Made available in DSpace on 2018-08-01T23:27:52Z (GMT). No. of bitstreams: 1 tese_11949_Dissertação Final - Brena Ramos Athaydes20180417-130407.pdf: 2565845 bytes, checksum: 072a11064c6f5b933c9397ac59474608 (MD5) Previous issue date: 2018-03-28 / A úlcera péptica é uma das doenças mais comuns que afetam a população mundial. É caracterizada por um desbalanço entre os fatores protetores (produção de muco e bicarbonato, antioxidantes e prostaglandinas) e agressores (estresse oxidativo, anti-inflamatórios não-esteroidais e Helicobacter pylori) da mucosa gástrica. O processo inflamatório ulceroso induz a liberação de citocinas pró-inflamatórias e o estresse oxidativo, que cronicamente podem levar ao câncer gástrico. No Brasil, há relatos do uso das sementes de Persea americana Mill. (abacateiro) para o tratamento de doenças gástricas, no entanto, sem evidências científicas. Algumas pesquisas também já destacaram seus efeitos antioxidante e antimicrobiano. Nesse contexto, avaliamos a atividade antioxidante, anti-H. pylori, o efeito imunomodulador e o efeito antitumoral em células de adenocarcinoma gástrico, do extrato hidroalcoólico (SCE) e das frações acetato de etila (SEAP) e hexânica (SHP) obtidos a partir das sementes de abacate. SEAP apresentou os melhores resultados; portanto, também realizamos o perfil químico e o estudo dos efeitos gastroprotetores em modelo agudo de úlcera gástrica induzida por indometacina da SEAP, através de análise histológica e quantificação dos parâmetros bioquímicos da inflamação. SEAP e SHP foram eficientes em inibir o crescimento de células tumorais gástricas e na atividade anti-H. pylori, confirmada por alterações na morfologia bacteriana. A SEAP apresentou os melhores resultados na captura de ABTS+, DPPH, O2-, H2O2, HOCl e inibição da enzima HRP, além de modular a inflamação por inibir significativamente a produção de IL-6. O estudo cromatográfico por ESI FT-ICR MS da SEAP revelou a presença de flavonoides, fenilpropanoides e taninos, como ácido cafeioilquínico, catequina e epicatequina (confirmados por CLAE-DAD) e derivados de quercetina e kaempferol. SEAP reduziu as características das lesões gástricas nas análises macroscópica e histológica, além de aumentar a produção de muco. Nos parâmetros do estresse oxidativo, houve redução significativa dos níveis de AOPP e MDA com aumento da atividade da SOD. Estes resultados mostram que as sementes de P. americana Mill. são capazes de inibir as vias envolvidas na formação de úlcera e câncer gástrico devido à presença de composto fenólicos, sendo uma alternativa estratégica no tratamento de doenças gástricas. Palavras-chaves: Helicobacter pylori, Estresse Oxidativo, Persea americana, Polifenois, Gastroproteção.

Submitted by Beatriz de Oliveira Garcia null (garciab@ibb.unesp.br) on 2017-03-27T14:51:06Z No. of bitstreams: 1 Dissertação - Garcia BO.pdf: 1426165 bytes, checksum: 3c7112c540967acea714ff22b90c3b9d (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-03-29T18:55:41Z (GMT) No. of bitstreams: 1 garcia_bo_me_bot.pdf: 1426165 bytes, checksum: 3c7112c540967acea714ff22b90c3b9d (MD5) / Made available in DSpace on 2017-03-29T18:55:42Z (GMT). No. of bitstreams: 1 garcia_bo_me_bot.pdf: 1426165 bytes, checksum: 3c7112c540967acea714ff22b90c3b9d (MD5) Previous issue date: 2017-02-16 / Diversas doenças gástricas estão associadas à presença do microrganismo Helicobacter pylori (H. pylori) na mucosa gástrica. O método de diagnóstico não invasivo, simples, seguro e bastante recomendado é o teste respiratório com ureia marcada com o isótopo estável do Carbono (¹³C-UBT). Existem consenso no protocolo de aplicação do ¹³C-UBT, contudo a necessidade do jejum ainda é alvo de controvérsias, além disso, o ácido cítrico utilizado junto a ureia marcada torna o substrato azedo, dificultando a aplicação do ¹³C-UBT em crianças e idosos. Dessa forma, o objetivo deste trabalho foi avaliar se o jejum influencia no diagnóstico da infecção pelo H. pylori pelo ¹³C-UBT, analisado por espectrômetro de massa de razão isotópica específico, e verificar a necessidade do ácido cítrico junto com a 13C-ureia. Para isso foi selecionado 40 pacientes, sendo que 35 estavam infectados e 5 não infectados pelo H. pylori. O ¹³C-UBT foi aplicado em 40 pacientes nos tempos de jejum de 1h, 2h, 4h e 6h com ácido cítrico. Por fim, 30 pacientes infectados foram submetidos ao ¹³C-UBT em 1h de jejum sem o ácido cítrico. Assim obtivemos o turnover do ¹³C da expiração do paciente durante aplicação do ¹³C-UBT em diferentes tempos de jejum. Com comportamento da curva do turnover foi possível observar que o jejum não interfere no resultado final do ¹³C-UBT, apesar do turnover do ¹³C em menores tempos de jejum atingiram menores intensidades, esse comportamento pode ser explicado pelo gasto energético. Verificou-se também que o ácido cítrico não é necessário, pois durante o período pós-prandial há produção de ácido clorídrico, que pode substituí-lo. Concluímos que em 1h de jejum, com e sem o ácido cítrico é possível aplicar o ¹³C-UBT sem alterar a eficácia do diagnóstico da infecção pelo H. pylori. / Several gastric diseases are associated with the presence of the Helicobacter pylori (H. pylori) microorganism in the gastric mucosa. The non-invasive, simple, safe and highly recommended method of diagnosis is the urea breath test with the stable isotope of Carbon (¹³C-UBT). There is a consensus in the ¹³C-UBT application protocol, however, the need of fasting is still controversial, and the citric acid used together with the labeled urea makes the substrate sour, making it difficult to apply ¹³C-UBT in children and the elderly. Thus, the objective of this study was to evaluate whether fasting influences the diagnosis of H. pylori infection by ¹³C-UBT, analyzed by a specific isotope ratio mass spectrometer, and to verify the need for citric acid together with 13C-urea. For this purpose, 40 patients were selected, 35 of whom were infected and 5 were not infected by H. pylori. ¹³C-UBT was applied in 40 patients in the fasting times of 1h, 2h, 4h and 6h with citric acid. Finally, 30 infected patients were submitted to ¹³C-UBT in 1h of fasting without citric acid. Thus, we obtained the turnover of ¹³C of patient expiration during the application of ¹³C-UBT in different fasting times. With behavior of the turnover curve, it was possible to observe that fasting does not interfere with the final result of ¹³C-UBT, although turnover of ¹³C in shorter fasting times reached lower intensities, this behavior can be explained by energy expenditure. It was also found that citric acid is not necessary, since during the postprandial period there is production of hydrochloric acid, which can replace it. We conclude that in 1h of fasting, with and without citric acid, it is possible to apply ¹³C-UBT without altering the efficacy of the diagnosis of H. pylori infection.

Orientador: Vladimir Eliodoro Costa / Resumo: Diversas doenças gástricas estão associadas à presença do microrganismo Helicobacter pylori (H. pylori) na mucosa gástrica. O método de diagnóstico não invasivo, simples, seguro e bastante recomendado é o teste respiratório com ureia marcada com o isótopo estável do Carbono (¹³C-UBT). Existem consenso no protocolo de aplicação do ¹³C-UBT, contudo a necessidade do jejum ainda é alvo de controvérsias, além disso, o ácido cítrico utilizado junto a ureia marcada torna o substrato azedo, dificultando a aplicação do ¹³C-UBT em crianças e idosos. Dessa forma, o objetivo deste trabalho foi avaliar se o jejum influencia no diagnóstico da infecção pelo H. pylori pelo ¹³C-UBT, analisado por espectrômetro de massa de razão isotópica específico, e verificar a necessidade do ácido cítrico junto com a 13C-ureia. Para isso foi selecionado 40 pacientes, sendo que 35 estavam infectados e 5 não infectados pelo H. pylori. O ¹³C-UBT foi aplicado em 40 pacientes nos tempos de jejum de 1h, 2h, 4h e 6h com ácido cítrico. Por fim, 30 pacientes infectados foram submetidos ao ¹³C-UBT em 1h de jejum sem o ácido cítrico. Assim obtivemos o turnover do ¹³C da expiração do paciente durante aplicação do ¹³C-UBT em diferentes tempos de jejum. Com comportamento da curva do turnover foi possível observar que o jejum não interfere no resultado final do ¹³C-UBT, apesar do turnover do ¹³C em menores tempos de jejum atingiram menores intensidades, esse comportamento pode ser explicado pelo gasto energético. Verific... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Several gastric diseases are associated with the presence of the Helicobacter pylori (H. pylori) microorganism in the gastric mucosa. The non-invasive, simple, safe and highly recommended method of diagnosis is the urea breath test with the stable isotope of Carbon (¹³C-UBT). There is a consensus in the ¹³C-UBT application protocol, however, the need of fasting is still controversial, and the citric acid used together with the labeled urea makes the substrate sour, making it difficult to apply ¹³C-UBT in children and the elderly. Thus, the objective of this study was to evaluate whether fasting influences the diagnosis of H. pylori infection by ¹³C-UBT, analyzed by a specific isotope ratio mass spectrometer, and to verify the need for citric acid together with 13C-urea. For this purpose, 40 patients were selected, 35 of whom were infected and 5 were not infected by H. pylori. ¹³C-UBT was applied in 40 patients in the fasting times of 1h, 2h, 4h and 6h with citric acid. Finally, 30 infected patients were submitted to ¹³C-UBT in 1h of fasting without citric acid. Thus, we obtained the turnover of ¹³C of patient expiration during the application of ¹³C-UBT in different fasting times. With behavior of the turnover curve, it was possible to observe that fasting does not interfere with the final result of ¹³C-UBT, although turnover of ¹³C in shorter fasting times reached lower intensities, this behavior can be explained by energy expenditure. It was also found that citric acid is n... (Complete abstract click electronic access below) / Mestre

Resumo não disponível

Estima la prevalencia del agente infeccioso H. pylori en casos diagnosticados con cáncer gástrico en el Instituto Nacional de Enfermedades Neoplásicas (INEN) y evalúa su relación con características clínico patológicas del tumor y datos clínicos de pacientes diagnosticados con cáncer gástrico en INEN entre 2015 y 2016. Se realiza un estudio observacional descriptivo, retrospectivo, transversal. El análisis de los datos obtenidos se lleva a cabo entre marzo del 2016 y abril del 2017. La encuesta se realiza en el piso en donde se encuentran internados los pacientes, las biopsias se realiza en el departamento de cirugía menor. Los ensayos para determinar H. pylori se lleva a cabo en el banco de tejidos tumorales y el procesamiento de los datos se lleva a cabo en el departamento de investigación del INEN. Entre los años 2015 y 2016 se toma las muestras de cirugía y biopsia de 200 pacientes con diagnóstico de adenocarcinoma gástrico que acuden al INEN para que se le realicen biopsia o cirugía en el departamento de abdomen y que cumplían con todos los criterios de inclusión para el estudio. De los 200 casos se excluye 17 casos porque la muestra es insuficiente para la determinación de ureasa (ureA) y proteína de shock térmico (hspA) mediante PCR (reacción en cadena de polimerasa) a tiempo real en el tejido. Finalmente se obtiene datos concluyentes para H. pylori en 183 casos. Los resultados muestran que de la población de estudio los casos positivos para H. pylori representa el 89.62 % (164 casos) y los casos negativos 10.38 % (19 casos) y que las variables relacionadas con la presencia de H. pylori no representan una relación estadísticamente significativa. La prevalencia de infección por H. pylori en pacientes con cáncer gástrico encontrada (89 %) representa una cantidad alarmante y muy por encima del promedio de diferentes poblaciones en el mundo. Lo que nos indica que existe una fuerte relación con el cáncer gástrico en la población peruana y es necesario tomar medidas para erradicar esta bacteria y disminuir el riesgo de desarrollar cáncer gástrico. Se debe desarrollar más estudios de investigación acerca de la etiología de la infección con H. pylori y métodos de erradicación como medida profiláctica para disminuir el riesgo de desarrollar cáncer gástrico. / Tesis

HpUreI is a pH-gated urea channel found in the pathogenic bacterium Helicobacter pylori. This protein is an essential component of the mechanism of acid acclimation, which allows Helicobacter pylori to survive in the acidic conditions of the stomach. HpUreI conducts urea into the cytoplasm, where it is hydrolyzed by urease into carbon dioxide and ammonia. These products then transit back into the periplasm, where they function as a buffer and proton consumer respectively. HpUreI is an attractive target for small molecule inhibitors for the treatment of H. pylori infections as mutant strains lacking this protein no longer survive under acidic conditions. Despite the importance of HpUreI, it remains biochemically uncharacterized and many questions remain as to how this channel performs its roles. We have solved many of the technical issues regarding the heterologous expression and purification of HpUreI, allowing us to investigate this protein in detail. A robust stopped-flow light-scattering assay was developed which was used to determine the permeability of urea (or other solute) through HpUreI reconstituted proteoliposomes. With slight modifications this assay was be used to measure a wide range of characteristics and variables. Our results show that HpUreI is a hexameric protein that has a relatively weak affinity for urea (˜160mM). Proteoliposome studies indicate that HpUreI is highly selective for urea and hydroxyurea, and is able to conduct water. Interestingly, water and urea conduction is pH-gated, suggesting that both solutes share a common conduction pathway. HpUreI displayed a pH-dependent activity profile with a pH of half maximum activity of ˜5.9. Based on these results an updated mechanism of acid acclimation was proposed. HpUreI is a pH-gated channel; only conducting urea under acidic conditions. The mechanism by which this occurs is not well understood, but can be localized to the periplasmic loops. Chimeric proteins were prepared by swapping the periplasmic loops of HpUreI and StUreI, a pH-independent UreI channel from Streptococcus thermophilus. Our results show that the pH-gating behavior of HpUreI was lost if either periplasmic loop was replaced with the corresponding loop from StUreI. Conversely, pH-gating was gained by StUreI when both periplasmic loops were swapped for those of HpUreI. A model of pH-gating was proposed which takes these findings into account. The mechanism of urea conduction was also examined. The recent crystal structure of HpUreI revealed a ladder of tryptophan residues lining one face of the conduction pathway. Mutation of these residues resulted in lower rates of urea conduction and reduced urea affinity. Our findings indicate that urea interacts directly with the tryptophan residues, via stacking and dipole-dipole interactions, to facilitate urea conduction. These studies have greatly increased our understanding of HpUreI and the role it plays in H. pylori. Further research is required to fully elucidate the mechanisms by which HpUreI operates. However, this is a starting point with which to pursue the ultimate goal of developing small molecule drugs to inhibit HpUreI, culminating in the eradication of H. pylori infections and prevention of gastric cancer.

Low pH is the main environmental stress encountered by Helicobacter pylori in the human stomach. To ensure its survival under acidic conditions, this bacterium utilizes urease (encoded by the ureAB operon), a nickel-activated metalloenzyme, which cleaves urea into ammonia to buffer the periplasmic space. Expression of the ureAB operon is tightly regulated at the transcriptional level. Moreover, the urease activity is modulated post translationally via the activity of nickel-binding proteins such as HP1432 that act as nickel sponges to either sequester or release nickel depending on the pH. However, little is known how the levels of these nickel-binding proteins are regulated at the post-transcriptional level. Interestingly, more than 60 candidate small regulatory RNAs (sRNAs) have been identified in a differential RNA-seq approach in H. pylori strain 26695, suggesting an uncharacterized layer of post-transcriptional riboregulation in this pathogen. sRNAs control their trans- or cis- encoded targets by direct binding. Many of the characterized sRNAs are expressed in response to specific environmental cues and are ideal candidates to confer post-transcriptional regulation under different growth conditions. This study demonstrates that a small RNA termed ArsZ (Acid Responsive sRNA Z) and its target HP1432 constitute yet another level of urease regulation. In-vitro and in-vivo experiments show that ArsZ interacts with the ribosome binding site (RBS) of HP1432 mRNA, effectively repressing translation of HP1432. During acid adaptation, the acid-responsive ArsRS two-component system represses expression of ArsZ. ArsRS and ArsZ work in tandem to regulate expression of HP1432 via a coherent feedforward loop (FFL). ArsZ acts as a delay mechanism in this feedforward loop to ensure that HP1432 protein levels do not abruptly change upon transient pH drops encountered by the bacteria. ArsZ “fine-tunes” the dynamics of urease activity after pH shift presumably by altering nickel availability through post transcriptional control of HP1432 expression. Interestingly, after adaptation to acid stress, ArsZ indirectly activates the transcription of HP1432 and forms an incoherent FFL with ArsRS to regulate HP1432. This study identified a non-standard FFL in which ArsZ can participate directly or indirectly in two different network configurations depending on the state of acid stress adaptation. The importance of ArsZ in the acid response of H. pylori is further supported by bioinformatics analysis showing that the evolution of ArsZ is closely related to the emergence of modern H. pylori strains that globally infect humans. No homologs of arsZ were found in the non-pylori species of Helicobacter. Moreover, this study also demonstrates that the physiological role of a sRNA can be elucidated without the artificial overexpression of the respective sRNA, a method commonly used to characterize sRNAs. Coupled with time-course experiments, this approach allows the kinetics of ArsZ regulation to be studied under more native conditions. ArsZ is the first example of a trans-acting sRNA that regulates a nickel storage protein to modulate apo-urease maturation. These findings may have important implications in understanding the details of urease activation and hence the colonization capability of H. pylori, the only bacterial class I carcinogen to date (WHO, 1994). / In der natürlichen Umgebung des menschlichen Magens ist Helicobacter pylori insbesondere niedrigen pH-Werten ausgesetzt. Um diese Bedingungen zu überleben, setzt das Bakterium das Enzym Urease ein (kodiert durch das ureAB Operon), ein Nickel-aktiviertes Metalloenzym, welches Urea zu Ammonium umsetzt um den pH-Wert des periplasmatischen Raums abzupuffern. Die Expression dieses Operons ist auf transkriptioneller Ebene streng reguliert. Zudem ist die Aktivität des Urease Enzyms auf post-translationaler Ebene moduliert. Dies geschieht durch die Aktivität von Nickel-Bindeproteinen wie HP1432, die in Abhängigkeit vom pH-Wert Nickelionen abfangen oder wieder freigeben. Allerdings ist nur sehr wenig darüber bekannt, wie diese Nickel-Bindeproteine auf post-transkriptioneller Ebene reguliert werden. Interessanterweise wurden mehr als 60 sRNA-Kandidaten (engl. small RNA für dt. kleine RNA) durch eine differentielle RNA-seq Methode im H. pylori Stamm 26695 identifiziert. Dies legt eine nicht charakterisierte Ebene post-transkriptioneller Riboregulierung in diesem Pathogen nahe. sRNAs kontrollieren ihre trans- oder cis-kodierten Zielgene durch direkte Interaktion. Viele der charakterisierten sRNAs werden als Antwort auf spezifische Umweltsignale exprimiert und stellen ideale Kandidaten für post-transkriptionelle Regulatoren unter verschiedenen Wachstumsbedingungen dar. In dieser Arbeit wird gezeigt, dass die kleine RNA ArsZ (engl. acid responsive sRNA Z für dt. säureabhängige sRNA Z) und ihr Zielgen HP1432 ein zusätzliches Level der Urease-Regulierung darstellen. In-vitro und in-vivo Experimente zeigen, dass ArsZ mit der Ribosomenbindestelle (RBS) der HP1432 mRNA interagiert, wodurch dessen Translation verhindert wird. Während der Säureanpassung verhindert das säureabhängige ArsRS Zweikomponentensystem die Expression von ArsZ. Zusammen regulieren ArsRS und ArsZ das Zielgen HP1432 in Form eines kohärenten Feed-forward-loops (FFL). ArsZ agiert hier als Verzögerungsmechanismus, um sicherzustellen, dass sich bei einem transienten Abfall des pH-Wertes das Proteinlevel von HP1432 nicht abrupt verändert. Nach pH-Änderungen vermittelt ArsZ eine Feinregulierung der Ureaseaktivität, vermutlich indem es durch die post-transkriptionelle Kontrolle der HP1432 Expression die Verfügbarkeit von Nickel verändert. Interessanterweise aktiviert ArsZ nach der Säureanpassung indirekt die Transkription von HP1432 und schließt dadurch einen inkohärenten FFL mit ArsRS zur Regulierung von HP1432. Diese Studie identifizierte einen Nicht-Standard-FFL, in dem ArsZ abhängig von dem Status der Säureadaptation in zwei verschiedenen Netzwerkkonfigurationen direkt oder indirekt agieren kann. Bioinformatorische Analysen unterstützen die Relevanz von ArsZ in der Säureantwort von H. pylori zusätzlich. Hierbei kann gezeigt werden, dass die Evolution von ArsZ mit dem Aufkommen moderner H. pylori Stämme einhergeht, die weltweit Menschen infizieren. In nicht-pylori Helicobacter Spezies konnten keine Homologe von arsZ gefunden werden. Zudem zeigt diese Studie, dass die physiologische Rolle einer sRNA ohne ihre artifizielle Überexpression aufgeklärt werden kann, eine standard-mäßige Herangehensweise zur Charakterisierung kleiner RNAs. In Kombination mit Zeitverlaufsexperimenten konnte die zeitabhängige Regulierung von Zielgenen durch ArsZ unter natürlicheren Bedingungen untersucht werden. ArsZ ist das erste Beispiel einer trans-agierenden sRNA die ein Nickel-Speicherprotein reguliert, um die Reifung der Apo-Urease zu modulieren. Diese Ergebnisse können wichtige Informationen liefern, um die Aktivierung des Urease Enzyms besser zu verstehen und um damit detailliertere Einblicke in die Kolonisierungsfähigkeit von H. pylori zu gewinnen, dem bislang einzigen bakteriellen Klasse-I-Karzinogen (WHO, 1994).

The Gram-negative, spiral-shaped, microaerophilic bacterium Helicobacter pylori is the causative agent of various disorders of the upper gastrointestinal tract, such as chronic superficial gastritis, chronic active gastritis, peptic ulceration and adenocarcinoma. Although many of the bacterial factors associated with disease development have been analysed in some detail in the recent years, very few studies have focused so far on the mechanisms that regulate expression of these factors at the molecular level. In an attempt to obtain an overview of the basic mechanisms of virulence gene expression in H. pylori, three important virulence factors of this pathogen, representative of different pathogenic mechanisms and different phases of the infectious process, are investigated in detail in the present thesis regarding their transcriptional regulation. As an essential factor for the early phase of infection, including the colonisation of the gastric mucosa, the flagella are analysed; the chaperones including the putative adhesion factors GroEL and DnaK are investigated as representatives of the phase of adherence to the gastric epithelium and persistence in the mucus layer; and finally the cytotoxin associated antigen CagA is analysed as representative of the cag pathogenicity island, which is supposed to account for the phenomena of chronic inflammation and tissue damage observed in the later phases of infection. RNA analyses and in vitro transcription demonstrate that a single promoter regulates expression of cagA, while two promoters are responsible for expression of the upstream divergently transcribed cagB gene. All three promoters are shown to be recognised by RNA polymerase containing the vegetative sigma factor sigma 80. Promoter deletion analyses establish that full activation of the cagA promoter requires sequences up to -70 and binding of the C-terminal portion of the alpha subunit of RNA polymerase to an UP-like element located between -40 and -60, while full activation of the major cagB promoter requires sequences upstream of -96 which overlap with the cagA promoter. These data suggest that the promoters of the pathogenicity island represent a class of minimum promoters, that ensure a basic level of transcription, while full activation requires regulatory elements or structural DNA binding proteins that provide a suitable DNA context. Regarding flagellar biosynthesis, a master transcriptional factor is identified that regulates expression of a series of flagellar basal body and hook genes in concert with the alternative sigma factor sigma 54. Evidence is provided that this regulator, designated FlgR (for flagellar regulatory protein), is necessary for motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR is shown to act as a repressor of transcription of the sigma 28-regulated promoter of the flaA gene, while changes in DNA topology are shown to affect transcription of the sigma 54-regulated flaB promoter. These data indicate that the regulatory network that governs flagellar gene expression in H. pylori shows similarities to the systems of both Salmonella spp. and Caulobacter crescentus. In contrast to the flagellar genes which are regulated by three different sigma factors, the three operons encoding the major chaperones of H. pylori are shown to be transcribed by RNA polymerase containing the vegetative sigma factor sigma 80. Expression of these operons is shown to be regulated negatively by the transcriptional repressor HspR, a homologue of a repressor protein of Streptomyces spp., known to be involved in negative regulation of heat shock genes. In vitro studies with purified recombinant HspR establish that the protein represses transcription by binding to large DNA regions centered around the transcription initiation site in the case of one promoter, and around -85 and -120 in the case of the the other two promoters. In contrast to the situation in Streptomyces, where transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible with thermal stimuli. Transcription of two of the three chaperone encoding operons is induced by osmotic shock, while transcription of the third operon, although HspR-dependent, is not affected by salt treatment. Taken together, the analyses carried out indicate that H. pylori has reduced its repertoire of specific regulatory proteins to a basic level that may ensure coordinate regulation of those factors that are necessary during the initial phase of infection including the passage through the gastric lumen and the colonisation of the gastric mucosa. The importance of DNA topology and/or context for transcription of many virulence gene promoters may on the other hand indicate, that a sophisticated global regulatory network is present in H. pylori, which influences transcription of specific subsets of virulence genes in response to changes in the microenvironment. / Das Gram-negative, spiralförmige Bakterium Helicobacter pylori verursacht verschiedene Krankheiten des oberen Verauungstraktes, wie z.B. chronische superfizielle Gastritis, chronische aktive Gastritis, Ulzera und Magenkarzinom. Obwohl viele der bakteriellen Faktoren, die zur Entwicklung dieser Krankheitsformen beitragen, in den letzten Jahren untersucht wurden, sind die molekularen Mechanismen, die die Expression dieser Faktoren regulieren, noch weitgehend unbekannt. Als Ansatz zur Untersuchung der grundlegenden Mechanismen der Virulenzgenexpression in H. pylori wurden in der vorliegenden Arbeit drei wichtige Virulenzfaktoren repräsentativ für die verschiedenen Phasen des Infektionsprozesses ausgewählt und in Bezug auf ihre transkriptionelle Regulation analysiert. Als essentielle Faktoren für die frühe Phase der Infektion, gekennzeichnet durch die Erstbesiedelung der Schleimschicht des Magens durch die Bakterien, wurden die Flagellen untersucht. Die Chaperone-Proteine mit den mutmaßlichen Adhärenzfaktoren GroEL und DnaK wurden stellvertretend für die Phase der Adhäsion an die Magenepithelzellen und die anschließende persistente Besiedelung der Magenschleimhaut analysiert. Als Verteter der cag Pathogenitätsinsel, die mit großer Wahrscheinlichkeit für die chronische Entzündung und Schädigung des Magengewebes in den späteren Phasen der Infektion verantwortlich ist, wurde schließlich das sogenannte Cytotoxin-assoziierte Antigen CagA untersucht. RNA-Analysen und in vitro-Transkriptionsstudien konnten zeigen, daß die Transkription des cagA-Gens von einem einzigen Promotor aus gesteuert wird, während die Expression des stromaufwärts gelegenen divergenten cagB-Gens von zwei Promotoren reguliert wird. Alle drei Promotoren werden von der vegetativen, sigma 80-enthaltenden RNA-Polymerase erkannt. Durch die Einführung spezifischer Deletionen zwischen cagA und cagB konnte weiterhin gezeigt werden, daß zur vollständigen Aktivierung des cagA-Promoters Sequenzen bis -70 sowie die Bindung der alpha-Untereinheit der RNA-Polymerase an ein UP-ähnliches Element zwischen -40 und -60 erforderlich sind, während die vollständige Aktivierung des wichtigsten cagB-Promotors Sequenzen oberhalb von -96 erfordert, die mit denjenigen des cagA Promoters überlappen. Diese Daten lassen darauf schließen, daß die Promotoren der Pathogenitätsinsel eine Klasse von Minimalpromotoren darstellen, die ein geringes Niveau an Transkription garantieren, für ihre volle Aktivierung aber regulatorische Elemente oder strukturelle DNA-bindende Proteine benötigen, die ein spezielles topologisches Umfeld produzieren. Bezüglich der Flagellen konnte ein zentraler Transkriptionsfaktor identifiziert werden, der in Kooperation mit dem alternativen Sigma-Faktor sigma 54 die Expression einer Serie von Genen kontrolliert, die für strukturelle Komponenten des Flagellenkörpers kodieren. Es konnte gezeigt werden, daß dieser Faktor (genannt FlgR für Flagellen-Regulator) für die Motilität der Bakterien notwendig ist und die Transkription von fünf Operonen reguliert, die für sieben strukturelle Gene des Flagellen-Basalkörpers und -Hakens kodieren. Darüberhinaus reprimiert FlgR die Transkription des sigma 28-regulierten flaA-Gens, während Änderungen der DNA-Topologie die Transkription des sigma 54-regulierten flaB-Promotors beeinflussen. Diese Ergebnisse deuten darauf hin, daß das regulatorische Netzwerk, welches die Expression der strukturellen Komponenten der Flagellen in H. pylori kontrolliert, Ähnlichkeiten zu den in Caulobacter crescentus und Salmonella spp. beschriebenen Systemen aufweist. Im Gegensatz zu den Flagellengenen, die von drei verschiedenen Sigma-Faktoren reguliert werden, konnte im Fall der drei Operone, die für die Haupt-Chaperone von H. pylori kodieren, eine sigma 80-abhängige Transkription nachgewiesen werden. Die Expression dieser Operone wird darüberhinaus negativ reguliert durch den transkriptionellen Repressor HspR, ein Homolog des gleichnamigen Hitzeschockrepressors von Streptomyces spp. In vitro-Experimente mit gereinigtem rekombinantem HspR konnten zeigen, daß das Protein die Transkription durch die Bindung an große DNA Regionen reprimiert, die in einem Fall mit dem Transkriptionsstart überlappen, und in den anderen beiden Fällen um -85 bzw. -120 lokalisiert sind. Im Gegensatz zur Situation in Streptomyces, wo die Transkription HspR-abhängiger Gene durch Hitzeschock induziert werden kann, ist die Transkription der HspR-regulierten Gene in H. pylori nicht mit Temperaturerhöhungen induzierbar. Die Expression zweier der drei Chaperone-kodierenden Operone kann dagegen durch osmotischen Schock induziert werden, während die Transkription des dritten Operons, trotz seiner HspR-Abhängigkeit, nicht durch osmotische Reize beeinflußbar ist. In ihrer Gesamtheit lassen die transkriptionellen Analysen der verschiedenen Virulenzfaktoren darauf schließen, daß H. pylori sein Repertoire an spezifischen regulatorischen Genen auf ein minimales Niveau reduziert hat, das die koordinierte Regulation derjenigen Faktoren sicherstellt, die für die Anfangsphase der Infektion, namentlich die Durchquerung des Magenlumens und die Erstbesiedelung des Magenepithels, notwendig sind. Die Bedeutung von DNA-Topologie und -Kontext für die Transkription vieler Virulenzgene könnte andererseits auf die Anwesenheit eines hochentwickelten globalen regulatorischen Netzwerkes hinweisen, welches die Transkription spezifischer Untergruppen von Virulenzgenen in Reaktion auf bestimmte Umweltfaktoren beeinflußt.

Die weltweit steigenden Antibiotikaresistenzen sind zu einer globalen Herausforderung geworden. Von dieser Entwicklung betroffen ist auch das Bakterium Helicobacter Pylori (H.p), welches in Ländern des afrikanischen Kontinents besonders hohe Prävalenzraten aufweist. In ressourcenschwachen Ländern wie Tansania ist aufgrund der begrenzten Verfügbarkeit diagnostischer Testverfahren die Identifizierung und Therapie von H.p. infizierten Personen oft unzureichend. Tansania weist im internationalen Vergleich bislang nur wenige Studien zur H.p.-Prävalenz und Antibiotikaresistenzlage auf. Um die Datenlage für Tansania zu verbessern wurden im Rahmen dieser Arbeit potentielle Risikofaktoren für sowie die Prävalenz und aktuelle Resistenzlage von H.p.-Infektionen bei Patientinnen und Patienten mit epigastrischen Beschwerden in Tansania untersucht. Darüber hinaus wurden diagnostische Schnelltestverfahren für H.p.-Infektionen im Hinblick auf ihre Testgenauigkeit und Anwendbarkeit in Tansania geprüft. Zur Identifizierung mögliche infektionsassoziierte Faktoren wurden mittels Fragebögen soziodemographische und klinische Merkmale sowie Aspekte der Lebensgewohnheiten und Lebensumstände der Probanden erhoben und statistisch ausgewertet. Die Prävalenzbestimmung des untersuchten Studienkollektivs erfolgte anhand der auf dem 23S-rDNA Gen basierten qRT-PCR, welche für diese Arbeit als Referenzverfahren definiert wurde. Die resistenzcodierenden DNA-Abschnitte wurden auf bekannte Resistenzmutationen gegen Clarithromycin- und Fluorchinolon-Antibiotika hin untersucht. Die Ergebnisse dieser Studie deuten auf eine weite Verbreitung von H.p.-Infektionen bei Patientinnen und Patienten mit epigastrischen Beschwerden in Tansania hin. Die darüber hinaus festgestellte hohe Anzahl molekulargenetisch nachgewiesener Resistenzraten von H.p.-Stämmen gegenüber Antibiotika verdeutlichen die Wichtigkeit einer sicheren Diagnostik und resistogrammgerechten Therapie im Klinikalltag. Die in dieser Arbeit untersuchten Schnelltestmethoden scheinen aufgrund der geringen Sensitivitäts- und NPW-Werte als Standardverfahren hierfür nicht geeignet. Die Etablierung einer routinemäßig durchgeführten prätherapeutischen Antibiogrammerstellung und eine daran angepasste Therapie wären wünschenswert. / Increasing antimicrobial resistance to commonly used antibiotics have become a global challenge. This development also affects the bacterium Helicobacter Pylori (H.p.), which shows high prevalence rates in African countries. The identification and therapy of H.p. infected people in resource-limited countries such as Tanzania is often insufficient due to the limited availability of diagnostic tests. By international comparison, there are only a few studies on the prevalence of antibiotic resistance rates of H.p. for Tanzania. In order to improve the data situation for Tanzania, this study investigates potential risk factors as well as the prevalence and current resistance situation of H.p. infections in Tanzanian patients with dyspeptic symptoms. In addition, rapid diagnostic test methods for H.p. infections were tested to identify their accuracy and applicability in Tanzania. Data about the patients’ sociodemographic situation, symptoms and aspects of lifestyle habits and living conditions were collected and statistically examined in order to identify possible risk factors for infection. The prevalence of H.p. is based on the results of the 23S-rDNA gene-based qRT-PCR. The resistance-encoding DNA segments: the 23S rDNA gene region and the gyrA gene were sequenced and evaluated for known resistance mutations to clarithromycin and fluoroquinolone antibiotics. The results of this study indicate that H.p. infections are widespread in patients with dyspeptic symptoms in Tanzania and that the resistance rates of H.p. strains to antibiotics are relatively high. This underlines the importance of reliable diagnostic tests and resistogram-based therapy in clinical practice. The establishment of a routine pretherapeutic antibiogram and an accordingly adapted therapy are desirable.

Helicobacter pylori is a unique bacterial pathogen which colonises the human stomach. Infection with H. pylori has been linked to several disease outcomes including gastric and duodenal ulcer, gastric cancer and MALT lymphoma. Considering the harsh environment in which it resides and the lack of competition from other bacteria, this host/pathogen relationship is particularly interesting. Microarray analysis is a new and powerful technique which can be used to investigate various aspects of these complex interactions. Expression profiling of bacteria using microarrays remains in its infancy and thus appropriate methods were developed herein for investigating the transcriptional responses of H. pylori to various environments in vitro. Studies showed the tight relationship between growth phase dependent expression of iron homeostasis, motility and virulence genes in H. pylori for the first time. Consequently, the late exponential phase of growth was implicated as the most virulent growth phase of this bacterium in vitro. In response to mammalian cell co-culture, induced expression of H. pylori metabolism/respiration genes, genes of unknown function and genes encoding the 2-component regulators, HP1021 and HP0166, were detected. These represent a set of genes likely to be important specifically in the context of infection. To investigate the host response to infection a new mouse colonising strain of H. pylori, the Sydney Strain 2000 (SS2000), was isolated for use in comparative studies with the established strain, Sydney Strain 1 (SS1). Both host and strain specific effects were studied in a 15 month colonisation experiment using C57BL/6 and BALB/c mice. Genomic typing was used to investigate dynamic changes that occurred in the mouse-adapted strains during colonisation. In these animals reponses relating to the severity of inflammation and to the infecting H. pylori isolate were revealed by gene expression profiling. Previously unrealised cellular responses were uncovered. These included the significant down-regulation of both ferritin and haemoglobin expression. This perhaps suggests a mechanism for H. pylori induced iron deficiency anaemia. Physiological connections between colonisation, acid secretion and expression of the endocrine hormones were also implicated. These experiments have shown the utility of microarray analysis in the investigation of pathogenesis and have highlighted many directions for further investigation.