteomanalyse hinaus gezeigt werden, dass die Adhäsine Opc und Opa, die unter der Kontrolle von NMB0573 stehen, im Biofilm vermindert exprimiert werden. / Neisseria meningitidis is a human pathogen that causes meningitis and sepsis. Asymptomatic nasopharyngeal carriage is crucial for the transmission of the bacterium and its interaction with the human host. Previous observations led to the assumption that meningococci persist in the tonsillar tissue in a biofilm-like state. Therefore in vitro biofilms are used to model carriage. Despite successful mapping of the meningococcal proteome and immuno-proteome, no proteome and only a few transcription analyses investigated the differences between biofilm and planktonic growth in pathogenic Neisseria. This study investigated the biofilm proteome of the unencapsulated N. meningitidis strain WUE3671 in comparison to planktonically grown bacteria. A continuous flow biofilm model based on silicone tubes was established. There was an accumulation of bacterial biomass over 48 h with the number of colony forming units reaching a plateau at 24 h. Light- and electron microscopy confirmed biomass accumulation and revealed a structuring of the 48 h biofilm in an apical region with predominantly vital meningococci and a basal region with increased numbers of bacteria with avital appearance. The proteomes of N. meningitidis biofilms grown for 24 or 48 h, respectively and of exponentially grown planktonic cultures were compared by 2D-gelelectrophoresis. Differentially expressed proteins were identified by mass spectrometry. The results were confirmed by spectral counting and, if available, with specific antibodies. Approximately 2 % of all protein spots in the biofilm were at least 2 fold differentially expressed in comparison to planktonic cells. Changes related to nutrient and oxygen limitation and increase of reactive oxygen species (ROS) were observed. There was an increased expression of SodC and MntC in the biofilm that supposedly is caused by ROS in the biofilm. MntC was specifically required for meningococcal biofilm formation, but not for planktonic growth. Data for SodC and MntC suggest that meningococci in the biofilm are trained to cope with mediators of the immune system like ROS. Moreover, NMB0573, an Lrp-like global regulator, was implicated as a possible mediator of metabolic adaption in the biofilm. Independent of the proteome analysis, the NMB0573-dependent adhesions Opc and Opa were in addition shown to be down-regulated within the biofilm.
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Molecular characterisation of Neisseria meningitidis serogroup B isolates in South Africa, 2002- 2006Moodley, Chivonne 17 October 2011 (has links)
MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2011 / Despite being a fulminant pathogen, Neisseria meningitidis (meningococcus) is
part of the commensal flora of the human nasopharynx. Globally, five
meningococcal serogroups (A, B, C, Y and W135) cause the majority of invasive
disease. Most serogroup B cases occur sporadically but may be endemic or
epidemic within a geographic region. In South Africa, there are limited data on
invasive serogroup B clones and the antigenic diversity of certain meningococcal
outer membrane proteins. This study examined the molecular epidemiology of
serogroup B meningococci in South Africa from 2002 through 2006.
Invasive meningococcal isolates were submitted to a national laboratory-based
surveillance system. For this study, serogroup B isolates were characterised by
pulsed-field gel electrophoresis (PFGE), PorA, FetA and multilocus sequence
(MLST) typing. PorA, FetA and multilocus sequence (MLST) typing were
performed on all 2005 isolates (n=58) and randomly selected isolates from other
years (n=25).
A total of 2144 invasive cases were reported over the study period. Of these, 76%
(1627/2144) had viable isolates available for serogrouping and 307 (19%) were
serogroup B. Serogroup B cases were reported from across the country however
the majority were from the Western Cape province. The highest incidence of
serogroup B was in children less than 5 years of age.
Isolates displayed a high level of diversity by PFGE. Despite this diversity the
majority of serogroup B meningococci collected over the 5-year period could be
grouped into several clonal clusters representative of global invasive MLST
clonal complexes. Overall, the most predominant MLST clones in South Africa
were ST-32/ET-5 and ST-41/44/lineage 3. In addition, at least 19 PorA types and
16 FetA types were determined among selected isolates.
Globally invasive serogroup B disease is caused by heterogeneous strains
however, prolonged outbreaks in several countries have been due to strains of
the ST-32/ET-5 and ST-41/44/lineage 3 clonal complexes. At present, serogroup
B disease in South Africa is not dominated by an epidemic clone, however, global
clonal complexes ST-32/ET-5 and ST-41/44/lineage 3 are circulating in Western
Cape and Gauteng, respectively.
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Potential coverage of an investigational, multi-component, meningococcal vaccine with a focus on the ST-269 clonal complexLucidarme, Jay January 2012 (has links)
Development of a broadly cross-protective capsular group B meningococcal (MenB) vaccine has been hampered by poor capsular immunogenicity and often diverse and poorly cross-protective subcapsular antigens. The MenB MC58 strain genome has facilitated the discovery of novel, relatively conserved vaccine candidates. The four-component MenB (4CMenB) investigational vaccine contains factor H-binding protein (fHbp; variant 1), neisserial heparin-binding antigen (NHBA), Neisserial adhesin A (NadA) and PorA P1.4-containing outer membrane vesicles. The latter are known to elicit protection against homologous strains. Clinical trials have demonstrated protective responses in infants and adults against isolates expressing homologous PorA or fHbp (subvariant 1.P1), or heterologous NadA (variant 2). Cross-protective responses have also been demonstrated in adults and, to a lesser extent, infants, against isolates expressing heterologous fHbp variant 1 subvariants. The contribution of NHBA is still poorly understood. MenB currently accounts for 87% of invasive meningococcal disease in England and Wales. The proportion of disease due to the ST-269 clonal complex (cc269) peaked at 45.6% in 2006 and is currently approximately 24.2%. The aims of this study were (i) to genotypically assess potential 4CMenB coverage against recent English and Welsh invasive disease isolates and, specifically, cc269 isolates from England and Wales and other countries, (ii) to compare phenotypic expression levels of the 4CMenB antigens (excluding PorA) among typical cc269 isolates, and (iii) to assess 4CMenB responses against typical cc269 isolates among healthy adults administered 4CMenB.Full length alleles for fHbp variant 1, NHBA and NadA variants 1, 2 and 3 were present in 64.6%, >99% and 7.1%, respectively, of English and Welsh invasive disease isolates from 2007/8. Between 67.5% and >99% (adults) or 25.7% and 43.5% (infants) of the isolates were predicted to be covered by 4CMenB. cc269 comprised two antigenically distinct lineages (clusters) centred around ST-269 and ST-275, respectively. These accounted for 57% and 40% of cc269 in 2007/8. Both clusters effectively lacked nadA and PorA P1.4. The predominant fHbp;NHBA profiles represented by the respective clusters were 1.P15;P0021 and (1.P13 or 2.P19);P0017. Between 77.4% and 100% (adults) or 2.2% and 27.1% (infants) of cc269 isolates from 2007/8 were predicted to be covered by 4CMenB. Estimates for infants were conservative due to e.g. the exclusion of NHBA. Serum bactericidal antibody (SBA) analyses targeting typical fHbp variant 1-expressing cc269 strains, indicated high levels of coverage among adults administered 4CMenB. Notable differences among genotypically matched isolates e.g. in terms of SBA geometric mean titres, were not reflected in the relative fHbp and NHBA expression levels. Such differences may lead to conflicting estimates of coverage in infant populations. Whilst these are investigated further it seems prudent to use typical isolates giving mid-range responses when assessing SBA, and therefore protection, among infants. Potential 4CMenB coverage of cc269 and the broader meningococcal population in England and Wales was high among adults and encouraging among infants when compared to that of existing MenB vaccines.
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The detection of meningococcal disease through identification of antimicrobial peptides using an in silico model creationAbdullah, Gadija January 2019 (has links)
Philosophiae Doctor - PhD / Neisseria meningitidis (the meningococcus), the causative agent of meningococcal disease (MD) was identified in 1887 and despite effective antibiotics and partially effective vaccines, Neisseria meningitidis (N. meningitidis) is the leading cause worldwide of meningitis and rapidly fatal sepsis usually in otherwise healthy individuals. Over 500 000 meningococcal cases occur every year. These numbers have made bacterial meningitis a top ten infectious cause of death worldwide. MD primarily affects children under 5 years of age, although in epidemic outbreaks there is a shift in disease to older children, adolescents and adults. MD is also associated with marked morbidity including limb loss, hearing loss, cognitive dysfunction, visual impairment, educational difficulties, developmental delays, motor nerve deficits, seizure disorders and behavioural problems. Antimicrobial peptides (AMPs) are molecules that provide protection against environmental pathogens, acting against a large number of microorganisms, including bacteria, fungi, yeast and virus. AMPs production is a major component of innate immunity against infection. The chemical properties of AMPs allow them to insert into the anionic cell wall and phospholipid membranes of microorganisms or bind to the bacteria making it easily detectable for diagnostic purposes. AMPs can be exploited for the generation of novel antibiotics, as biomarkers in the diagnosis of inflammatory conditions, for the manipulation of the inflammatory process, wound healing, autoimmunity and in the combat of tumour cells. Due to the severity of meningitis, early detection and identification of the strain of N. meningitidis is vital. Rapid and accurate diagnosis is essential for optimal management of patients and a major problem for MD is its diagnostic difficulties and experts conclude that with an early intervention the patient’ prognosis will be much improved. It is becoming increasingly difficult to confirm the diagnosis of meningococcal infection by conventional methods. Although polymerase chain reaction (PCR) has the potential advantage of providing more rapid confirmation of the presence of the bacterium than culturing, it is still time consuming as well as costly. Introduction of AMPs to bind to N. meningitidis receptors could provide a less costly and time consuming solution to the current diagnostic problems. World Health Organization (WHO) meningococcal meningitis program activities encourage laboratory strengthening to ensure prompt and accurate diagnosis to rapidly confirm the presence of MD. This study aimed to identify a list of putative AMPs showing antibacterial activity to N. meningitidis to be used as ligands against receptors uniquely expressed by the bacterium and for the identified AMPs to be used in a Lateral Flow Device (LFD) for the rapid and accurate diagnosis of MD.
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