Die transkriptionelle Aktivierung des HDC Gens sowie des Chromogranin A Gens in ECL-Zellen der Magenmucosa repräsentiert einen zentralen Mechanismus der Säureregulation durch Gastrin und scheint ausserdem Bedeutung für die Pathogenese der gastroduodenalen Ulkuskrankheit zu haben. Unsere Untersuchungen identifizieren erstmals die molekularen Mechanismen der Gastrin-abhängigen Regulation beider Gene und definieren die beteiligten Transkriptionsfaktoren, regulatorischen DNA-Elemente und intrazellulären Signalwege. Des weiteren wurde durch transgene Untersuchungen die transkriptionelle Regulation des ChromograninA Gens in vivo bestätigt und die neuroendokrin-spezifische Expression eines 4.8kB-langen CgA-Promotorfragmentes demonstriert. Als pathobiologisch relevante Aktivatoren des HDC Gens konnten oxidativer Stress sowie die H. pylori Infektion identifiziert und hinsichtlich ihrer molekularen Wirkungen auf das Schlüsselgen der Histaminsynthese im Magen charakterisiert werden. Diese Ergebnisse dokumentieren einen potentiellen Mechanismus für die Interaktion beider Stimuli mit den physiologischen Regelkreisen der Magensäureregulation und können durch die Definition neuer molekularer Angriffspunkte möglicherweise zur Entwicklung innovativer Therapieansätze beitragen. / Transcriptional activation of the genes encoding histidine decarboxylase and chromogranin A represents a key mechanism of gastrin-dependent acid regulation and also appears to be involved in the pathogenesis of gastroduodenal ulcer disease. Our results for the first time identify the molecular mechanisms underlying gastrin-dependent activation of both genes, and define the transcription factors, regulatory DNA elements and signal transduction pathways involved in this process. Furthermore, transgenic studies confirmed the principle of gastrin-dependent transcriptional activation of the chromogranin A gene in vivo, and demonstrated neuroendocrine-specific expression of a 4.8kB-CgA promotor fragment. In addition, the pathobiological stimuli oxidative stress and H. pylori were molecularly characterized regarding their activating effects on the key gene of gastric histamine sythesis. These results provide potential mechanisms for the interaction of both stimuli with regulatory circuits of gastric acid secretion, and can probably contribute via definition of new molecular targets to the development of inovative therapeutic strategies.

In der humanen Magenschleimhaut regulieren eine Vielzahl von Interaktionen zwischen verschiedenen Zellpopulationen die Verdauung und die Überwachung von Infektionen. Epithelzellen in der Schleimhaut differenzieren in spezialisierte Zelltypen, die schützenden Mukus, Magensäure, Verdauungsenzyme oder Hormone produzieren. Eine Infektion mit Helicobacter pylori kann die Gewebehomöostase fehlregulieren, was die Wahrscheinlichkeit erhöht, dass an der Infektionsstelle ein Magengeschwür, ein Adenokarzinom oder letztendlich Magenkrebs auftritt. In dieser Arbeit wird die Entwicklung eines neuartigen in vitro Kulturmodells für humane primäre Magenepithelzellen, die sogenannte Mukosoidkultur, gezeigt. Die Mukosoidkulturen sind repräsentativ für Epithelbarrieren und rekapitulieren die meisten Funktionen der menschlichen Magenschleimhaut in vivo, einschließlich der Schleimproduktion, und ermöglichen eine langfristige und stabile Kultivierung von Epithelzellen sowie Infektionsstudien mit H.pylori. Mukosoidkulturen aus Corpus wurden verwendet, um die Nischenfaktoren zu untersuchen, die die Differenzierung von Oberflächenepithelzellen, Hauptzellen und Parietalzellen fördern. EGF erwies sich zusammen mit BMP/Noggin als ein wichtiger Regulator bei der Differenzierung. Stromazellen sind Teil der Lamina propria der Magenschleimhaut. Über die Wechselwirkung mit dem Epithel unter homöostatischen Bedingungen und bei bakteriellen Infektionen mit H.pylori ist nur sehr wenig bekannt. Die Co-Kultur von humanen primären Stromazellen des Magens mit Epithelzellen unter Verwendung des Mukosoidkultur-Modells zeigte die aktive Signalübertragung zwischen beiden Zelltypen auf. Darüber hinaus wurden Mukosoidkulturen erfolgreich mit H.pylori infiziert. Die Ergebnisse zeigen, dass Stromazellen aktiv mit Cytokin- und Chemokinexpression auf eine epitheliale Infektion reagieren. Gleichzeitig erhöhten Stromazellen die NFκB-gesteuerte Entzündungsreaktion in Epithelzellen. / In the human gastric mucosa, multiple interactions between different cell populations regulate digestion and surveillance of infections. Epithelial cells in the mucosa differentiate into specialized cell types to produce protective mucins, gastric acid, digestive enzymes or hormones. Infection with Helicobacter pylori dysregulates the tissue homeostasis increasing the chance to develop a gastric ulcer, adenocarcinoma or ultimately gastric cancer at the site of infection. In this thesis, the development of a novel in vitro culture model for human primary gastric epithelial cells, called the mucosoid culture, is shown. The mucosoid cultures are representative of epithelial barriers and recapitulate most of the functions of the human gastric mucosa in vivo, including mucus production, and allow long-term and stable cultivation of epithelial cells as well as infection studies with H.pylori. Corpus derived mucosoids were used to investigate the niche factors that promote the differentiation of foveolar cells, chief cells, and parietal cells. EGF was found to be a major regulator in differentiation together with BMP/Noggin. Stromal cells are part of the lamina propria of the gastric mucosa. Very little is known about the interaction with the epithelium under homeostatic conditions and during bacterial infections with Helicobacter pylori. The co-culture of human primary gastric stromal cells with epithelial cells using the mucosoid culture model demonstrated the active signaling between both cell types. Furthermore, mucosoid cultures were successfully infected with H. pylori. The results revealed that stromal cells actively respond to epithelial infection with cytokine and chemokine expression. Concurrently stromal cells increased the NFκB-driven inflammatory response in epithelial cells.

Das humane Magenpathogen Helicobacter pylori (H. pylori) besiedelt den menschlichen Magen und kann zu der Entstehung schwerwiegender Krankheiten wie Magenkrebs und Magengeschwüren führen. Die Pathogenese ist eng mit dem bakteriellen Typ IV Sekretionssystems (T4SS) assoziiert, das die Translokation des Effektorproteins CagA in die Wirtszelle vermittelt. Bisher ist noch unbekannt, in welchem Ausmaß wirtszellspezifische Faktoren die T4SS induzierte Pathogenese beeinflussen. Dieser Aspekt wurde in dieser Arbeit durch die Analyse verschiedenster Zelllinien das erste Mal systematisch untersucht. Interessanterweise unterschied sich die zelluläre Antwort auf die T4SS spezifische Infektion erheblich in Abhängigkeit der verwendeten Zelllinie. Die Ergebnisse beweisen, dass Wirtszellfaktoren eine ebenso große Rolle in der H. pylori induzierten Pathogenese spielen wie bakterielle Effektoren. Zusätzlich wurde in dieser Arbeit eine genomweite Screening-Methode etabliert, die es ermöglicht, neue Komponenten des T4SSs, translozierte NF-B Effektoren und bakterielle Adhäsine zu identifizieren. Auch der Einfluss von CagA auf den EGF-Rezeptor wurde hier näher untersucht. Der Rezeptor steht ebenfalls eng mit der Entstehung von Krebs in Verbindung. Hierbei stellte sich heraus, dass CagA die Endozytose des EGF-Rezeptors durch die Aktivierung der Nicht-Rezeptor Tyrosinkinase c-Abl hemmt und dadurch die Rezeptorpopulation auf der Wirtszelloberfläche erhöht. Interessanterweise führt dieser Effekt jedoch nicht zu einer Verstärkung der EGF-Rezeptor Signaltransduktion. Vielmehr kommt es zu einer Hemmung der EGF-Rezeptor Transaktivierung und zu einer Blockade der EGF vermittelten Wundheilung. Die Daten weisen auf eine Rolle des EGF-Rezeptors in der H. pylori induzierten Geschwürbildung hin. Auch der zu Grunde liegende molekulare Mechanismus der Rezeptor-Inhibierung konnte hier entschlüsselt werden, der sowohl von CagA als auch von der Phosphatase SHP-2 gesteuert wird. / The human gastric pathogen Helicobacter pylori (H. pylori) elicits a tremendous medical burden because of its causative association with peptic ulcer disease and gastric cancer. The pathogenic potential of H. pylori is intricately linked to the expression of a pathogenicity island encoded type IV secretion system (T4SS), which translocates the bacterial effector protein CagA into the eukaryotic host cell. The role of host cell determinants in T4SS mediated pathogenesis has not yet been systematically examined. To elucidate the role of host cell factors within T4SS induced host cell responses, different eukaryotic cell lines were analyzed systematically for respective phenotypes. Remarkably, T4SS mediated host responses among these cell lines varied considerably, thereby demonstrating the importance of host cell components in H. pylori induced pathogenesis. In addition, a H. pylori genome wide bacterial screen for factors important in pathogenesis, such as unknown T4SS components or novel NF-kappaB effector molecules, was developed and optimized. The precise function of the prominent effector protein CagA remains unclear. To functionally characterize the role of CagA, its impact on the epidermal growth factor (EGF)-receptor pathway was analyzed. The results suggest a mechanism where EGF-receptor endocytosis is completely blocked by a CagA induced activation of c-Abl, leading to an elevated receptor surface exposition. Surprisingly, EGF-receptor transactivation and EGF-dependent wound healing are selectively blocked during prolonged infections as well, indicating that an increased receptor-population on the cell surface does not necessarily promote signaling. This data suggests a role for the EGF-receptor in H. pylori- induced ulcer disease. The underlying molecular mechanism was identified as being SHP-2 and CagA dependent.

Die kontinuierliche Rezirkulation von Immunzellen durch periphere und sekundäre lymphatische Organe (SLOs) ist Bestandteil der Immunüberwachung und wichtig für die Aufrechterhaltung und Funktionsbereitschaft des Immunsystems. Der homöostatische Chemokinrezeptor CCR7 vermittelt dabei nicht nur die Rezirkulation von Lymphozyten durch SLOs, sondern scheint auch an der homöostatischen Rezirkulation von Lymphozyten durch nicht-lymphoide periphere Gewebe beteiligt zu sein. Im Rahmen dieser Arbeit wurde mithilfe von CCR7-defizienten Mäusen die funktionale Bedeutung von CCR7 für die homöostatische Rezirkulation von Lymphozyten durch das Peritoneum untersucht und nachgewiesen, dass CCR7 der dominante Chemokinrezeptor ist, der unter physiologischen Bedingungen die Transitzeit von Lymphozyten durch das Peritoneum festlegt. Die gestörte Rezirkulation von Lymphozyten begünstigte außerdem die Entstehung von tertiären lymphoiden Organen (TLOs) in der Magenschleimhaut von CCR7-defizienten Mäusen. Untersuchungen zur zellulären und molekularen Grundlage dieser und weiterer pathomorphologischer Veränderungen in der Magenschleimhaut von CCR7-defizienten Mäusen verdeutlichten die Funktion von CCR7 für die Etablierung von zentraler und peripherer Toleranz gegenüber gastrischen Antigenen. Fehlt CCR7, dann entwickelten Mäuse eine spontane Autoimmungastritis, welche durch gastritogene CD4+ T-Zellen verursacht wurde, deren Aktivierung auch unabhängig von Lymphknoten und TLOs erfolgte. Die Entstehung von TLOs wird auch bei einer durch Helicobacter pylori ausgelösten chronischen Gastritis beobachtet. Die Expression des homöostatischen Chemokinrezeptors CXCR5 und seines Liganden CXCL13 ist mit der Entwicklung dieser TLOs korreliert worden. Unter Verwendung eines Mausmodells für H. pylori-induzierte chronische Gastritis konnte gezeigt werden, dass CXCR5 die Ausbildung von TLOs vermittelt und eine Rolle für die Induktion von H. pylori-spezifischen T-Zell- sowie humoralen Immunantworten spielt. / Homeostatic recirculation of immune cells through peripheral and secondary lympoid organs (SLOs) is required for immune surveillance and the maintenance and functionality of the immune system. The homeostatic chemokine receptor CCR7 controls not only lymphoid cell trafficking to and within SLOs, but also seems to be involved in the homeostatic recirculation of lymphocytes through non-lymphoid peripheral tissues. Within the scope of this work we investigated the functional relevance of CCR7 for the homeostatic recirculation of lymphocytes through the peritoneal cavity and could show, that CCR7 is the dominant chemokine receptor which defines the transit time of lymphocytes in the peritoneal cavity under physiological conditions. Impaired recirculation of lymphocytes also promoted the development of tertiary lymphoid organs (TLOs) in the gastric mucosa of CCR7-deficient mice. Analysis of the cellular and molecular mechanisms underlying these and other pathomorphological alterations in the gastric mucosa of CCR7-deficient mice provided further evidence regarding the function of CCR7 for the establishment of central and peripheral tolerance towards gastric antigens. Mice that lack CCR7 spontaneously developed autoimmune gastritis, which was caused by gastritogenic CD4+ T-cells. Such autoreactive T cell responses were also initiated in the absence of lymph nodes and TLOs in CCR7/LT-alpha double-deficient mice. Development of TLOs is also observed during chronic gastritis induced by Helicobacter pylori. The expression of the homeostatic chemokine receptor CXCR5 and its ligand CXCL13 has been correlated with the development of these TLOs. Using a mouse model for H. pylori-induced chronic gastritis, we could show that CXCR5 is responsible for the development of TLOs and also plays a role for the induction of H. pylori-specific T and B cell responses.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-23T18:02:31Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PrevalenciaHelicobacterPylori.pdf: 934699 bytes, checksum: b0ff88938ac2eac1e23b4faf11e82eb3 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-24T14:45:38Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PrevalenciaHelicobacterPylori.pdf: 934699 bytes, checksum: b0ff88938ac2eac1e23b4faf11e82eb3 (MD5) / Made available in DSpace on 2013-04-24T14:45:38Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PrevalenciaHelicobacterPylori.pdf: 934699 bytes, checksum: b0ff88938ac2eac1e23b4faf11e82eb3 (MD5) Previous issue date: 2013 / Introdução: Infecções por Helicobacterpylori(HP) e vírusEpstein-Barr (VEB) são comuns no mundo todo, embora o HP seja o maior fator em doenças gastroduodenais, seu percentual de associação com VEB é incerto. Tanto o VEB quanto o HP são classificados como carcinógenos classe 1 pela Organização Mundial de Saúde, e uma substancial fração de indivíduos se tornam co-infectados na adultice. Esses dois patógenos podem potencializar sinergicamente para causar gastrite crônica perpetua. O objetivo deste trabalho foi verificar a prevalência de HP e do vírus Epstein-Barr em crianças e adolescentes. Material e Método: Estudo descritivo, do tipo transversal. Foram analisadas amostras de mucosa gástrica de 64 crianças e adolescentes através do Teste da Urease para diagnóstico do HP, da técnica de PCR para detecção da cepa cagA de H. pylori, da técnica de hibridização in situ para detecção do EBV e da análise patológica para determinação de características histopatológicas. Resultados: A prevalência de HP nas crianças e adolescentes em estudo foi de 53,1% enquanto a prevalência de VEB foi 3,1%. Entre os pacientes infectados por HP, a maioria (94,3%) apresentava gastrite a endoscopia digestiva alta, sendo gastrite enantemática a mais comumente encontrada. Na análise histopatológica, também a maioria (97,1%) dos pacientes apresentava algum grau de gastrite, com 80% classificados com gastrite crônica moderada. Cepas cagA positivas foram encontradas em 64,7% dos infectados com HP e entre estes todos tinham gastrite, com predomínio de gastrite crônica moderada (54%), no entanto não se observou correlação com significância estatística entre esses achados. Em adição, também não houve significância estatística para a associação entre infecção por HP e por VEB na população estudada, a baixa prevalência de VEB nesta análise sugere que esse vírus não é um agente etiológico das lesões da mucosa gástrica. No nosso conhecimento, este é o primeiro estudo que relaciona estes dois agentes infecciosos na mucosa gástrica de crianças e adolescentes do norte do Brasil. Conclusão: A maioria dos achados deste estudo se assemelha aos relatos da literatura, contudo evidenciou-se a necessidade de estudos com maior casuística, envolvendo a população pediátrica imunocompetente afim de melhor esclarecer se há ou não correlação entre a infecção por HP e VEB em nossa região. / Introduction: Infections by Helicobacter pylori (HP) and Epstein-Barr virus (EBV) are common worldwide, although HP is the highest factor in gastroduodenal diseases, its percentage of association with EBV is uncertain. Both EBV and HP are classified are class 1 carcinogens by the World Health Organization, and a substantial number of individuals become co-infected in adulthood. These two pathogens may have synergic potential to cause perpetual chronic gastritis. The purpose of this paper was to verify the prevalence of HP and Epstein-Barr virus in children and adolescents. Material and Methods: Transversal descriptive study. The gastric mucosa of 64 children and adolescents was analyzed through the Urease Test to diagnose HP and the PCR technique to detect H. pylori’s cagA strain, the in situ hybridization technique to detect EBV and the pathological analysis to determine the histopathological characteristics. Results: The prevalence of HP and EBV found by this study was 53.1% and 3.1, respectively. Most of the patients infected by HP (94.3%) presented gastritis in the upper gastrointestinal endoscopy, with enanthemathous gastritis being the most commonly found type. In the histopathological analysis, most patients (97.1%) presented some level of gastritis, 80% of which classified as moderate chronic gastritis. Positive cagA strains were found in 64.7% of the patients infected with HP and all of them had gastritis, with predominance of moderate chronic gastritis (54%); however, there was no statistically significant correlation between these findings. There was also no statistically significant association between infection by HP and EBV in the studied population. The low prevalence of EBV in this analysis suggests that this virus is not an etiological agent in gastric mucosa lesions. To our knowledge, this is the first study that relates these two infectious agents in the gastric mucosa of children and adolescents in northern Brazil. Conclusion: Most of the findings in this study are in line with the literature; however, it is necessary to conduct larger studies, involving aimmunocompetent pediatric population in order to determine whether there is a correlation between infection by HP and EBV in our region.

Tese de Mestrado em Nutrição Clínica apresentada à Faculdade de Ciências de Nutrição e Alimentação da Universidade do Porto / Resumo da dissertação: A infecção por Helicobacfer pylori (H. pylorg afecta 50 % da população mundial e está associada a gastrite crónica, a úlcera péptica e ao cancro gástrico. A aquisição da infecção por H. pylori e a sua erradicação espontãnea parece ser comum na infância. Na fase adulta, a infecção é mais persistente. Alguns autores defendem que o consumo de bebidas alcoólicas pode contribuir para a erradicação espontãnea de H. pylori, nos adultos. Esta hipótese é apoiada por vários estudos epidemiológicos e experimentais. No presente trabalho foi testada, in vifro, a actividade anti H. pylori de vinho tinto e branco, de diferentes tipos de cerveja (branca com e sem álcool, preta com álcool e preta com álcool enriquecida com xantohumol e iso-xantohumol), assim como do resveratrol e do xantohumol, compostos fenólicos presentes no vinho e na cerveja, respectivamente. Onze estirpes clínicas e uma estirpe de referência (CCUG 15818) de H. pylori foram inoculadas em agar Columbia suplementado com 10 % de sangue, ao qual foram adicionadas as várias bebidas e os compostos fenólicos, em diferentes concentrações, e incubadas em condições de microaerofilia, a 36+-1ºC durante 48 horas.

<p>Prokaryotes are continually exposed to variations in their environment. Survival in unstable milieu requires a wide range of transcriptional regulators (TRs) that respond to specific environmental and cellular signals by modulating gene expression and provide an appropriate physiological response to external stimuli. These adaptive responses to environmental signals are mostly mediated by TRs from one of two families: the single or the two component signal transduction systems (1CSTS; 2CSTS). In this thesis the structural analysis of two 1CSTS – DntR and NikR − are presented. One study was carried out to try to develop a bacterial biosensor for synthetic dinitrotulenes compounds, the other to characterise the Ni-sensing mechanism that contributes to the acid adaptation of the human pathogen<i> Helicobacter pylori.</i> DntR belongs to the LysR family and the crystal structures obtained have allowed the proposal a model of the interaction of DntR with salicylate inducer as well as giving insights into the signal propagation mechanism in LysR-type transcription factors (<b>paper I</b>). DntR mutant crystal structures combined with the modelling of DntR-2,4-dnt interactions led to the design of a DntR mutant that has a limited response to 2,4-dnt in a whole cell biosensor system (<b>paper 2</b>). Crystal structures of apo-NikR from <i>H. pylori </i>(HpNikR) and of Ni-bound intermediary states of the protein were obtained. The latter have helped in unravelling the Ni incorporation and selectivity mechanisms of NikRs and have shown a strong cooperativity between conformational changes in the Ni binding domain with movements of the DNA binding domain (<b>paper 3</b>). Biochemical studies and comparisons of the HpNikR crystal structures with those of NikR homologues strongly suggest that HpNikR has evolved different surface properties (<b>paper 4</b>) and a new mode of DNA binding. </p>

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

Prokaryotes are continually exposed to variations in their environment. Survival in unstable milieu requires a wide range of transcriptional regulators (TRs) that respond to specific environmental and cellular signals by modulating gene expression and provide an appropriate physiological response to external stimuli. These adaptive responses to environmental signals are mostly mediated by TRs from one of two families: the single or the two component signal transduction systems (1CSTS; 2CSTS). In this thesis the structural analysis of two 1CSTS – DntR and NikR − are presented. One study was carried out to try to develop a bacterial biosensor for synthetic dinitrotulenes compounds, the other to characterise the Ni-sensing mechanism that contributes to the acid adaptation of the human pathogen Helicobacter pylori. DntR belongs to the LysR family and the crystal structures obtained have allowed the proposal a model of the interaction of DntR with salicylate inducer as well as giving insights into the signal propagation mechanism in LysR-type transcription factors (<b>paper I</b>). DntR mutant crystal structures combined with the modelling of DntR-2,4-dnt interactions led to the design of a DntR mutant that has a limited response to 2,4-dnt in a whole cell biosensor system (<b>paper 2</b>). Crystal structures of apo-NikR from H. pylori (HpNikR) and of Ni-bound intermediary states of the protein were obtained. The latter have helped in unravelling the Ni incorporation and selectivity mechanisms of NikRs and have shown a strong cooperativity between conformational changes in the Ni binding domain with movements of the DNA binding domain (<b>paper 3</b>). Biochemical studies and comparisons of the HpNikR crystal structures with those of NikR homologues strongly suggest that HpNikR has evolved different surface properties (<b>paper 4</b>) and a new mode of DNA binding.

ABSTRACT Protein-protein interactions play a central role in most biological processes. One such biological process is the maintenance of a reducing environment inside the cell. To maintain an internal reducing environment, living cells have evolved two enzymatic systems (glutathione and thioredoxin (Trx) systems). The Trx system is composed of the enzyme TrxR and its substrate Trx. The two proteins constitute an important thiol-dependent redox system that catalyzes the reduction of many proteins that are responsible for a variety of cellular functions. The system relies on transient protein-protein interactions between Trx and TrxR for its function. Cross-reactivity of components of the Trx system between species has been shown to be medically relevant. For example, Helicobacter pylori Trx (HP Trx) is thought to mediate catalytic reduction of human immunoglobulins and thus facilitate immune evasion. It has also been proposed that Helicobacter pylori gains access to the impenetrable gastric mucous layer by using secreted HP Trx to reduce the disulfide bonds present in the cysteine-rich mucin regions that are responsible for cross-linking mucin monomers. Therefore, disruption of secreted HP Trx-host protein interaction may result in restoration of the viscoelastic and hydrophobic protective properties of mucus. Previous studies aimed at understanding the nature of cross-reactivity of Trx system components among various species have shown that Trxs have higher affinity for cognate TrxRs (same species), than for TrxRs from different species. However, the basis for this specificity is not known. A growing body of evidence suggests that most protein-protein interactions are mediated by a small number of protein-protein interface residues, referred to as hot spot residues or binding epitopes. Therefore, understanding the biochemical basis of the affinity of proteins for their partners usually begins by identifying the hot spot residues responsible for the protein complex interactions. In this study, the crystal structures of Deinococcus radiodurans thioredoxin reductase (DR TrxR) and Helicobacter pylori TrxR (HP TrxR) were determined at 1.9 Å and 2.4 Å respectively. Analysis of the Trx-binding sites of both structures suggests that the basis of affinity and specificity of Trx for TrxR is primarily due to the shape rather than the charge of the surface. In addition, the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (EC Trx) was used to identify residues that are responsible for TrxR-Trx interface stability. Using computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface, 22 EC TrxR side chains were shown to make contact across the TrxR-Trx interface. Although more than 20 EC TrxR side chains make contact across the TrxR-Trx interface, our results suggest that only 4 residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent DR TrxR residues (M84, K137, F148, F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. These hot spot residues are surrounded by less important residues (hydrophobic and hydrophilic) that are also predicted to contribute to interface stability. F148 and F149 are invariant across bacterial TrxRs, however other residues that contact Trx are less conserved including M84 and K137. When M84 and K137 were changed to match equivalent E. coli TrxR residues (K137R, M84F); D. radiodurans TrxR substrate specificity was altered from its own Trx to that of E. coli Trx. The results suggest that a small subset of the TrxR-Trx interface residues are responsible for the majority of Trx binding affinity and specificity, a property that has been shown to general to protein-protein interfaces.