Dissertations / Theses on the topic 'Pylor'

Thesis (Ph. D.)--Ohio State University, 2003.<br>Title from first page of PDF file. Document formatted into pages; contains xiii, 145 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Kathryn Eaton, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 130-145).

From 1994, Helicobacter pylori was classified by WHO (World Health Organization) as a class I carcinogen and its infection has been associated to gastroduodenal disease. It colonizes more than half of worldwide population, with a prevalent infection rate in developed countries. In spite of the majority of infected people are asymptomatic, around 20% develop severe pathologies like peptic ulcers and the 1% lymphoma of the mucosa-associated lymphoid tissue (MALT) and stomach cancer. This significant epidemiological study both of the unique characteristics of H. pylori inspired many scientists, as bacteriologist, gastroenterologists, cancer and pharmaceutical scientists to understand physio-pathological aspects of this bacterium, and also microbiologist, taxonomist, microbial ecologist and molecular biologist, for a more detailed molecular approach. H. pylori, a Gram negative, microaerophilic bacteria that colonize human gastric mucosa. It is not an acidophilus bacterium and even if the stomach lumen presents inhospitable condition for most microbes, it is able to survive for a short period, sufficient to enter in the highly viscous mucosa, reach gastric epithelium, and colonize the gastro-enteric tract. H. pylori colonization is mediated by a predominant virulence factor, the flagellar motility associated to chemotaxis. To avoid its discharge in the intestinal tract by peristalsis, the bacteria establish a persistent infection inside the viscous gastric mucus film that covers the gastric epithelium. A nickel containing enzyme, the urease, hydrolyzes the urea present in the stomach to ammonia and CO2, buffering the pH of the periplasm. The most severe clinical outcomes are always associated to cag+ strains. cag-PAI is defined as the “Cytotoxic Associated Genes Pathogenicity Island” and it consists of a characteristic chromosome, flanked by transposable elements. Another important virulent factor is the vacuolating cytotoxin A, known as VacA, which induces the formation of large cytoplasmic vacuoles in gastric cultured cell lines. Moreover the iron and nickel acquisition is essential grow factors and a large number of genes are responsible of this mechanism. While the development of an efficient vaccine against H. pylori is now the aim of many researchers, the search for new specific antibiotics as a new pharmaceutical target is required for the complete eradication of H. pylori. In this thesis has been investigate the structural and function role of different pathogenic proteins involved in the H. pylori colonization of human gastric mucosa. These potential drug targets have been cloned, 8 out of 11 were expressed in a heterologous expression system, after purification, 2 of them generate protein crystals and only one was possible to characterize the molecular structure. In particular it has been elucidated a possible physiological role of CeuE (HP1561), a Class III SPB (Substrate Binding Protein), crystalized with Ni(His)2 complex and it was determined its affinity to the complex by an in vitro approach. The H. pylori flagella play a key role during infection allowing the bacterium to move through the mucous layer. The H. pylori hook scaffolding protein FlgD were cloned, expressed, purified and crystalized. A study of other purified pathogenic H. pylori factors belonging to flagellar component apparatus and transcriptional factors involved in cellular stress response has been reported. To obtain these results, different experimental approaches has been used. Bioinformatics analysis of target proteins has been performed to predict the best candidates for a crystallographic study and for genetic construction design. Molecular cloning in plasmid vectors has been performed from PCR amplification. The expression conditions were optimized and performed in E. coli, a heterologous system. The solubility of recombinant proteins were checked and obtained also with protein refolding methods. Different purification techniques were used in order to obtain pure protein. Target characterization was performed due analytical gel filtration, UV spectroscopy, DLS (Dynamic Light Scattering) and CD (Circular Dichroism). The proteins were concentrated to crystallization trials. The protein crystals obtained were analyzed at ESRF synchrotron (Grenoble, France). Functional in vitro approaches were performed using fluorescence spectroscopy, SPR (Surface Plasmon Resonance) and Mass spectroscopy. In the second chapter is described the three dimensional structure of a H. pylori pathogenic protein crystalized in presence with its possible physiological substrate. HP1561 (CeuE) is a H. pylori protein predicted to be an ABC transporter component, periplasmic iron-bind transporter. Recently it was published that CeuE and fecDE genes of H. mustelae encode for a nickel and cobalt acquisition system. In Gram-negative bacteria, nickel uptake is guaranteed by multiple and complex systems that operate at the membrane and periplasmic level. H. pylori employs other yet uncharacterized systems to import the nickel required for the maturation of key enzymes, such as urease and hydrogenase. To understand this contradiction of the data about Ni2+ acquisition system in H. pylori CeuE was cloned, expressed, purified, crystallized and its structure determined. Identity between the sequences of the two Helicobacter is 44%. The two Histidine residues (H103 and H197), potentially involved in Siderophores/Ni2+ binding coordination in H. pylori CeuE, are partially conserved. The His corresponding to H. pylori position 103 is conserved, whilst His197 is replaced by a Leucine. In order to check, if this substitution influence the binding of siderophores/Ni2+, the mutant of H. pylori CeuE H197L was than produced and purified. The crystal structure of H. pylori CeuE has been determined at 1.65Å resolution using the SAD method, in Apo-form and in complex with Ni(His)2. It comprises two structurally similar globular domains, each consisting of a central five-stranded β-sheet surrounded by α-helices, an arrangement commonly classified as a Rossmann-like fold. Structurally, H. pylori CeuE belongs to the class III periplasmic substrate-binding protein. Crystallographic data, fluorescence binding assays and SPR analysis allow to exclude a role of the protein in the transport of VitB12, heme, enterobactin and isolated Ni2+ ions. On the contrary, the crystal structure of the protein/Ni(L-His)2 complex and dissociation constant obtained by SPR technique suggests that H. pylori CeuE binds and transport nickel in vivo thanks to the formation of a Ni2+/histidine complex or to some ligand that mimics it. In the third chapter is presented the study of FlgD, a flagellar component involved in the formation the extracellular complex, the flagellar hook. The motility of H. pylori is considered a colonization factor, due the fact that less motile strains are less able to colonize or survive in the host than full motile strains. In the flagellum machinery are involved more than 50 genomic genes for regulation and assembly. The three major components are the filament, the hook and the basal body. FlgD is not present when the flagellum is completed, but plays a key role during the assembly. Therefore, it has been classified as the hook-scaffolding protein, considering it also as the hook capping protein, interacting with FlgL and FlgK and the basal body rod – modification protein. In H. pylori G27 strain FlgD correspond to the gene hp0858 that was amplified from purified genomic DNA and cloned in an expression plasmid vector. The protein was produced in E. coli BL21 in reach medium ad it resulted to a soluble protein. DLS and analytical gel filtration confirm the oligomeric state of FlgD that resulted to be a tetramer in solution. The protein was concentrated to 30g/l and crystalized after a couple of month of incubation. The crystals had diffracted at 2.7Å of maximum resolution. For molecular replacement approach was used homology modeling. Different molecular models were built to fit experimental diffraction data. The secondary structure of the generated models was fitted with experimental CD spectra, where FlgD resulted to have around 12% of helices and 45% of β-sheets (190-260nm). Crystallographic statistics do not properly converged to a positive molecular refinement with the tested models. To solve FlgD structure are necessary crystals of recombinant Selenomethionine FlgD that was expressed, purified and crystalized. In the fourth chapter are reported H. pylori pathogenic proteins that had been characterized. These proteins could be divided in two groups, the first one of flagellar proteins and the second of cellular stress response factors, in collaboration with Professor V. Scarlato of the department of Biology of Bologna University. FliN is a cytosolic protein, localized in the C ring of the flagellar basal body. It interacts with the other two components FliM and FliG. Missense or mutation of fliN had been associated to non-motile strains. It has been reported that regulates the clockwise/counterclockwise switching of flagella. H. pylori FliN was cloned, expressed and purified from the inclusion body after refolding. Oligomerization after refolding was tested by DLS and analytical gel filtration. The protein resulted to be poly-disperse in solution and no protein crystals have been obtained. FliD is the filament capping protein and it was observed that interact with FliT that is not only a flagellar type III substrate specific export chaperone but also inhibits the expression of fliD thought its specific interaction with the master regulator FlhD4C2 complex. In order to analyze possible structure of the co-crystalized FliD-FliT, it was plan to co-express these proteins. Both were cloned with a different affinity purification system, but only FliT was possible to express and purify from inclusion bodies. The CD spectra presented a strong β-sheet component in the secondary structure. DLS and analytical gel filtration revealed that this protein is poly-disperse in solution and no protein crystals were be obtained. FlgN is a type III secretion chaperone and it has been reported to interact with the two hook junction protein FlgK and FlgL preventing the protein proteolysis when the flagellum is not assembled. These proteins have been cloned in different type of plasmid vectors for a co-expression experiment, but only FlgN was properly expressed in E. coli. Recombinant FlgN was purified by Ni-IMAC and resulted to be soluble in solution. The protein was characterized by analytical gel filtration, DLS and CD. The protein resulted to be a monomer in solution with a 30% of not defined secondary structure (190-260nm). FlgN was concentrated and different crystallization conditions were tested. In the latter group there are three proteins related to Heat shock response, produced when bacteria encounter stress such as the elevated temperatures, ethanol, H2O2 and acid. It was demonstrated that H. pylori Hsps play an important role during the host infection. HrcA and HspR are negative repressor of groESL and dnaK machinery. HrcA activity depends by the presence of HspR, because it is demonstrated that HrcA is not able to bind DNA in absence of HspR. These two proteins were expressed in E. coli and purified by Ni-IMAC affinity. During the concentration step, these proteins present a solubility limit influenced by the concentration. Mutagenesis of a Cys in HspR and detergent solubility screening with HrcA has been performed, but no suitable protein for crystallization trials has been obtained. Hp1026 is a gene present in the same operon of HspR (hp1025). The function of this gene has not been reported. From sequence homology was possible to identify a helicase domain and ATP-binding domain. This protein, ORF, has been expressed in E. coli and purified by Ni-IMAC affinity. Analytical gel filtration and CD has been performed to characterize this protein. The protein was a dimer in solution with a 35% of α-helices component. Crystallization trials have been performed at different protein concentrations and also in presence of its possible cofactor, ATPγS. No crystals have been obtained in tested condition. Appendix: Structural and functional study on a human protease S1P/ SKI1 The study of human S1P/SKI1 protease was performed in collaboration with Professor S. Kunz of the Institute of Microbiology, University Hospital Center and university of Lausanne, Switzerland. S1P/SKI-1 is a serine protease that belongs to the mammalian family of Proprotein Convertases (PC). The aim of this family member is to mediate the activation of different important substrates for cell live. Among these proteases, S1P has been shown to have unique substrate specificity, preferring cleavage after non-basic amino acids. Known S1P cellular targets are SREBP-2, involved in the biosynthesis and uptake of lipids and cholesterol, BDNF, ATF-6 and the surface glycoprotein of viruses belonging to the family of Arenaviridae. S1P is 118 kDa multi-domain protein; two regions of S1P have been investigated, the "Prodomain", involved in the regulation of S1P catalytic activity, and the so called "catalytic domain", which include the residues responsible for the cleavage reaction itself. Moreover it was analyzed an inactive mutant of cS1P: H249A. Also for ProD was chosen one constructs (ProD_AB and ProD_AC) involved in the affinity of the protease substrate. Hence, the sequences corresponding to the domains were synthesized as optimized genes for the expression in E. coli and sub-cloned in expression plasmids in order to obtain C-term His-tagged fusion proteins. These constructs have been expressed in E. coli, purified by Ni-IMAC and positive fractions have been collected and concentrated in order to perform crystallization trials. Unfortunately no protein crystals have been obtained in tested condition. To elucidate the role of a mutated variant of the cleavage site “C” of Pro Domain, it was performed a mass spectrometry analysis. Secreted S1P/SKI1 mutant C was purified from culture medium of HEK293 cell line was isolated by IMAC-Co. The sample, loaded in RP-HPLC, was denatured in 6 M Guanidine-HCl. The chromatographic fractions corresponding to the major HPLC peaks were dried out in a speed-vac concentrator and directly injected in the ESI source. Mass measurements were performed with a quadrupole-TOF spectrometer. Analysis of mass spectra, compared with wild-type form of S1P, allows generating a preliminary Pro Domain auto-processing profile.<br>Dal 1994 il batterio Helicobacter pylori è stato classificato come organismo cancerogeneno di prima classe e la sua infezione è associata a patologie gastroduodenali. Più di metà della popolazione mondiale ne è infettata con una maggiore prevalenza nei paesi sviluppati. Nonostante la maggior parte dei casi le infezioni sono asintomatiche, il 20% sviluppa gravi patologie come ulcere peptiche e nell’1% dei casi genera linfomi e gastro carcinomi. L’incidenza e le caratteristiche di questo batterio hanno ispirato batteriologi, gastroenterologi, oncologi e farmacologi per indagare gli aspetti fisiopatologici legati all’infezione, così come microbiologi, ecologi, biologi molecolari hanno cercato i fattori di virulenza coinvolti in nell’infezione. H. pylori è un batterio microaerofilico Gram negativo che colonizza la mucosa gastrica. Non è un batterio acidofilo, anche se è in grado di sopravvivere nel lume dello stomaco per un breve periodo necessario per raggiungere le cellule epiteliali spostandosi attraverso la mucosa gastrica. La colonizzazione è mediata da fattori di virulenza predominanti come la motilità flagellare associata alla chemiotassi. Per evitare che sia espulso dal tratto intestinale dalla peristalsi, il batterio H. pylori stabilisce un’infezione cronica. L’ureasi, che è un enzima nickel dipendente, che idrolizza l’urea presente in ammoniaca e CO2 tamponando il pH acido dello stomaco. I casi più gravi sono associati ai ceppi che esprimono l’isola di patogenicità cag-PAI, che consiste in un cromosoma delimitato da elementi trasponibili. Un altro importante fattore di virulenza è la tossina vacuolizzante VacA, che induce la formazione di vacuoli citoplasmatici. Anche il meccanismo di acquisizione di ferro e nickel è fondamentale per la colonizzazione batterica e dunque finemente regolata da un gran numero di geni. Lo sviluppo di un vaccino e nuovi antibiotici nutrono una costante ricerca di nuovi possibili bersagli farmacologici, necessari per completa ed efficiente eradicazione del batterio H. pylori. In questa tesi sono stati analizzati il ruolo e la struttura di alcune proteine patogenetiche del H. pylori. Questi potenziali target farmacologici sono stati clonati, otto su undici sono stati espressi in un sistema eterologo, due proteine di quelle purificate hanno generato cristalli e di una sola ne è stata definita la struttura molecolare. In particolare è stato definito un possibile ruolo della proteina CeuE (HP1561), appartenete alla famiglia delle proteine che legano un substrato, cristallizzata in presenza del complesso Ni(His)2 e definita l’affinità con lo stesso in vitro. Del flagello, che svolge un ruolo chiave durante l’infezione, ne è stata studiata la proteina coinvolta nella formazione dell’uncino FlgD che è stata clonata, espressa, purificata e cristallizzata. Inoltre è stato riportato anche uno studio di altri fattori del flagello e di alcune proteine coinvolte nella risposta allo stress cellulare. Per ottenere tali risultati sono stati utilizzati approcci differenti. Per individuare le migliori proteine candidate per uno studio cristallografico e progettare costrutti funzionali sono state effettuate predizioni bioinformatiche. Gli amplificati di PCR sono stati clonati in vettori plasmidici. Le condizioni di espressione sono state ottimizzate e fatte in E. coli, un sistema di espressione eterologo. La solubilità delle proteine ricombinanti è stata analizzata e ottenuta anche mediante refolding. Sono stati usati diversi sistemi di purificazione per ottenere un buon grado di purezza. Per la caratterizzazione proteica sono state usate come tecniche la gel filtrazione analitica, spettroscopia UV, DLS (Dynamic Light Scattering) e dicroismo circolare. Le proteine sono state concentrate e sottoposte a esperimenti di cristallizzazione. I cristalli sono stati analizzati al sincrotrone ESRF (Grenoble, France). Spettroscopia di fluorescenza, SPR (surface plasmon resonance) e spettroscopia di massa sono le tecniche utilizzate per la caratterizzazione In Vitro. Nel secondo capitolo viene decritta la struttura tridimensionale di una proteina patogenetica di H. pylori, cristallizzata in presenza del suo possibile substrato fisiologico. HP1561 (CeuE) è una proteina di H. pylori annotata come componente periplasmatico di un trasportatore ABC che lega e trasporta il ferro. Recentemente è stato pubblicato chele ceuE e fecDE di H. mustelae codificano per proteine coinvolte nel acquisizione del nickel e cobalto. Nei Gram negativi, l’acquisizione del nickel è garantita da sistemi di proteine che operano a livello di membrana e periplasmatico. Per l’acquisizione del nickel, l’ H. pylori integra diversi sistemi non ancora caratterizzati, necessari per la maturazione di enzimi chiave come l’ureasi e l’idrogenasi. Per chiarire tale contraddizione nel sistema di acquisizione del nickel nell’H. pylori, CeuE è stata clonata, espressa, purificata, cristallizzata e la sua struttura è stata risolta. L’identità di sequenza tra i due Helicobacter (pylori e mustelae) è del 44%. Le due Istidine (H103 e H197), potenzialmente coinvolte nel legame di coordinazione del sistema sideroforo/Ni2+ nel H. pylori CeuE, risultano essere parzialmente conservate. L’His corrispondente alla His103 di H. pylori è conservata, mentre His197 è sostituita da una Leucina. Al fine d’identificare se tale mutazione possa influenzare il legame sideroforo/Ni2+, è stato prodotto e purificato il mutante H. pylori CeuE H197L. La struttura molecolare di H. pylori CeuE è stata determinata con una risoluzione di 1.65 Å mediante metodo SAD, sia nella forma apo, che in complesso col Ni(His)2. Essa è costituita da due domini globulari simili, ognuno costituito da cinque foglietti-β circondati da α-eliche, comunemente classificato come Rossman fold. Strutturalmente H. pylori CeuE appartiene alla Classe III della famiglia di proteine che legano un substrato specifico (SBPs). Dati cristallografici, saggi di fluorescenza e analisi all’SPR ci permettono di escludere il coinvolgimento della proteina nel trasporto della VitB12, eme, entrobactina, e ioni Ni2+ isolati. Al contrario la struttura della proteina/complesso Ni(His)2 e le costanti di dissociazione ottenute mediante SPR suggeriscono che H. pylori CeuE lega e trasporta il nickel in vivo mediante il complesso Ni2+/His o altro ligando che lo mima. Nel terzo capitolo viene presentato lo studio su FlgD, una proteina flagellare fondamentale nella formazione di un complesso extracellulare, l’uncino del flagello. La motilità dell’H. pylori è considerata un fattore di colonizzazione, attraverso il quale ceppi meno motili hanno minori possibilità di colonizzare e sopravvivere nell’ospite di ceppi più motili. Per la formazione del flagello sono coinvolti più di 50 geni per la regolazione e l’assemblaggio delle varie componenti. Le tre componenti principali sono il filamento, l’uncino e il corpo basale. FlgD non è presente quando il flagello è maturo, ma ha un ruolo chiave durante l’assemblaggio. Perciò, è stato classificato come proteina necessaria per l’impalcatura dell’uncino (hook scaffolding protein), considerata anche proteina di testa dell’uncino (capping protein) in quanto interagisce con FlgL, FlgK e le proteine del corpo basale. Nel ceppo H. pylori G27, FlgD corrisponde al gene hp0858 che è stato amplificato dal DNA genomico purificato e clonato in un vettore plasmidico. La proteina è stata prodotta in E. coli BL21 e la proteina è risultata essere solubile. Gel filtrazione analitica e misure al DLS confermano il suo stato di oligomerizzazione, che risulta essere un tetramero in soluzione. La proteina è stata concentrata fino a 30 g/l e cristallizzata dopo un paio di mesi d’incubazione. I cristalli hanno diffratto a una risoluzione massima di 2.7 Å. Per la sostituzione molecolare è stata usata la tecnica del homology modelling. Sono stati costruiti diversi modelli molecolari per fittare i dati sperimentali. La struttura secondaria dei modelli generati è stata comparata con gli spettri di dicroismo circolare, dove FlgD è risultata essere composta da un 12% di eliche e complessivamente da un 45% di foglietti beta (190-260nm). Le statistiche cristallografiche non hanno dato convergenza positiva negli esperimenti di sostituzione molecolare con i modelli testati. Per risolvere la struttura di FlgD sono necessari cristalli di FlgD derivatizzata con Selenometionine, che è stata espressa, purificata e cristallizzata. Nel quarto capitolo sono riportate le proteine patogenetiche di H. pylori che sono state caratterizzate in questa tesi. Queste proteine possono essere divise in due gruppi, il primo delle proteine flagellarli ed il secondo delle proteine coinvolte nella risposta allo stress cellulare in collaborazione con il Prof. V. Scarlato del dipartimento di Biologia dell’università di Bologna. FliN è una proteina citosolica localizzata nell’anello C del corpo basale del flagello ed interagisce con altri due componenti FliM e FliG. Mutazioni missenso di fliN sono state associate a ceppi non-motili ed è stato riportato che regola la rotazione oraria/antioraria del flagello. H. pylori FliN è stata clonata, espresso e purificata dai corpi d’inclusione dopo refolding. Lo grado di oligomerizzazione è stato analizzato mediante DLS e gel filtrazione analitica. La proteina è risultata essere polidispersa i soluzione e non sono stati ottenuti cristalli di proteina. FliD è la proteina “capping” del filamento cellulare ed è stato osservato che interagisce con FliT, che non è solo un chaperon substrato specifico del sistema III di esporto flagellare, ma inibisce anche l’espressione di fliD attraverso l’interazione con il complesso FlhD4C2. Al fine di analizzare la struttura del complesso FliD-FliT, è stata pianificata la co-espressione di queste proteine. Entrambe sono state clonate con un sistema di purificazione differente, ma solo la purificazione di FliT è stata possibile dai corpi d’inclusione. Lo spettro di dicroismo circolare ha rivelato una forte componente di foglietti-β nella struttura secondaria. Secondo le misure di DLS e gel filtrazione analitica FliT è polidispersa in soluzione e perciò non stati ottenuti cristalli della stessa. FlgN è una proteina del sistema secrezione tipo III ed è stato osservato che interagisce in maniera specifica con le proteine di giunzione dell’uncino con il filamento FlgK ed FlgL, prevenendone la proteolizzazione prima della maturazione del flagello. Queste proteine sono state clonate in differenti tipi di vettori plasmidici, ma solo FlgN è stata efficacemente espressa in E. coli. FlgN ricombinante è stata purificata mediante Ni-IMAC è risultata essere solubile. La proteina è stata caratterizzata con gel filtrazione analitica, DLS e CD. La proteina è un monomero in soluzione con un 30% di struttura secondaria non definita (190-260 nm). FlgN è stata concentrata e sottoposta a test di cristallizzazione. Nell’ultimo gruppo ci sono tre proteine HSPs (Heat Shock Response), prodotte dal batterio quando incontra stress come elevate temperature, etanolo, H2O2 e acidi. E’ stato accurato che le HSPs di H. pylori svolgono un ruolo importante durante l’infezione dell’ospite. HrcA e HspR reprimono la trascrizione di groESL e dnaK. L’attività di HrcA è influenzata dalla presenza di HspR, in quanto è stato dimostrato che HrcA non è in grado di legare il DNA in assenza di HspR. Queste due proteine sono state espresse in E. coli e purificate con Ni-IMAC. Durante le fasi di concentrazione hanno mostrato un limite di solubilità. Mutagenesi mirata sul costrutto di HspR e screening di detergenti su HrcA sono hanno migliorato il sistema, senza però riuscire ad ottenere una condizione ottimale per la formazione di cristalli di proteina. HP1026 (ORF) è un gene presente nello stesso operone di HspR (hp1025), ma con funzione non nota. Dall’analisi della sequenza è stato identificato un dominio con attività elicasica ed un dominio legante l’ATP. La proteina è stata espressa in E. coli e purificata con Ni-IMAC. Per la caratterizzazione sono state effettuate gel filtrazione analitica e dicroismo circolare. La proteina risulta essere un dimero in soluzione con un 35% di α-elica. I test di cristallizzazione son stati effettuati scrinando diverse concentrazioni e anche in presenza del possibile cofattore, ATPγS in forma non idrolizzabile. Nessun cristallo è stato ottenuto dalle condizioni testate. Appendice: Studio strutturale e funzionale della proteasi umana S1P/SKI1 Lo studio di questa proteasi umana è stato effettuato in collaborazione con il Prof. S. Kunz dell’Istituto di Microbiologia, del Centro Universitario Ospedaliero e dall’ Univ. Di Lausanne, Svizzera. S1P/SKI1 è una serina proteasi della famiglia delle Proprotein Convertasi (PCs). Lo scopo di membri di questa famiglia è quello di mediare l’attivazione di diversi importanti substrati per la vita cellulare. Tra queste proteasi, S1P presenta una specificità di substrato, con un sito di taglio dopo un residuo non basico. Tra i target cellulari di S1P sono stati identificati SREBP-2, coinvolto nella biosintesi dei lipidi e del colesterolo, BDNF, ATF-6 e glicoproteine superficiali di virus appartenenti alla famiglia delle Arenaviridae. S1P pesa 118kDa ed è una proteina multidominio; quindi 2 regioni di S1P sono state studiate, il “Prodomain” (ProD) che regola l’attività catalitica, ed il “cathalytic domain” (cS1P) che include i residui responsabili per la reazione proteasica. Inoltre è stato analizzato un mutante inattivo (cS1P_H249A) e due costrutti per il dominio di regolazione (ProD_AB e ProD_AC). Le sequenze nucleotidiche dei corrispettivi costrutti sono state sintetizzate come geni ottimizzati per l’espressione in E. coli e subclonati in vettori plasmidici per l’espressione ottenendo proteine in fusione con una coda di 6-His. Questi costrutti sono stati espressi in E. coli, purificati con Ni-IMAC e le frazioni positive sono state raccolte e concentrate per test di cristallizzazione. Sfortunatamente non sono stati ottenuti cristalli di proteina nelle condizioni testate. Per chiarire il ruolo di una variante mutata nel sito di taglio “C” del dominio di regolazione è stata effettuata una analisi di spettrometria di massa. La proteina secreta S1P mut C (sS1P_MutC, 116kDa) è stata purificata dal medium di coltura di una linea di HEK293 trasfettate e isolata con Co-IMAC. Il campione è stato denaturato in Guanidinio 6M e caricato in HPLC. Le frazioni corrispondenti ai picchi predominanti sono stati essiccati ed iniettati in spettrometro di massa (ESI-TOF). L’analisi delle masse, confrontate con la forma nativa (sS1P_WT) ha permesso di generare un profilo preliminare del pattern di processamento del dominio di regolazione (ProD) with a quadrupole-TOF spectrometer. Analysis of mass spectra, compared with wild-type form of S1P, allows generating a Pro Domain auto-processing profile.

Thesis (Ph. D.)--University of Oregon, 2006.<br>Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80 - 90). Also available for download via the World Wide Web; free to University of Oregon users.

Les microARN sont de petits ARN non codant régulant post-transcriptionnellement l’expression de certains gènes. Du fait de leur fort potentiel régulateur, une modification de leur expression peut conduire à l’apparition de pathologies telles que le cancer ou l’inhibition des mécanismes de défense contre des pathogènes. Notre objectif est de caractériser le rôle de certains miARN dans la formation de cancer gastrique dû à Helicobacter pylori. En effet, cette bactérie peut conduire à l’apparition d’adénocarcinome gastrique et de lymphome du MALT. Sa virulence est essentiellement due à la protéine CagA, injectée dans les cellules de la muqueuse gastrique. Par séquençage à haut débit du contenu en miARN d’une lignée épithéliale gastrique humaine, co-cultivée ou non avec H. pylori, nous avons observé que les niveaux de miR-200b/c sont augmentés par l’infection. Ces miARN sont des inhibiteurs puissants de la transition épithélio-mésenchymateurse (TEM), modification morphologique promotrice d’invasion. Ils ciblent les facteurs de transcription ZEB1/2 avec lesquels ils sont impliqués dans une boucle de rétro-action mutuellement répressive. Le niveau basal élevé de miR-200b/c dans ces cellules réprime totalement ZEB1, tandis que l’infection par H. pylori, sous la dépendance de CagA, promeut une TEM en induisant ZEB1. Paradoxalement, les miR-200b/c sont aussi augmentés lors de l’infection transcriptionnellement. Nous avons pu démontrer que l’augmentation des miR-200b/c dans les cellules infectées a pour rôle de modérer l’induction de ZEB1 via l’activation de NF-kB, constituant ainsi un mécanisme de défense des cellules hôte contre la perte de leur identité épithéliale<br>MicroRNA are small noncoding RNA that post-transcriptionally regulate gene expression. Due to their high regulator potential, a change in their expression may lead to the emergence of diseases such as cancer or inhibition of defense mechanisms against pathogens. Our aim is to characterize the role of miRNA in the response of gastric eptithelial cells to Helicobacter pylori (H. pylori). Indeed, H. pylori promote gastric adenocarcinoma and MALT lymphoma. Its virulence is essentially mediated by CagA, injected into cells of the gastric mucosa. Thanks to high throughput sequencing of miRNA content of a gastric epithelial cell line, infected or not with H. pylori: miR-200b and -200c appeared up-regulated upon infection. These miRNA are potent inhibitors of the “epithelial-to-mesenchymal transition” (EMT), a process that drastically alters cell morphology and promotes cell invasion. MiR-200b/c target the transcription factors ZEB1 and ZEB2, with which they are involved in a mutually repressive feedback loop. In basal conditions, the high levels miR-200b/c in gastric epithelial cells totally silence ZEB1 mRNA whereas H. pylori promotes EMT via ZEB1 expression, on the dependence of CagA translocation into host cells. But, paradoxically, miR-200b/c levels were also up-regulated upon infection. The increased miR-200b/c levels in infected cells moderate ZEB1 induction thanks to NF-kB activation and constitute a self-defense mechanism to thwart the loss of their epithelial phenotype upon infection

Contexte : Amélioration de la prise en charge de l’infection à H. pylori. Matériels et Méthodes : Détection de H. pylori, la résistance à la clarithromycine, la tétracycline, la lévofloxacine, et la détermination des gènes de pathogénicité ont été réalisées par PCR en temps réel, du gène de l’ARNr 23 S, de l’ARNr 16S, PCR classique et séquençage. L’évaluation de la stabilité du mutant résistant par rapport à l’isolat sensible était obtenue par compétition en culture sur cellules gastriques AGS sur une longue période, suivie du séquençage du génome entier. L’évaluation de l’effet de l’extrait de Ceiba pentandra sur H. pylori était réalisée par la détermination de la concentration minimale inhibitrice. Résultats : Prévalence de l’infection à H. pylori : 75.52%, résistance à la clarithromycine et tétracycline : 4,2% et 1,2%, résistance à la lévofloxacine : 57%. Gène CagA : 92,2%. Gène Vac As1m1 : 82%. Absence de stabilité du mutant résistant dans le couple de souches 3695 R/S (ratio R / S 0,1), à 30 jours de la co-culture (p &lt;0.05) ; ce mutant présentait la mutation A2142G, conférant la résistance à la clarithromycine. On notait la stabilité du mutant résistant dans l’autre couple de souches 3657R/S (ratio R / S ratio : 1,7) à 40 jours de la co-culture (p &lt;0.05), avec développement des mutations compensatoires ; ce mutant présentait la mutation A2143G. L’activité modérée à faible était notée avec les extraits hydroéthanolique et butanolique de Ceiba pentandra, avec une concentration minimale inhibitrice de 50 à 80 μg / ml.Conclusion : il est possible de traiter l’infection à H. pylori avec une thérapie à base de clarithromycine au Congo. L’absence d’une activité forte ne permet pas de recommander Ceiba pentandra dans le traitement de l’infection à H. pylori . La réversion de la résistance dans le cas de H. pylori peut être envisagée<br>Context: The objective of this work was to improve Helicobacter pylori infection management. Materials and methods: H. pylori detection, it’s resistance to clarithromycine, tetracyclin, levofloxacin, and determination pathogenic genes were done by real-time PCR on 23 S rRNA, on 16 S rRNA gene, classic PCR, sequencing. Evaluation of the resistant mutant stability to its sensitive isolate was carried out by competing them over a long period in culture on AGS gastric cells and whole sequencing genome. The evaluation of Ceiba pentandra extract effect on H. pylori was carried out by determining the minimum inhibitory concentration. Results: Prevalence of H. pylori infection: 75.52%, resistance to clarithromycin and tetracycline: 4.2% and 1.2%, levofloxacin resistance: 57%. CagA gene: 92.2%. Vac As1m1 gene: 82%. Lack of stability of the resistant mutant in a 3695 R/S pair of isolates (R/S ratio 0.1), at the 30 day of the co-culture (p &lt;0.05); this mutant had an A2142G mutation conferring resistance to clarithromycin. Stability of the resistant mutant in the other 3657 pair of isolates (R/S ratio of 1.7) at the 40 day of the co-culture (p &lt;0.05), with development of compensatory mutations; this mutant had an A2143G mutation conferring resistance to clarithromycin. The moderate to low activity was noted with the hydroethanol extract and the butanol extract: minimum inhibitory concentration: 50 to 80 μg / ml. Conclusion: It’s possible to treat H. pylori infection with therapy based on clarithromycin in Congo. The absence of a strong activity does not make it possible to recommend Ceiba pentandra in the treatment of H. pylori infection. Reversion resistance is possible with H. pylori

Aquesta Tesi Doctoral té com objectius principals l’estudi d’Helicobacter pylori en mostres aquàtiques de Catalunya, en aigües procedents de sistemes dentals de consultes de dentistes, en saliva i en femtes de pacients amb símptomes gastrointestinals mitjançant el mètode de la PCR. També es va estudiar la supervivència d’Helicobacter pylori en aigua dolça usant un model de laboratori aplicant diferents tècniques d’anàlisi i es va interpretar el canvi de morfologia, la viabilitat i la culturabilitat de la bactèria així com la detecció i quantificació del seu ADN durant un període de temps concret. Per aconseguir aquests objectius, primer es va escollir la llet descremada al 40% v/v com a millor crioprotector i el medi agar Columbia suplementat amb 5% de sang desfibrinada de cavall com a medi de cultiu. Durant el desenvolupament de la tesi es va millorar la tècnica de PCR escollida inicialment basada amb l’estudi de Clayton i col•laboradors (1992) on s’usaven els iniciadors HPU1 i HPU2 en la PCR pel gen ureA, gen estructural de l’enzim ureasa. La tècnica de la hibridació seguida per aquests investigadors es va substituir per una segona PCR ja que aquesta permetria obtenir resultats més ràpidament. En aquesta PCR semiimbricada es va introduir un tercer iniciador intern, HPUI1, i mantenint HPU2 com a extern. Pel gen 16S rRNA, gen usat per a la detecció del gènere Helicobacter, es van usar els iniciadors 1F i 1R per a la primera PCR i els 1F i 2R per a la PCR semiimbricada. El mètode d’extracció escollit en aquest treball basat en partícules de sílice i tiocianat de guanidina descrit per Boom i col., (1990) i l’optimització de les barreges de reacció de les PCR semiimbricades pel gen ureA i 16S rRNA van permetre detectar 50 UPF/mL i 2-20UFC/mL, respectivament. Es va determinar la presència de H.pylori en mostres fecals humanes aplicant dos mètodes: mètode antigènic HpSA i l’amplificació del gen ureA. Amb el mètode molecular es va detectar la presència de ADN de H. pylori en 12 mostres i va mostrar un 75% de coincidència amb els resultats obtinguts amb el mètode antigènic HpSA. També es va determinar la presència de H. pylori a aigües amb diferent de nivell de contaminació fecal. Es va amplificar ADN d’aquesta espècie en un 30% de les mostres d’aigua residual, un 8,34% de les mostres d’aigua de riu de Catalunya i no es va detectar en aigua de font. L’estudi de supervivència de H. pylori a l’aigua en la foscor i a 7ºC es va demostrar que el recompte de bacteris i el número de genomes es manté constant durant tot el període d’estudi, 21 dies. La detecció per PCR també va ser positiva i constant durant aquest període. No obstant això, les cèl•lules només es van mantenir cultivables fins al sisè dia d’emmagatzematge. També es va observar una conversió morfològica de les cèl•lules bacterianes passant de la forma espiral a cocal amb el pas del temps. L’estudi de la morfologia cel•lular en un tall vertical d’una colònia d’H. pylori de 4 dies va mostrar la coexistència de cèl•lules amb morfologia bacil•lar i coccal. L’anàlisi de la saliva de 31 persones sanes va evidenciar la presència d’aquest bacteri en la boca de 6% de la població analitzada. La anàlisi feta amb la PCR semiimbricada per al gen 16S rRNA va identificar la presència d’aquest gen en 19 persones però la seqüenciació dels amplicons del gen ureA només va confirmar la presència de H. pylori en tres mostres. L’anàlisi de 31 mostres d’aigua procedents de les corresponents cadires no va mostra la presència del patogen.<br>This thesis has as main objectives the study of Helicobacter pylori in water samples from Catalonia, saliva and human feces using the PCR method. Also studied survival, morpholgy, viability and culturability of Helicobacter pylori in water. We first chose skim milk at 40% v / v as cryoprotectant and Columbia agar supplemented with 5% horse blood desfibrinada as a medium. We used a seminested PCR for ureA gene with HPU1 and HPU2 primers for the first PCR and HPUI1 and HPU2 for the second PCR. By gene 16S rRNA, we used the 1F and 1R primers for the first PCR and 1F and 2R for the second PCR. Using a silica and guanidine extraction method and optimization of the seminested PCR reaction mixtures for urea and 16S rRNA genes were detected 50 UPF / mL and 2-20UFC/mL respectively. H.pylori was detected in 12 human fecal samples. DNA of H.pylori was amplified in 30% of samples of wastewater, a 8.34% of the samples of river water in Catalonia and was not detected in spring water. The bacterial count and the number of H.pylori genomes remains constant throughout the study period, 21 days water in the dark at 7 ° C. The detection by PCR was also positive and constant during this period. The cells remained culturable only until the sixth day of storage. We observed morphological conversion of bacterial cells through the spiral to cocal over time. The study of cell morphology in a vertical section of a colony of H.pylori showed the coexistence of cells with bacillary and coccal morphology. The analysis of saliva from 31 healthy individuals showed the presence of this bacterium in the mouth of 6% of the analyzed population but we urea only confirmed the presence of H.pylori in three samples by sequencing.

Helicobacter pylori is a gram negative, microaerophilic bacteria that plays an important role in chronic gastritis and peptic ulcer disease. Multiple antimicrobial therapies including combinations of clarithromycin, metronidazole or amoxycillin and proton pump inhibitor have been used to eradicate H. pylori. Antimicrobial resistance in H. pylori has been associated with treatment failure. In this study, antimicrobial susceptibility patterns of amoxycillin, ciprofloxacin, clarithromycin, erythromycin, tetracycline and metronidazole were evaluated in 110 H. pylori strains isolated from 454 antral biopsies of patients undergoing endoscopy at the Royal Infirmary, Edinburgh. The MICs were determined by E-test. Resistance to clarithromycin and erythromycin was found in 8.2% (9/110) and 9.1% (10/110) respectively. Metronidazole resistance was demonstrated in 8 isolates (7.3%). Tetracycline resistance was found in one of the isolates (0.9%). Two isolates were resistant to ciprofloxacin (1.8%). Resistance to amoxycillin was not detected. Molecular mechanisms of fluoroquinolone, macrolide and metronidazole resistance in H. pylori were investigated. Resistant to fluoroquinolone has been associated with alterations in the Quinolone Resistance-Determining Region (QRDR) of gyrA gene. Mutation at position 91, leading to an amino acid change from Aspartic acid to Asparagine was found in 2 ciprofloxacin-resistant isolates. One isolate had a mutation at Asparagine-87 to Lysine. Mutations in the 23S rRNA conferring macrolide resistance were investigated. Mutations at position 2143 (A to G) were shown in seven of the ten macrolide-resistant isolates. Two of the seven isolates carried an additional T to C mutation at either position 2182 or 1934. Of the ten macrolide-resistant isolates, two had a single mutation at either position 2182 or 2195. Mutation at position 2182, however, has previously been identified not to be associated with macrolide resistance. The mutations at position at 1934 (T to C) and position 2195 (C to T) have not previously been reported. One of the ten isolates (MIC > 256 mg/L) had no alteration in the 23S rRNA. The results indicate that different mechanisms play a role in macrolide resistance in these H. pylori strains. Metronidazole resistance has been reported to be associated with mutations in the rdxA gene, encoding oxygen-insensitive nitroreductase. To investigate the role of rdxA, sequencing analysis of rdxA of metronidazole-resistant isolates was determined. The results showed that no particular amino acid substitution was associated with metronidazole resistance. One isolate contained a nonsense mutation, generating a stop codon. However, two metronidazole-sensitive strains had alterations in rdxA by insertions of a mini-IS605 sequence. These results suggest that alterations in rdxA are not the sole mechanism of metronidazole resistance and other mechanisms are required in the development of resistance. Since the prevalence rate of metronidazole resistance is variable, ranging from 11 to 70%, it is possible that some variation in reported resistance levels derives from difficulties in the method of sensitivity testing. To set a standard for susceptibility testing for metronidazole in H. pylori, the optimum conditions for sensitivity testing were evaluated. Activation of metronidazole requires an anaerobic environment. It was found that incubation under microaerophilic conditions elevated metronidazole MIC, suggesting that microaerophilic conditions cannot activate metronidazole to its active form. Pre-incubation of H. pylori in anaerobic conditions for 24 hours prior to incubation under microaerophilic conditions for 72 hours was found to be necessary to achieve accurate susceptibility results. This can explain why some centres report high levels of metronidazole resistance. To investigate the development of fluoroquinolone resistance in H. pylori, ciprofloxacin-resistant mutants were selected in vitro by exposing sensitive strains to serial increments of ciprofloxacin in Columbia blood agar plate. The QRDR of gyrA gene was analysed for mutations. Reduced susceptibility to ciprofloxacin was associated with either a single or double amino acid changes in gyrA gene. Mutations at position 85, 87 and 91 were found to be associated with ciprofloxacin resistance. The results also demonstrated that gyrA mutations are not sole contributors for the mechanism of ciprofloxacin resistance in H. pylori. As no parC gene has not yet been identified in H. pylori, any contribution from topoisomerase IV cannot be quantified. To investigate the DNA gyrase activity in H. pylori, gyrA and gyrB were separately cloned and overexpressed by using a T7 promoter vector, which contain a fusion tag of six histidine residues. GyrA and GyrB were purified by affinity chromatography using nickel-chelating resins. The ability of DNA gyrase to supercoil relaxed DNA was determined.

In the past decades, Helicobacter pylori has received the attention of many researchers because of its known relation with gastric cancer. Although many studies have tried to decipher the exact relation between the bacteria and cancer state, and several virulence factors have been discovered, an exact answer has not been found yet. Further work should be made in order to study more accurately the genome of this bacterium and to understand its precise involvement. The bacterium is characterised for a highly genetic diversity, meaning it is continuously changing in order to adapt itself to its hostile niche, the human stomach. Infection by H. pylori is estimated to affect half of the world’s population, being more extended in developing countries than in developed ones, possibly due to the high consumption of antibiotics and the increased level of sanitation in the latest. It has been demonstrated that the gastric lumen can be colonized by more than just one strain of the bacterium, sometimes these strains could have evolved from the same ‘mother’ strain, or they could come from unrelated strains. The study of these situations is important in order to elucidate if there is just one strain who is responsible for starting the pathogenic cascade, and what are the specific differences between the different strains that inhabit the human stomach. On the first work of this thesis, our group studied the usefulness of six housekeeping genes for the detection of H. pylori infection and the characterization of various strains isolated from gastric isolates, studying as well their phylogeny. In some cases, the distance value between the strains was high, indicating and event of multiple infection. In other cases, small differences were found between clones, suggesting events of microevolution rather than multiple infection. This work was further extended with the study of the usefulness of amplicon sequencing of these housekeeping genes in the detection of microevolution and mixed infections from gastric biopsies of patients with dyspeptic symptoms and different histopathological findings (from atrophy to adenocarcinoma). Five gastric biopsies from four patients infected by H. pylori were involved in this study. We detected in all the analyzed gastric biopsies multiple H. pylori infections with a predominant strain. These results suggest that H. pylori colonizes the human stomach through diverse infection circumstances that lead to a gastric multi-infection with a predominant strain together alongside other minority strains. Furthermore, it was shown that mixed infections are the main status in the colonization of the human gastric mucosa. The last part of this thesis started with a preliminary study of 51 complete sequenced H. pylori genomes and further focused on three genomes obtained from the same patient in order to analyse and compare them. Particularly, these isolates were sampled at the same time from a stomach with adenocarcinoma, one strain was from the non- tumoral tissue, and the other two were isolated from the tumoral tissue. They all lacked from the most noticeable virulence factor, the cag pathogenicity island; one of the most studied and the main factor related to the malignancy of the bacterium. On the other hand, we found differences in the genotype of the vacuolating cytotoxin gene (vacA) and in genes related with urease, the outer membrane and flagella. Despite the contributions made in this thesis, further studies are needed to find better genetic markers of H. pylori related to virulence and progression to gastric cancer.<br>Helicobacter pylori és el focus d’atenció de molts estudis a causa de la seva coneguda relació amb el càncer gàstric. Encara que molts estudis han tractat de desxifrar la relació exacta entre el bacteri i la progressió del càncer, i s'han descobert diversos factors de virulència, no s'ha trobat una resposta exacta. En la primera part d'aquesta tesi, es va estudiar la utilitat de sis gens conservats per la detecció de la infecció per H. pylori i la caracterització de diverses soques aïllades de biòpsies gàstriques, estudiant-se també la seva filogènia. En alguns casos, el valor de la distància entre les soques era alt, fet indicatiu d’un esdeveniment d'infecció múltiple. En altres casos, es van trobar petites diferències entre els clons, el que suggereix esdeveniments de microevolució en lloc d’infecció múltiple. Aquesta tesi es va ampliar amb l'estudi de la utilitat de la seqüenciació d'amplicons d'aquests gens conservats en la detecció de microevolució i infeccions múltiples en biòpsies gàstriques de pacients amb símptomes dispèptics. En aquest estudi es van analitzar cinc biòpsies gàstriques de quatre pacients infectats per H. pylori. En totes les biòpsies gàstriques analitzades es van detectar infeccions múltiples per H. pylori amb una soca predominant. Aquests resultats suggereixen que H. pylori colonitza l'estómac humà mitjançant diverses circumstàncies d'infecció que condueixen a una infecció múltiple gàstrica amb una soca predominant juntament amb altres soques minoritàries. L'última part d'aquesta tesi va començar amb l’estudi preliminar de 51 genomes complets d’H. pylori i es va centrar després en l’estudi i comparació de tres genomes obtinguts del mateix pacient. Específicament, aquestes soques van ser aïllades alhora d’un estómac amb adenocarcinoma, on una soca provenia del teixit no tumoral i les altres dues del teixit tumoral. Totes mancaven del factor de virulència més destacat, l'illa de patogenicitat cag; un dels factors més estudiats i el més relacionat amb la malignitat del bacteri. Per una altra banda, vam trobar diferències en el genotip del gen vacA i en gens relacionats amb la ureasa, la membrana externa i els flagels.

Helicobacter pylori infects the human stomach and triggers an inflammatory response that damages the gastric tissue. This host-pathogen interplay has dire consequences as up to 20 % of infected individuals develop peptic ulcer disease or gastric cancer. Given that half of the world’s population is infected, the number of afflicted humans is staggering and also tells that H. pylori is extremely efficient in spreading and maintaining infection. To enable persistent infection many factors play a role, but one important feature of H. pylori is its impressive ability to adhere to the slimy gastric mucus layer and the underlying epithelial cells. This occurs mainly via the BabA and SabA proteins that bind ABO/Leb- and sLex/sLea-antigens. I have in my thesis studied how these two proteins are utilized and regulated. H. pylori transcription is in part controlled by two-component systems (TCSs) that use a sensor protein and a DNA-binding response regulator. We have studied how these systems control sabA and to some extent babA and indeed found a better map of how sabA and babA is regulated at the transcriptional level. We also found that variations in a polynucleotide T-tract located in the sabA promotor could fine-tune SabA expression/ sLex-binding. Thus we have exposed how strict regulation by TCSs combined with stochastic processes together shapes attachment in the bacterial population. As the buffering mucus layer is constantly exfoliated, placing H. pylori in bactericidal acid, we hypothesized that low pH should abrogate adhesion. SabA expression was indeed repressed in low pH, however BabA expression remained unaffected. The BabA/ Leb-binding was instead directly reversibly hampered by low pH and the degree of pH sensitivity was strain dependent and encoded in the BabA sequence. We believe that the pH dependent loss of binding is one key factor H. pylori utilizes to maintain persistent infection. BabA is divided in generalists that bind ABO antigens and specialists that only bind blood group (bg) O. We co-crystalized BabA bound to these receptors and established the structural basis for generalist vs. specialist discrimination. We furthermore found a disulfide-clasped loop (CL2) in the center of the binding domain crucial for binding. Breaking CL2 with N-Acetylcysteine (NAC) disrupted binding and H. pylori infection mice experiments revealed inflammatory reduction upon NAC-treatment. In sum, I have in my thesis dissected how H. pylori controls its adhesive abilities and how intrinsic properties in binding can be exploited for therapeutic purposes.

Nous avons construit plusieurs plasmides intégratifs et réplicatifs pour appliquer la méthode TAP-tag à l'étude du protéome de la bactérie Helicobacter pylori. Le transfert des plasmides chez H. Pylori a échoué cependant les derniers résultats obtenus montrent que l'intégration d'ADN exogène par double recombinaison homologue dans le génome bactérien semble être une alternative. Une seconde approche utilisant la méthode BN-PAGE d'électrophorèse en conditions natives couplée à une analyse en LC-MS/MS a permis l'identification de 91 complexes membranaires et cytoplasmiques dont 36 complètement nouveaux et 55 déjà connus, validant l'intérêt d'utiliser cette technique pour l'étude du complexome de H. Pylori. Ces complexes impliquent des facteurs de virulence ainsi que de nombreuses protéines de surface. De tels résultats ouvrent de nouvelles voies dans la compréhension de la pathogénicité de H. Pylori et devraient définir de nouvelles stratégies thérapeutiques et/ou vaccinales<br>Several integrative and replicative plasmids have been built to study the proteome of the bacterium Helicobacter pylori using the TAP-tag method. The transfer of these plasmids in H. Pylori have failed, however the last results obtained show that the exogenic DNA integration by double homologous recombination in the bacterial genome seems to be an alternative way. A second approachj using BN-PAGE method of electrophoresis performed in native conditions coupled to an analysis in LC-MS/MS was used. It allowed the identification of 91 membrane and cytoplasmic protein complexes including 36 entirely new and 55 already described, validating the interest to use this technique for the study of the complexome of H. Pylori. These complexes involve virulence factors as well as many proteins of cell surface. Such innovating results open new ways in the comprehension of the pathogenicity of H. Pylori and should allow the definition of new therapeutic and/or vaccine strategies

Helicobacter pylori exhibits tropism for the human stomach causing a spectrum of complications ranging from gastritis to gastric cancer in susceptible individuals. The mechanism(s) that allow the bacteria to persist and cause disease are unfolding. p-defensins are a family of endogenous, epithelial anti-microbial peptides that engage in host defense most prominently at mucosal surfaces. We and others have previously shown that human p-defensin (hBD)-2 and -3 are potent bactericidal agents against H. pylori. At present the identity of signalling pathways involved in host-bacterial cross talk leading to modulation of host antimicrobial immunity are unknown. The present study firstly investigated the potential role of bacterial virulence factors in mediating human p-defensin gene expression during H. pylori infection. AGS gastric epithelial cells were infected with cytotoxic H. pylori strains (60190, 84-183) and isogenic mutant strains (cagA-, cagE-, vacA- and CagPAl-). Human p-defensin (hBD2 & -3) gene expression quantified by RT-PCR and p-defensin transcriptional regulation was followed by transient transfection studies utilising hBD2 and -3 promoter luciferase constructs. We found hBD2 induction was dependent upon an intact cagPAI and minimal involvement was observed for the bacterial virulence factors CagA and VacA in modulating P-defensin expression. We sought to investigate the bacterial component responsible for instigating epithelial innate immune responses. Through the use of siRNA for NODI we determined a role for NODI-dependent NF-kB activation in mediating hBD2 but not hBD3 expression. Experiments utilising specific inhibitors of the MAP Kinase pathways directed us to delineate the role of each pathway in modulating p-defensin expression by the activation of stably transfected conditional MAP Kinase mutants. These studies revealed critical involvement of ERK pathway in the regulation of hBD3 but not hBD2 gene expression. Signalling upstream of ERK was explored and revealed EGFR as the host receptor responsible for detection and initiation of hBD3 gene and peptide production. Our studies demonstrated a crucial role for NODI in H. /Ty/ort-mediated hBD2 but not hBD3 expression and implicate EGFR transactivation in mediating hBD3 but not hBD2 expression, thus indicating two distinct regulatory mechanisms at play during innate immune host response to H. pylori infection.

Cordia lutea Lam. (Flor de Overo) est une plante utilisée en médecine traditionnelle péruvienne pour le traitement des troubles gastro-intestinaux, des hépatites et des douleurs rénales. Onze composés ont été isolés à partir de l'extrait éthanolique des fleurs de cette plante médicinale sur la base d'un fractionnement bioguidé. Dix de ces composés (cordiasecosides A-J), représentent les premiers exemples de 9,10-seco-29-norcycloartane glycosides. Leurs structures ont été déterminées par des analyses de données RMN et MS ainsi que des analyses cristallographiques aux rayons X. Ces composés ont montré une activité in vitro anti-Helicobacter pylori significative et aucune activité contre Escherichia coli ou Pseudomonas aeruginosa. Une activité significative a été observée pour les cordiasecosides E et F contre Staphylococcus aureus. Tous les composés présentent une faible cytotoxicité contre les cellules RAW 264,7. Les activités antileishmaniales et antiplasmodiales in vitro des cordiasecosides A à F ont également été évaluées. Le présent travail de recherche comprend aussi l'isolement de deux composés précédemment décrits dans la littérature : l'acide isorinique et la quercétine 3-robinobioside. Enfin, l'étude botanique et chimique de différents lots de matériel végétal achetés sur différents marchés a également été réalisée, dans le but d'évaluer la variabilité de la drogue et de proposer des pistes pour contrôler sa qualité<br>Cordia lutea Lam. (Flor de Overo) is a plant used in Peruvian traditional medicine as a remedy for the treatment of gastrointestinal disorders, hepatitis and kidney pain. Eleven compounds were isolated from the ethanol extract of the flowers of this medicinal plant on the basis of bioassay-guided fractionation. Ten of these compounds (cordiasecosides A-J), represent the first examples of 9,10-seco-29-norcycloartane glycosides. Their structures were determined by the application of NMR and MS data analysis together with X-ray crystallographic analyses. These compounds showed significant in vitro anti-Helicobacter pylori activity, and no activity against Escherichia coli or Pseudomonas aeruginosa. Significant activity was observed for cordiasecosides E and F against Staphylococcus aureus. All compounds displayed weak cytotoxicity against RAW 264.7 cells. The in vitro antileishmanial and antiplasmodial activities of cordiasecosides A -F were also evaluated. This research work also includes the isolation of two compounds previously described in the literature: isorinic acid and quercetin 3-robinobioside. Finally, the botanical and chemical study of different batches of plant material purchased from different markets was also carried out, with the aim of assessing the variability of the drug and proposing ways to control its quality

Les systèmes Toxine Antitoxine (TA) sont présents chez la plupart des génomes bactériens. Nous rapportons dans cette étude la présence de tels systèmes chez la bactérie pathogène Helicobacter pylori. Ce nouveau système TA de type I, de la même manière que les autres systèmes de type I décrits, est composé d’une toxine peptidique membranaire (AapA) dont la traduction est inhibée par un ARNnc (IsoA) suivant une interaction côté 5’ non traduit de l’ARNm. La structuration particuliere de l’ARNm a permit l’identification d’orthologue de ce système dans les chromosomes des genres Helicobacter et Campylobacter mais aussi sur leur patrimoine génétique mobiles, tels les plasmides. Ceci impliquant leur potentielle acquisition dans le génome par transfert génétique horizontal. La deuxième partie de l’étude se focalise sur la résolution par RMN du liquide de la structure atomique de la toxine AapA1 dans un environnement pseudo-membranaire. Une approche par mutation révèle les determinants structuraux de la toxicité, pointant la présence d’une empreinte de charge dans la partie helicoidale transmenbranaire de la toxine présente aussi chez d’autres toxines de Type I. La dernière partie de l’étude se centre sur l’expression et la purification de la toxine afin d’en étudier la structure en environnement membranaire complexe par RMN du solide. Les résultats prometteurs ouvrent la voie à une caractérisation de la toxine par l’expérience de PISEMA<br>Toxin-antitoxin (TA) systems are present in almost all bacterial genomes. Here we report that the genome of the major human gastric pathogen, Helicobacter pylori, is hosting several copies of a new family of type I TA systems. Similarly to other type I TA systems, the toxin (AapA) is a small membrane protein whose expression is controlled by a small antisense RNA (IsoA, antitoxin) that binds to the 5’ untranslated region (UTR) of the mRNA. In addition we used the strong conservation of the mRNA folding to identify homologs of this TA system not only in other Helicobacter and Campylobacter chromosomes but also on plasmids, indicating that this new TA system might have been spread over different genomes via horizontal gene transfer.The second part of the study take account of the AapA toxin itself. We acutely determine the structure of AapA1 by liquid state NMR in membrane mimicking environment. We then probe first structural insight on atomic structural determinants of its toxicity following a mutation studies.These results reveal a particular charge pattern on the transmembrane α helix domain of AapA toxin similary to other Type I toxin. A third part of the studies is based on expression and purification of the toxin in order to determine the structure in complex membrane environment by solid state NMR. The promosisng result open the way to characterize the toxin by PISEMA experiment

Thesis (Ph. D.)--University of Oregon, 2006.<br>Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-91). Also available for download via the World Wide Web; free to University of Oregon users.