Reactivation of latent infection is the major cause of tuberculosis (TB). Cholesterol is a critical carbon source during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the sterol-ring degradation and is essential for intracellular survival. NAT from M. tuberculosis (TBNAT) can utilise propionyl-CoA and therefore was proposed as a target for TB-drug development. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. NAT inhibitors, including the piperidinol class, were identified by high-throughput screening. The insolubility of recombinant TBNAT has been a major limitation in pursuing it as a drug target. Subcloning tbnat into a pVLT31 vector resulted in a yield of 6-16 mg/litre-bacterial-culture of pure-soluble recombinant TBNAT. The increased yield allowed for extensive screening for crystallisation conditions. However, since a structure was not obtained, the model NAT from M. marinum (MMNAT) was employed to further understand NAT as a target. Screening against a panel of Acyl-CoA cofactors showed that MMNAT can also utilise propionyl-CoA. The MMNAT structure in complex with the high affinity substrate hydralazine was determined (2.1 Å) and the architecture of the arylamine pocket was delineated. A novel mechanism for the acetylation reaction of hydralazine has emerged. It is proposed that the acetyl group is transferred from acetyl-CoA to the heterocyclic aromatic nitrogen of hydralazine, which explains the immediate cyclisation of the acetylated metabolite into an N-methyltriazolophthalazine. By employing mass spectroscopy, enzyme assays, computational docking and structural studies, a covalent mechanism of inhibition by the piperidinol class was established, and the inhibitor-binding pocket was identified. Inhibitors with new scaffolds were identified using the in silico 3D-shape screening and thermal shift assay.

Le TMC207 est un nouvel antituberculeux appartenant à la famille des diarylquinolines qui inhibe très efficacement l'ATP synthase des mycobactéries. Dans le but de cartographier les interactions entre le TMC207 et sa cible et de comprendre le mécanisme d'action exact de cette nouvelle drogue, nous avons sélectionné in vitro des mutants résistants au TMC207 à partir de plusieurs espèces mycobactériennes. Six mutations distinctes ont été identifiées dans l'anneau c de l'ATP synthase: D28G, D28A, L59V, E61D, A63P et I66M. L'effet de ces mutations dans la résistance a été évalué en mesurant le niveau de résistance conféré dans les clones résistants, ainsi que dans un système de complémentation chez M. smegmatis. Les résultats ont été interprétés grâce à la construction d'un modèle structural de l'anneau c, utilisé pour faire des expériences de docking avec le TMC207. Nos résultats montrent que les résidus substitués dans les clones résistants définissent une poche localisée entre deux sous-unités c adjacentes dans l'anneau, englobant le site de fixation du proton, qui permet la stabilisation du TMC207. La drogue bloque ainsi le transfert des protons et la synthèse d'ATP. Pour finir, nous avons mis au point l'expression et la purification de l'ATP synthase mycobactérienne afin d'initier l'étude structurale de ce macro-complexe en microscopie électronique et en cristallographie des protéines. Les résultats obtenus en microscopie électronique en coloration négative nous ont permis d'obtenir les premières images de l'ATP synthase de M. smegmatis

Submitted by Diogo Misoguti (diogo.misoguti@gmail.com) on 2018-04-18T13:18:24Z No. of bitstreams: 1 Tese-15860.pdf: 879437 bytes, checksum: c869f5350ca0a0b0f13be8ba348f8fa5 (MD5) / Approved for entry into archive by Mariusa Loução (mariusa.loucao@unifesp.br) on 2018-04-18T13:20:15Z (GMT) No. of bitstreams: 1 Tese-15860.pdf: 879437 bytes, checksum: c869f5350ca0a0b0f13be8ba348f8fa5 (MD5) / Made available in DSpace on 2018-04-18T13:20:15Z (GMT). No. of bitstreams: 1 Tese-15860.pdf: 879437 bytes, checksum: c869f5350ca0a0b0f13be8ba348f8fa5 (MD5) Previous issue date: 2016 / O uso da biologia molecular como ferramenta de diagnóstico microbiológico vem se expandindo no setor da medicina laboratorial. Novas plataformas aparecem com a prerrogativa de facilitar e acelerar o processo de análise. O uso de sistemas automatizados, que realizam extração, amplificação e detecção de ácidos nucleicos dentro da mesma plataforma, permite maior precisão e facilidade ao desenvolvimento de um novo teste por apresentarem todos os processos acoplados e reagentes disponíveis para o processo. Dentre as plataformas automatizadas, uma das que se destacam é o BD Max™ (Becton Dickinson Diagnostics). O sistema BD Max é uma plataforma automatizada aberta, que combina a extração de ácidos nucleicos, PCR (Reação de Polimerização em Cadeia) em tempo real e detecção dentro do mesmo instrumento, oferecendo a opção de usar os testes aprovados pelo Food and Drug Administration (FDA) e também, testes desenvolvidos pelo usuário. Com o objetivo de testar os recursos que a modalidade aberta da plataforma BD Max oferece, foram desenvolvidos três estudos distintos para o diagnóstico molecular de agentes infecciosos direto de amostras clínicas. Estudo 1: O objetivo deste estudo foi validar um teste multiplex usando a tecnologia PCR em tempo real no sistema aberto BD MAX™, para detectar o complexo Mycobacterium tuberculosis (CMT), complexo Mycobacterium avium (CMA) e Mycobacterium spp. (PAN) diretamente de amostras clínicas. Quando os resultados do novo teste foram comparados com os resultados da cultura, a reação de PCR apresentou especificidade de 97,1%, 100% e 100% para CMT, CMA e PAN, respectivamente. Estudo 2: O objetivo deste estudo foi validar um teste multiplex usando a tecnologia PCR em tempo real no sistema aberto BD MAX™ para detectar o grupo Mycobacterium abscessus (GMA), complexo Mycobacterium fortuitum (CMF) e Mycobacterium chelonae (MC), diretamente de amostras clínicas. Quando os resultados do novo teste foram comparados com os resultados da cultura, uma concordância de 97%, 100% e 99% para GMA, CMF e MC, respectivamente foi observada. Estudo 3: O objetivo deste estudo foi validar um teste multiplex usando a tecnologia PCR em tempo real no sistema aberto BD MAX™ para detectar e identificar Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) e Stenotrophomonas maltophilia (SM) diretamente de amostras respiratórias de pacientes portadores de Fibrose Cística (FC). Quando os resultados do novo teste foram comparados com os resultados da cultura, uma alta concordância foi observada entre as duas metodologias. Conclusão: Os 3 testes desenvolvidos provaram ser específicos e sensíveis para detectar por PCR em tempo real microrganismos causadores de infeção direto da amostra clínica. A plataforma automatizada BD Max provou ser uma excelente ferramenta para a realização de testes moleculares automatizados. / The use of molecular biology as a tool for microbiology diagnostic has been expanding in the laboratory routine. Despite of the strong growth of the area, companies can not afford the demand about epidemiological changes around the world and for this reason laboratories opt to develop their own methods. New platforms appear with the prerogative to facilitate and accelerate the analysis process. The use of automated sample-in results-out platforms allows higher precision and facilitates the development of a new test by presenting all attached processes and reagents available for the test. Among platforms that best fits this profile, the BD Max™ (BD Diagnostics) is one of the most used ones. The BD Max system is an automated open platform that combines extraction and real time PCR in the same instrument, offering the option of using tests approved by the FDA or the open platform mode for userdeveloped test. In order to explore the BD Max open mode platform, three differents studies were developed to detect the microorganisms that causes infection directly from clinical samples. Study 1: A multiplex real time PCR was validated on the BD MAX™ open mode system to detect Mycobacterium tuberculosis complex (MTC), Mycobacterium avium complex (MAC) and Mycobacterium spp. (PAN) directly from clinical specimens. When compared to culture results, the new BD MAX PCR test presented specificities of 97.1%, 100% and 100% for MTC, MAC and PAN, respectively. Study 2: A multiplex real time PCR was validated on the BD MAX™ open mode system to detect Mycobacterium abscesses Group (MAG), Mycobacterium fortuitum complex (MFC) and Mycobacterium chelonae (MC) directly from clinical specimens. When compared to culture results, the new BD Max PCR test presented an overall agreement of 97%, 99% and 100% for the detection of MAG, MFC and MC, respectively. Study 3: A multiplex real time PCR was validated on the BD MAX™ open mode system to detect Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) directly from clinical specimens collected from Cystic Fibrosis patients. When culture results were compared to the new BD Max PCR test results, a high overall agreement were observed between both methodologies. Conclusion: All 3 tests proved to be specific and sensitive to detect different microorganisms associated with infections, directly from clinical samples. The BD Max proved to be an excellent tool for automated molecular tests.

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Bovine tuberculosis (BTB) is a zoonotic disease that affects domestic and wild animals, and humans. It is caused by Mycobacterium bovis (M. bovis) and has a wide host range. The effective control of BTB is of paramount importance and this can be achieved through the use of accurate and comprehensive diagnostic tests. The most widely used methods to detect BTB are the skin test and in vitro gamma interferon assay which do not detect anergic animals, but serological tests such as ELISA and fluorescence polarization assay (FPA) have been found promising in ancilliary tuberculosis diagnosis. The overall aim was to study M. tuberculosis complex (MTBC) protein, mycobacterial protein bovis 70 (MPB70) as a target for serological assays in the detection of antibodies to bovine tuberculosis. The MPB70 protein was expressed, purified and labeled with fluorescein (FITC). The mpb70 gene was fragmented into three regions without disrupting predicted epitopes. The resulting protein Fragments were expressed as fusion proteins with the monster green fluorescent protein (MGFP). The recombinant MPB70 (rMPB70) and the expressed gene fragments 2&3 were tested in immunoblots and ELISAs. The rMPB70 and fragment 2-MGFP reacted with chicken antibodies raised against rMPB70 and immune sera from BTB infected buffaloes. MPB70 peptides were synthesized as an approach to identify even smaller antigenic regions. The peptides BT1G (residues 31-45) and BT51L (residues 81-95) were recognised by anti-MPB70 chicken antibodies in the ELISA and fall within fragment 1 and 2, respectively. The tracers (rMPB70-FITC, fragment 2-MGFP fusion and peptides BT1G&BT51L) were tested in the FPA, but the results failed to distinguish between immune sera from chickens immunized with rMPB70 and negative control sera. Even though the FPA was not successful, the MPB70 fragment 2-MGFP fusion protein, which was recognized by sera from BTB infected buffaloes, was tested in an ELISA using panels of sera from uninfected and naturally M. bovis infected buffaloes and cattle. The diagnostic performance of the ELISA was, however, overall unsatisfactory and hence of very limited use as a serological test to detect antibody responses to BTB as a stand-alone assay. Sera from some of the animals gave false positive reactions indicating that MPB70 was not sufficiently specific for serodiagnosis of M. tuberculosis complex infections. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Gegenstand dieser Arbeit ist die Untersuchung zellulärer Testsysteme bei Patienten mit Verdacht auf latente Infektion mit M. tuberculosis. Eine latente Tuberkulose kann unter Immunsuppression zu einer aktiven Tuberkulose werden. Deshalb wird bei immunsuppressiven Therapien insbesondere mit TNF-alpha-Blockern eine Chemoprävention empfohlen. Daher ist es sehr wichtig, latente Infektionen zu erkennen. Ist ein Patient mit M. tuberculosis infiziert, reagieren seine T-Zellen auf Stimulation mit Antigenen wie ESAT-6 und CFP-10. Diese Immunantwort ist die Grundlage der modernen IFN-γ release Assays (IGRA). ESAT-6 und CFP-10 fehlen bei allen BCG-Stämmen und bei den meisten nicht-tuberkulösen Mykobakterien mit Ausnahme von M. kansasii, M. szulgai und M. marinum (Andersen et al. 2000; Behr et al, 1999; Lalvani 2003). Im Gegensatz dazu haben Personen, die mit M. tuberculosis-Komplex-Organismen infiziert sind, in der Regel T-Zellen im Blut, die diese und andere mykobakterielle Antigene erkennen. Ziel der vorliegenden Arbeit war es, die Nutzung von IGRA in der medizinischen Labordiagnostik dahingehend zu analysieren, ob es Unterschiede zwischen ELISPOT-basierten Tests und Röhrchen-Tests als Testformat gibt, inwieweit diese Tests im klinischen Alltag verlässlich sind und ob sich mit einem anderen Auswertealgorithmus eine sicherere Aussage zum Vorliegen einer latenten Tuberkulose treffen lässt. Im Einzelnen wurden dabei drei Ansätze verfolgt: 1. In einer retrospektiven Studie wurden die Ergebnisse von 2686 Patienten ausgewertet, die im Labor des Instituts für Klinische Immunologie des Universitätsklinikums Leipzig von 2013 bis 2016 als Routineuntersuchungen erhoben wurden. Bei klinisch unplausiblen Ergebnissen wurden bei einem Teil der Patienten eine Wiederholungsuntersuchungen durchgeführt. Die analytische Sensitivität und Spezifität sowie den positiven und negativen prädiktiven Wert konnten wir nur unter der Annahme abschätzen, dass der Ausfall in der Wiederholungsuntersuchung einen Hinweis auf falsch- oder richtig-positive oder -negative Werte zulässt. Wir kommen damit zu einer Sensitivität von nur 28 %, einer Spezifität von immerhin 91 %, einem positiven prädiktiven Wert von 32 % und einem negativen prädiktiven Wert von 90 %. Damit sind 68 % der positiven Werte falsch positiv und 10 % der negativen Werte falsch negativ. Unsere Untersuchungen zur Wiederholbarkeit der ELISPOT-Tests im eigenen Labor bestätigen, dass negative Ergebnisse meist wiederholbar sind, positive Werte jedoch skeptisch betrachtet werden müssen. 2. Seit 2012 sendet Instand e.V. zweimal jährlich Ringversuchsproben für den IGRA aus (Ringversuch 650, https://www.instand-ev.de/). Wir analysierten, wie viele Labors bei Teilnahme an der externen Qualitätssicherung (sogenannten Ringversuchen) für IGRA ein korrektes Ergebnis erzielt hatten und ob Unterschiede zwischen ELISPOT-Assay und Röhrchen-Test bestehen. Im Ringversuch waren die Ergebnisse von ELISPOT (z.B. TB-Spot, Oxford Immunotec) und Röhrchentest (Quantiferon Gold bzw. Gold-Plus, Qiagen oder Diasorin) bis auf den 2. Ringversuch 2019 vergleichbar und unterschieden sich nicht. 3. Obwohl die meisten Labore an den Ringversuchen erfolgreich teilnehmen, ist die recht häufige Anzahl vor allem falsch positiver Ergebnisse im diagnostischen Alltag problematisch. Wir haben daher in einem dritten Untersuchungsschritt die Validierungsdaten eines Labors detailliert untersucht, um ein definiertes Verfahren zur Ermittlung positiver Testergebnisse vorzuschlagen. Zwischen 2011 und 2013 erfolgte im Labor Ettlingen eine umfangreiche Qualitätssicherung des ELISPOT unter Nutzung von 70 Proben in Doppelbestimmung. Dabei wurden jeweils die Messungen für die Positiv- und die Negativkontrolle sowie die Werte nach Stimulation mit ESAT-6 und CFP-10 analysiert. Um relevante Unterschiede zwischen Negativkontrolle und der eigentlichen Messung zu bewerten, wurden die Unterschiede mit der Wiederholpräzision in Beziehung gesetzt: Die Unterschiede sollten größer sein als die daraus abgeleitete Ungenauigkeit (Impräzision) der Messungen. Daraus wurde ein Cut-off-Wert kalkuliert, der unmittelbar auf den Daten des Labors beruht. Hierzu ist die Homogenität der Standardabweichungen über alle Proben erforderlich. Sie wird durch die Wurzeltransformation aller Daten erreicht. Für die verwendeten Daten ist sie anwendbar (Altman 1991, Bland 2000). Dies bedeutete bei den untersuchten Daten, dass bei Doppelbestimmungen die Unterschiede zwischen dem Testergebnis immer um den Faktor 0,76 größer sein muss als die Negativkontrolle. Dazu sind allerdings zwei Voraussetzungen zu erfüllen: 1. die Werte müssen vor Analyse wurzeltransformiert werden. 2. benötigt werden mindestens Doppelbestimmungen. Da keine Doppelbestimmungen vorliegen, konnte das Verfahren nicht an einem eigenen Datensatz verifiziert werden. Wir schlussfolgern daraus zusammenfassend: 1. in der Praxis gibt es keine wesentlichen Unterschiede zwischen ELISPOT und Röhrchen-Test als Testformat, was Sensitivität und Spezifität anbelangt 2. IGRAs sind ein verlässliches Werkzeug im klinischen Alltag, insbesondere wenn es um den Ausschluss einer latenten Tuberkulose geht 3. die Auswertung von ELISPOT-Daten lässt sich über Mehrfachbestimmung und quadratwurzeltransformierte Auswertung optimieren (wobei das noch in einer Folgearbeit zu beweisen ist).:Inhalt Abkürzungsverzeichnis 3 1 EINFÜHRUNG 5 1.1 Epidemiologie der Tuberkulose 5 1.2 Pathogenese der Tuberkulose 8 1.3 Prävention der Tuberkulose 10 1.4 Diagnostik der Tuberkulose 11 1.4.1 Radiologische Untersuchungen 12 1.4.2 Mikrobiologische Untersuchungen 12 1.4.3 Molekularbiologischer Nachweis 13 1.5 Nachweis der Immunreaktion gegenüber M. tuberculosis 14 1.5.1 Tuberkulin-Hauttest 14 1.5.2 Serologische Tests auf M. tuberculosis-Infektion 16 1.5.3 Zelluläre Labortests auf M. tuberculosis-Reaktivität 18 2 AUFGABENSTELLUNG 22 3 MATERIAL UND METHODEN 23 3.1 Patienten 23 3.2 Blutentnahme und Zellpräparation 23 3.3 ELISPOT 25 3.4 Ringversuch zur externen Qualitätssicherung 26 3.5 Retrospektive Analyse der Daten des eigenen Labors und der Ringversuchsdaten 26 3.6 Analyse der Validierungsdaten aus Ettlingen 27 4 ERGEBNISSE 29 4.1 Analyse der ELISPOT-Daten des eigenen Labors 29 4.2 Ergebnisse des Instand-Ringversuchs 33 4.3 Analyse der ELISPOT-Wiederholungsmessungen 35 5 DISKUSSION 41 6 Zusammenfassung 48 Literaturverzeichnis 52 Lebenslauf 63 Danksagung 64 Erklärung über die eigenständige Abfassung der Arbeit 65

No / BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is considered to be less transmissible due to the fitness cost associated with drug resistance-conferring mutations in essential genes. OBJECTIVE: To test the hypothesis that TB drug resistance-conferring mutations with fitness cost are more frequent among human immunodeficiency virus (HIV) positive than among HIV-negative patients. DESIGN: We analysed all strains from the two TB drug resistance surveys conducted in Uganda between 2008 and 2011. Strains phenotypically susceptible to rifampicin and/or isoniazid were assumed to be wild-type; in all other cases, we performed whole-genome sequencing. Mutations at the rpoB531 and katG315 codons were considered without fitness loss, whereas other rpoB codons and non-katG were considered with fitness loss. RESULTS: Of the 897 TB patients, 286 (32.1%) were HIV-positive. Mutations with fitness loss in HIV-positive and HIV-negative patients were respectively as follows: non-531 rpoB: 1.03% (n = 3), 0.71% (n = 4) (OR 1.46, 95%CI 0.58-3.68); non-katG: 0.40% (n = 1), 1.0% (n = 6) (OR 0.40, 95%CI 0.07-2.20); rpoB531: 1.49% (n = 4), 0.69% (n = 4) (OR 2.29, 95%CI 0.83-5.77); katG315: 3.86% (n = 11), 2.55% (n = 15) (OR 1.54, 95%CI 0.81-2.90). The odds of mutations with and without fitness cost were higher for patients with a history of previous anti-tuberculosis treatment. CONCLUSIONS: Our data do not support the hypothesis that resistance-conferring mutations with fitness cost are likely to be often present in HIV-positive individuals.

Made available in DSpace on 2016-12-23T13:56:05Z (GMT). No. of bitstreams: 1 Maria Carolina Campana - texto completo.pdf: 1739324 bytes, checksum: 2c77a0541ea8a062d1765a13e6b4e9e5 (MD5) Previous issue date: 2010-09-02 / Immunity against tuberculosis depends on the establishment of an effective Th1 response, with IFN- production, a pivotal cytokine to the activation of macrophages and bacilli destruction. It is known that several factors are related to the host s resistance to tuberculosis. In the present work, the influence of T regulatory cells (Tregs) in the immune response against Mtb was evaluated in whole blood and PBMC cultures from tuberculin skin test positive (TST+) and tuberculin skin test negative (TST-) individuals. Paralelly, we also evaluated if different levels of resistance to anti-mycobacterial drugs displayed by different Mtb clinical isolates could interfere with the cytokine profile, Tregs frequency, and with its proliferative capacity on both PBMC and whole blood cultures from TST+ and TST- subjects. Twenty-two healthy volunteers were invited to participate in our study, 11 TST+ and 11 TST-. Twelve Mtb clinical isolates were used, 6 drug-resistant and 6 drug-susceptible. Our data shows that differences in drug susceptibility did not affected any of the evaluated parameters. In the other hand, TST status correlated with differences in Treg frequencies, which was significantly elevated among TST+ when compared to TSTsubjects. The increase in Tregs was also associated with a decrease in the microbicidal effect observed in whole blood cultures from TST+, when compared to TST-. This association was confirmed through the depletion of Tregs, which restored the microbicidal activity and an increase in IFN-, IL-2, IL10, and TNF- when compared to undepleted cultures. Data presented here supports the hypothesis that pre-exposure to MTB, represented by a positive TST result, may increase the number of Treg cells, which in turn could affect negatively the establishment of a protective immunity against TB in TST+ individuals / A resposta imune contra a tuberculose é mediada por uma resposta do tipo Th1, com a produção de IFN-γ, citocina fundamental para a ativação dos macrófagos e, consequentemente para a eliminação do bacilo. Vários fatores podem estar relacionados com a resistência a tuberculose. Neste trabalho, foi avaliado o impacto das células T reguladoras na resposta imune contra o Mtb em infecções de amostras de sangue total e CMSP de indivíduos positivos para o teste com PPD (PPD+) e negativos ao teste com PPD (PPD-). Além disso, avaliamos também se o perfil de resistência dos isolados de Mtb tem relação com o perfil de citocinas secretadas, com a frequência de células T reguladoras e com sua capacidade proliferativa em culturas de sangue total e CMSP dos indivíduos estudados. Para isso, foram arrolados 22 voluntários saudáveis. Desses indivíduos, 11 eram PPD+ e 11 eram PPD-. Foram também utilizados 12 isolados de Mtb, sendo 6 sensíveis e 6 resistentes a drogas antituberculose. Não foram encontradas diferenças entre os perfis de resistência dos isolados de Mtb e os parâmetros avaliados. O número de células T reguladoras encontrado nos indivíduos PPD+ foi maior em relação aos PPD- e a presença dessas células afetou negativamente o efeito microbicida nas culturas de sangue total de indivíduos PPD+, nas quais os isolados de M. tuberculosis proliferaram mais. Quando foram feitas infecções com isolados de Mtb em culturas de CMSP nas quais as células T reguladoras foram depletadas uma diminuição na proliferação do Mtb foi encontrada tanto nas amostras de indivíduos PPD+ quanto PPD-. Essa alteração na proliferação do Mtb foi acompanhada de maiores níveis de INF-γ, IL-2, IL-10 e TNF-α em relação às culturas de CMSP na presença das células T reguladoras. Os resultados desse trabalho sugerem que a pré-exposiçã o ao Mtb, evidenciada pelo teste positivo com o PPD, pode aumentar o número de células T reguladoras que, por sua vez, podem prejudicar a resposta imune protetora contra a tuberculose nos indivíduos PPD+