Rozprawy doktorskie na temat „Helicobacter hepaticus”

Thesis (M.S.) University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (June 27, 2007) Includes bibliographical references.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.
Includes bibliographical references (leaves 112-118).
Recognition of polymicrobial infections is becoming important for understanding differential host responses to environmental exposures, vaccines, as well as therapeutics. Citrobacter rodentium is a well-characterized model of infectious colitis with particular usefulness for modeling human diarrheal disease or inflammatory bowel disease. Infection with Helicobacter hepaticus is subclinical and persistent in C57BL/6 mice, but causes disease in susceptible strains and immunodeficient mice. To test the hypothesis that subclinical persistent infection modulates the host response to diarrheal disease a polymicrobial mouse model utilizing H. hepaticus and C. rodentium was developed and characterized. Concurrent infection has been shown to modulate disease outcome through several mechanisms including: cross-reactivity between viral antigens; shifting T cell response from Th1 to Th2 by helminth infection; and induction of regulatory T cells that suppress host response. In this new model of polymicrobial infection, a new paradigm in which persistent infection prolonged the course of acute colitis associated with a deviation from Thl-biased disease to Th17 was observed.
(cont.) In addition, Foxp3+naturally-occurring regulatory T cells (nTre,) were markedly increased during active colitis. The accumulation of nTreg was sustained when mice were persistently infected with H. hepaticus, indicating on-going active colitis. Although persistent infection was able to modulate host response, protective immunity to a subsequent C. rodentium infection was not compromised. Persistent infection also modulated host response to soluble antigen by preventing induction of oral tolerance to single bolus, but not to continuous, high-dose antigen feeding. Using H. hepaticus infection of C57BL/6 mice, models to investigate the immunomodulatory potential of persistent infection on immunogenic responses of protective immunity to enteric infection, host response to polymicrobial enteric infection, as well as tolerogenic responses to soluble antigen were developed. These models establish baselines for further investigation into the influences of persistent infection on host immune responses.
by Megan Earley McBee.
Ph.D.

Thesis (Ph. D. in Molecular and Systems Bacterial Pathogenesis)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references.
Helicobacter hepaticus infection of A/JCr mice is a model of liver cancer resulting from chronic active inflammation. We monitored hepatic global gene expression profiles and correlated them to histological liver lesions in H. hepaticus infected and control male A/JCr mice at 3 months, 6 months, and 1 year of age. We used an Affymetrix-based oligonucleotide microarray platform on the premise that a specific genetic expression signature at isolated time points would be indicative of disease status. Model based expression index comparisons generated by dChip yielded consistent profiles of differential gene expression for H. hepaticus infected male mice with progressive liver disease versus uninfected control mice within each age group. Linear discriminant analysis and principal component analysis allowed segregation of mice based on combined age and lesion status, or age alone. Up-regulated genes present throughout the 12 month study involved inflammation, tissue repair, and host immune function. Upregulation of putative tumor and proliferation markers correlated with advancing hepatocellular dysplasia. Transcriptionally down-regulated genes in mice with liver lesions included those related to peroxisome proliferator, cholesterol, and steroid metabolism pathways. Transcriptional profiling of hepatic genes documented gene expression signatures in the livers of H. hepaticus infected male A/JCr mice with chronic progressive hepatitis and preneoplastic liver lesions, complemented the histopathological diagnosis, and suggested molecular targets for the monitoring and intervention of disease progression prior to the onset of hepatocellular neoplasia. Our laboratory, in collaboration with Professors Suerbaum and Schauer, recently identified a
(cont.) 70kb genomic island in Helicobacter hepaticus strain ATCC 51488 as a putative pathogenicity island (HhPAI) (Suerbaum et al, PNAS, 2003). This region within H. hepaticus contains genes HH0233-HH0302, a differential GC content, several long tandem repeats but no flanking repeats, and three components of a type IV secretion system (T4SS). A/JCr mice were experimentally infected with three naturally occurring strains of H. hepaticus including the type strain H. hepaticus ATCC 51488 strain (Hh 3B1) isolated from A/JCr mice, MIT 96-1809 (Hh NET) isolated from mice shipped from the Netherlands, and MIT-96-284 (HhG) isolated from mice acquired from Germany.4 HhNET (missing most of the HhPAI) infected male A/JCR mice exhibited a significantly lower prevalence (p<.05) of hepatic lesions at 6 months post infection than Hh 3B1 with an intact HhPAI. Hh G also has a large segment of the genomic island deleted, but not as many genes are deleted as compared to Hh NET. Hh G also demonstrated a lower prevalence of hepatic lesions. This variable pathological effect was evident in male mice only. The severity of chronic active inflammation in the liver of the H. hepaticus infected A/JCr mice depended on H. hepaticus liver colonization levels. The in vivo results support the presence of the HhPAI as a legitimate virulence determinant and predictor of severity of liver lesions in H. hepaticus infected A/JCr male mice. To further determine the differences in virulence of the H. hepaticus strains Hh 3B1, Hh NET, Hh G and an isogenic mutant H. ...
by Samuel R. Boutin.
Ph.D.in Molecular and Systems Bacterial Pathogenesis

Enterohepatic Helicobacter species (EHS) are emerging infectious disease agents. Infection of the enterohepatobiliary tract of several mammals by this group of bacteria results in various pathological disorders. The availability of the Helicobacter hepaticus sequenced and annotated genome, allowed molecular characterisation of the responses of H. hepaticus to host factors such as bile. The adaptation/responses of the bacterium to bovine, porcine and human bile were investigated using proteomics and transcriptomics. Ninety-one different proteins were identified in the responses of H. hepaticus response to the three types of bile. These proteins participate in several key cellular processes including DNA replication; protein transcription, translation and folding; oxidative stress response; motility; virulence; and metabolism. In particular, the bacteria deployed several strategies such as inhibition of the TCA cycle and the electron transport chain as well as iron sequestration to ensure control of the levels of hydroxyl radicals. The results of this study revealed also the modulation by bile of the expression of H. hepaticus genes involved in response to oxidative stress and virulence. The responses of human HEp-2 and Huh7-derived cell-lines to H. hepaticus and Helicobacter bilis, respectively, were investigated employing proteomics and transcriptomics. One-hundred and twenty different proteins were differentially expressed in the responses of the human cells to the presence of Helicobacter spp. in the cell cultures. These proteins are involved in regulation of cell proliferation and structure; metabolism; protein transcription, translation and modification; stress response; and tumour induction. For example, in co-cultures of Huh7-derived cells and H. bilis, the activation of several mitochondrial and endoplasmic reticulum stress-related proteins and the dysregulation of several apoptosis effectors were suggested as mechanisms that could result in the death of the liver cells. Importantly, the differential expression of several tumour-related proteins by the Huh7 cells supported a possible role for Helicobacter spp. in liver cancer.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastro-intestinal tract that usually manifests as either ulcerative colitis or Crohn's disease. Helicobacter hepaticus (Hh)-induced colitis is a mouse model of intestinal inflammation whereby IL-10 knock out mice are infected with Hh, resulting in pathology similar to that seen in Crohn's disease. Following the Complex Systems Modelling and Simulation (CoSMoS) process, a principled approach to the engineering of simulations of complex systems, we have developed IBDSim, a computational model of the processes active in the intestinal tract during Hh-induced colitis. IBDSim is a hybrid agent-based model (ABM) that combines agent-based modelling with systems biology and quantitative systems pharmacology approaches, to capture both cell- and system-level behaviours. In combining these approaches, the Automated Simulation Parameter Alteration and SensItivity Analysis toolkit (ASPASIA) was developed to aid in the calibration and analysis of models written in Systems Biology Mark-up Language (SBML), where the addition of an intervention is required for key behaviours to emerge. SBML models generated using this toolkit could then be incorporated into IBDSim.

In the Helicobacter hepaticus (Hh) colitis model, Hh infection of either wild-type mice treated with a blocking antibody to the IL-10 receptor (anti-IL-10R) or IL-10 KO mice results in intestinal inflammation associated with inflammatory Th1 and Th17 responses. Recent findings suggest that altered expression of the post-transcriptional gene regulators microRNAs contribute to pathogenic immune responses during intestinal inflammation. Here, examination of microRNA expression during Hh colitis showed that microRNAs are differentially expressed in the inflamed large intestine of Hh+ IL-10 KO mice compared to uninfected controls, both at the tissue-level and the CD4+ T-cell level. Kinetic examination of the cecal and colonic levels of miR-155, miR-326 and miR-132 (microRNAs previously shown to augment Th1 and/or Th17 responses) demonstrated that miR-155 was up-regulated and miR-326 and miR-132 were down-regulated at different time points post Hh infection. Furthermore, the change in expression of these microRNAs coincided with inflammation development. Microarray profiling of large intestine LP CD4+ T cells revealed that two microRNAs were significantly up-regulated (miR-21a and miR-31) and seven microRNAs were significantly down-regulated (miR-125a, miR-125b, miR-139, miR-181a, miR-192, miR-30a and miR-467c) in colitic IL-10 KO mice compared to uninfected controls. The anti-inflammatory cytokine IL-10 is necessary for protection against intestinal inflammation. Here, the phenotype of IL-10-producing LP CD4+ T cells was examined in a non-inflammatory immune response (Hh+ WT mice) and in an inflammatory immune response (Hh+/anti-IL-10R-treated WT mice). Compared to uninfected controls, the Hh+ mice showed a slight expansion in IL-10+ IL-17A+FoxP3+/- cells whereas the Hh+/anti-IL-10R-treated mice showed a significant expansion in all the IL-10+ LP CD4+ T cells co-expressed both inflammatory cytokines IL-17A and/or IFN-γ and/or the Treg transcription factor FoxP3. The experiments carried out in this thesis demonstrate that the profile of two important regulatory factors, microRNAs and IL-10, is markedly different in LP CD4+ T cells from the colitic setting compared to uninfected controls.

L’homme est fréquemment exposé à des génotoxines bactériennes impliquées dans les cancers digestifs, comme la colibactine et la toxine CDT (Cytolethal Distending Toxin). Ces toxines endommagent l'ADN des cellules hôtes et constituent un facteur prédisposant au développement de cancers. Les Helicobacter entérohépatiques, tels que Helicobacter hepaticus et Helicobacter pullorum, sont associés à plusieurs maladies intestinales et hépatiques. Leur principal facteur de virulence est la toxine CDT. Nous avons étudié les mécanismes d'action de la génotoxine CDT dans l'activation des processus procancéreux et la survie cellulaire, en utilisant l'infection à Helicobacter hepaticus comme modèle. Nous avons démontré que le remodelage nucléaire induit par la sous-unité active CdtB de la CDT de Helicobacter implique la surexpression de l'oncoprotéine MAFB qui est ensuite associée à une forte localisation nucléaire et périnucléaire dans les noyaux distendus en réponse à la CDT/CdtB, ainsi qu’à la formation de gros lamellipodes, des extensions cellulaires connues pour être impliquées dans la migration cellulaire et la production de métastases. Nous avons également montré que le remodelage nucléaire induit par CDT/CdtB peut être associé à la formation de réticulum nucléoplasmique (NR) enrichi en particules ribonucléoprotéiques. Ces structures dynamiques et transitoires induites par la génotoxine pourraient correspondre à une passerelle privilégiée pour la synthèse d'ARNm sélectionnés, qui seraient préférentiellement transportés du noyau à travers les pores nucléaires et traduits à l'intérieur du NR. Le NR induit par la CDT (et aussi par la colibactine) pourrait permettre à la cellule de s'arrêter et de réparer les dommages à l'ADN causés par les génotoxines bactériennes afin de maintenir la survie cellulaire. L'identification à base de MicroArrays des gènes régulés par la CdtB a aussi montré une régulation de l'autophagie par cette toxine. Nous avons montré que la CdtB stimulait le flux autophagique et induisait une autophagie de survie suite aux dommages à l'ADN induits par la CDT/CdtB. Nous avons montré que la CDT/CdtB favorise la formation d'agrégats cytoplasmiques SQSTM1/P62 parfois profondément invaginés dans les noyaux distendus ainsi que des vésicules extra-cellulaires SQSTM1/P62-positives. La formation de NR suite aux dommages à l'ADN induits par la CDT est associée à l'induction de l'autophagie, qui joue un rôle de survie dans ce contexte. Les particules SQSTM1/P62 ont un rôle clé dans ces effets. Ces résultats offrent de nouvelles perspectives dans le contexte de la formation du NR et de la survie cellulaire en réponse aux dommages à l'ADN, une caractéristique commune à de nombreux cancers, qui apparaissent non seulement en réponse aux dommages à l'ADN induits par les thérapies mais aussi plus tôt en réponse aux bactéries génotoxiques. De nouvelles études sont nécessaires pour identifier les structures cytosoliques entourées et englouties par P62/SQSTM1 ainsi que pour déchiffrer le rôle de cette protéine cargo dans la survie des cellules dont l'ADN est endommagé et, plus particulièrement, son rôle dans la formation de NR à la lumière de sa fonction de protéine de liaison à l’ARN récemment démontrée. Les résultats issus de cette thèse confortent l’idée que les génotoxines bactériennes pourraient être à l’origine de cancers
Humans are frequently exposed to bacterial genotoxins, colibactin and Cytolethal Distending Toxin (CDT), involved in digestive cancers. These toxins damage the DNA of host cells and constitute a predisposing factor for the development of cancer. Enterohepatic Helicobacters, such as Helicobacter hepaticus and Helicobacter pullorum, are associated with several intestinal and hepatic diseases. Their main virulence factor is CDT.We studied the mechanisms of action of CDT genotoxin in the activation of pro-cancerous processes and cell survival, using Helicobacter hepaticus infection, as a model. We demonstrated that the nuclear remodeling induced by Helicobacter CdtB active subunit of CDT involves MAFB oncoprotein overexpression which is subsequently associated with a strong nuclear and perinuclear localization of MAFB protein in CDT/CdtB-distended nuclei, and with the formation of large lamellipodia, corresponding to cellular extensions known to be involved in cell migration and arousal of metastases. We also showed that the nuclear remodeling induced by CDT/CdtB could be associated to the formation of transient rich-messenger ribonucleoprotein nucleoplasmic reticulum (NR). These genotoxin-induced dynamic structures may correspond to a privileged gateway for the synthesis of selected mRNA, preferentially transported from the nucleus, through pores and translated therein. CDT-induced NR may allow the cell to pause and repair DNA damage caused by bacterial genotoxins in order to maintain cell survival. Microarray-based identification of differentially-expressed genes pointed a CdtB regulation of autophagy. We showed that CdtB stimulated autophagic flux and enhanced pro-survival autophagy following CDT-induced DNA damage. CDT promoted the formation of cytoplasmic SQSTM1/P62 aggregates, sometimes deeply invaginated in distended nuclei, as well as SQSTM1/P62-positive extra-cellular like vesicles. NR formation following DNA damage induced by CDT is associated with the induction of autophagy, which plays a survival role in this context. SQSTM1/P62 have a key role in these effects.These findings offer new insights into the context of NR formation and cell survival in response to DNA damage, a common feature of many cancers, which not only appears in response to therapies-induced DNA damage, but also earlier in response to genotoxic bacteria.Further studies are required identify the cytosolic structures surrounded and engulfed by P62/SQSTM1 and to decipher the role of this cargo protein in the prosurvival of cells whose DNA is damaged, and more particularly its role in NR formation in light with its recently demonstrated RNA-binding protein function.The results of this thesis support the idea that bacterial genotoxins could cause cancer

Colorectal cancer (CRC) is one of the most treatable cancers, with a 5-year survival rate of similar to 64%, yet over 50,000 deaths occur yearly in the United States. In 15% of cases, deficiency in mismatch repair leads to null mutations in transforming growth factor beta (TGF-beta) type II receptor, yet genotype alone is not responsible for tumorigenesis. Previous work in mice shows that disruptions in TGF-beta signaling combined with Helicobacter hepaticus cause tumorigenesis, indicating a synergistic effect between genotype and microbial environment. Here, we examine functional shifts in the gut microbiome in CRC using integrated - omics approaches to untangle the role of host genotype, inflammation, and microbial ecology. We profile the gut microbiome of 40 mice with/without deficiency in TGF-beta signaling from a Smad3 (mothers against decapentaplegic homolog-3) knockout and with/without inoculation with H. hepaticus. Clear functional differences in the microbiome tied to specific bacterial species emerge from four pathways related to human colon cancer: lipopolysaccharide (LPS) production, polyamine synthesis, butyrate metabolism, and oxidative phosphorylation (OXPHOS). Specifically, an increase in Mucispirillum schaedleri drives LPS production, which is associated with an inflammatory response. We observe a commensurate decrease in butyrate production from Lachnospiraceae bacterium A4, which could promote tumor formation. H. hepaticus causes an increase in OXPHOS that may increase DNA-damaging free radicals. Finally, multiple bacterial species increase polyamines that are associated with colon cancer, implicating not just diet but also the microbiome in polyamine levels. These insights into cross talk between the microbiome, host genotype, and inflammation could promote the development of diagnostics and therapies for CRC. IMPORTANCE Most research on the gut microbiome in colon cancer focuses on taxonomic changes at the genus level using 16S rRNA gene sequencing. Here, we develop a new methodology to integrate DNA and RNA data sets to examine functional shifts at the species level that are important to tumor development. We uncover several metabolic pathways in the microbiome that, when perturbed by host genetics and H. hepaticus inoculation, contribute to colon cancer. The work presented here lays a foundation for improved bioinformatics methodologies to closely examine the cross talk between specific organisms and the host, important for the development of diagnostics and pre/probiotic treatment.

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The mucosa is the most vulnerable gastric layer to damage, as it is the interface between the external environment and the body. For this reason, it presents specific mechanisms to maintain the integrity, but, when the triggering agent persists or is very aggressive, the defense mechanisms are overcome and mucosal injuries manifest. The endoscopy is the main technique for identifying this type of injury and it has the advantage of facilitating the material collection for the bacteriological and histological confirmation of the causing agent. The present study aimed to evaluate the gastroscopic and histological aspects of the gastric mucosa of dogs experimentally intoxicated with carbon tetrachloride and to identify the most efficient technique for detecting the bacteria of the genus Helicobacter in the stomach of this animals species by comparing the techniques of cytology, fast urease test and histological sections stained with HE, Giemsa and Warthin-Starry (WS). In poisoned dogs there were hyperemia and hemorrhage by gastroscopy and histology edema, hyperemia and hemorrhage. The Helicobacter detecting prevalence was higher in the cytology exam and the sensitivity was higher in histological sections stained with WS. It was concluded that orally administration of CCl4 to dogs cause diffuse circulatory changes in gastric mucosa and the most efficient technique to Helicobacter spp research is the cytology, being gastric fundus the most suitable region for sampling.
A mucosa é a camada gástrica mais vulnerável a lesões, pois representa a interface entre o meio externo e o organismo. Por esse motivo, apresenta mecanismos específicos que visam a manutenção da integridade, entretanto, quando o agente desencadeante persiste ou é muito agressivo, os mecanismos de defesa são suplantados e ocorrem as lesões na mucosa. O exame de endoscopia é a principal técnica para a identificação dessas lesões e apresenta a vantagem de facilitar a colheita de material para confirmações bacteriológicas e histológicas do agente agressor. O presente estudo teve por objetivos avaliar os aspectos gastroscópicos e histológicos da mucosa gástrica de cães intoxicados experimentalmente com tetracloreto de carbono e identificar a técnica mais eficiente em detectar bactérias do gênero Helicobacter no estômago de animais desta espécie, por meio da comparação das técnicas de citologia, teste rápido de urease e cortes histológicos corados por HE, Giemsa e Warthin-Starry (WS). Nos cães intoxicados verificou-se à gastroscopia hiperemia e hemorragia e à histologia edema, hiperemia e hemorragia. A prevalência em detectar helicobactérias foi maior no exame de citologia e a sensibilidade mais elevada nos cortes histológicos corados por WS. Conclui-se que a administração de CCl4 por via oral aos cães causa alterações circulatórias difusas na mucosa gástrica e que a técnica mais eficiente para a pesquisa de bactérias do gênero Helicobacter é a citologia, sendo o fundo gástrico a região mais apropriada para colheita das amostras.

Abbreviations ALT – alanine aminotransferase AST – aspartate aminotransferase BMI – body mass index CHC – chronic hepatitis C EBR – early biochemical response EHR – early histological response EIA – enzyme immunoassay EVR – early virological response HAI – hepatitis activity index HCV – hepatitis C virus HCV RNA - hepatitis C virus ribonucleic acid Helicobacter spp. – Helicobacter species H. pylory – Helicobacter pylori IFN – interferon α-2b PCR – polymerase chain reaction PEG IFN – peginterferon RBV – ribavirin SVR – sustained virological response SBR – Sustained biochemical response INTRODUCTION Chronic hepatitis due to hepatitis C virus (HCV) infection is a worldwide disease representing a serious public health problem. Chronic hepatitis C (CHC) infection affects nearly 170-200 million people worldwide. The prevalence of anti-HCV at this time in the general adult population of Lithuania is nearly 50 thousand (0.9%). Without effective treatment strategies, hepatitis C - related morbidity and mortality is expected to increase nearly 3-fold by the year 2015. Current hepatitis C therapies are aimed at achieving eradication of HCV infection as a means of delaying progression to end-stage liver disease and preventing the development of hepatocellular carcinoma. The treatment options include interferon (IFN), ribavirin (RBV) and peg interferon’s a-2a and a-2b (PEG IFN). IFN-based regiments for the treatment of CHC have become increasingly effective and are to eradicate virus... [to full text]

Vertebrates are host for a very large number of bacteria, notably in the intestinal lumen. This complex microbiota encompasses microorganisms that can cause pathology in immunocompromised individuals, but not in healthy hosts who remain silent carriers. The reciprocal interactions between the host and such microbes must involve components of active immune tolerance maintaining at check protective immune responses of the ridding type. We addressed this hypothesis by studying mouse-Helicobacter hepaticus interactions. H. hepaticus is a gut bacteria commonly found in mouse facilities and in the wild. Mice can be persistently colonized with this microbe, even from the first days of life, without developing signs of pathology or decrease in breeding efficiency. However, immunocompromised animals can develop colitis when colonized with H. hepaticus. In this work, we sought to identify the immunological mechanisms triggered upon colonization that ensure a stable relationship between healthy mice and H. hepaticus.(...)

Helicobacter hepaticus stellt den Prototyp der enterohepatischen Helicobacter dar und führt zu einer persistenten Infektion von Mäusen. In immundefizienten Tieren kann er eine chronische Entzündung des Darmtraktes auslösen, welche den chronisch entzündlichen Darmerkrankungen des Menschen, Morbus Crohn und Colitis Ulcerosa, ähnelt. Deshalb wird H. hepaticus bevorzugt als Modellorganismus zur Untersuchung der immunologischen Ursachen von chronisch entzündlichen Darmerkrankungen im Tiermodell eingesetzt. Ebenfalls kann eine Infektion mit H. hepaticus in suszeptiblen Mäusestämmen (z.B. Balb/c, C3H/An) zu Entzündungen der Leber und Gallengänge führen, welche sich bis zu einer Hepatitis und Leberkarzinomen ausweiten können. In den meisten Studien wurde H. hepaticus bisher aber hauptsächlich als Auslöser dieser Erkrankungen eingesetzt, während die bakterielle Seite kaum betrachtet wurde. Im Rahmen dieser Arbeit wurde in einer Kooperation mit MWG Biotech, GeneData und dem Massachusetts Institute of Technology (MIT) die Gesamtgenomsequenz des H. hepaticus Referenzstammes ATCC 51449 bestimmt und annotiert. Das Genom hat eine Größe von 1.799.146 bp und kodiert für 1.875 Proteine. Die globale Ähnlichkeit des Genoms von H. hepaticus ist etwa gleich groß zu den sequenzierten Genomen von H. pylori und C. jejuni. Es fehlen H. hepaticus aber die meisten Virulenzfaktoren von H. pylori wie Adhäsine (SabA, BabA, AlpA), VacA und die meisten Proteine der cag-Pathogenitätsinsel, während Homologe zu Pathogenitätsfaktoren von C. jejuni wie CDT und Peb1 vorhanden sind. Das Genom von H. hepaticus enthält neben vielen kleineren genomischen Inseln eine Genominsel mit einer Größe von 71 kb, welche als HHGI1 benannt wurde. Sie kodiert mutmaßlich für ein TypIV-Sekretionssystem und enthält weitere Virulenzfaktoren. In Microarray- basierten Gesamtgenomvergleichen konnte gezeigt werden, dass die Insel in sieben von 13 untersuchten Stämmen großteils oder komplett fehlt. Während Mäuse, aus denen HHGI1-positive Stämme isoliert wurden, pathologische Veränderungen der Leber aufwiesen, wies keine von den Mäusen, aus denen HHGI1-negative Stämme isoliert wurden, Auffälligkeiten in der Leber oder dem Gallentrakt auf. In einem Tiermodell wurde in Kooperation mit dem MIT gezeigt, dass zwei Insel-negative Stämme zu einer geringeren Besiedlung und einer schwächeren Entzündung der Leber als der Insel-positive Referenzstamm ATCC 51449 führen. Durch die Genomvergleiche konnte auch gezeigt werden, dass verschiedene H. hepaticus-Stämme trotz einer niedrigen Sequenzvariabilität eine hohe Variation des Genomgehalts aufweisen und dass neben der HHGI1-Insel weitere kleinere Inseln in einzelnen Stämmen fehlen. Es wurden in der vorliegenden Arbeit erstmals verschiedene isogene Mutanten von H. hepaticus in der HHGI-1-Insel hergestellt, die in vitro eine verringerte Immunstimulation in Makrophagen zeigten. Der Mechanismus dieser Immunsuppression konnte noch nicht vollständig aufgeklärt werden, sie werden jedoch derzeit in Mausmodellen weiter auf ihre krankheitsauslösenden Eigenschaften untersucht. Da bisher keine gut charakterisierten Zellkulturmodelle für die in vitro-Untersuchung von H. hepaticus vorlagen, wurden solche im Rahmen dieser Arbeit etabliert. Dazu wurden die intestinale murine epitheliale Zelllinie m-ICcl2, welche das primäre Habitat von H. hepaticus (Krypten im Dünndarm) imitiert, die murine Hepatozytenzelllinie NCTC Klon 1469, welche ein mögliches sekundäres Habitat (Lebercanaliculi) imitiert und die murine Makrophagenzelllinie J774 benutzt. Während J774 und NCTC Klon 1469 durch die meisten Liganden für Mustererkennungsrezeptoren stimuliert werden konnten, reagierten m-ICcl2- Zellen substantiell nur auf den TLR4-Liganden E. coli-LPS. Dementsprechend induzierte H. hepaticus in J774 und NCTC Klon 1469 eine starke proinflammatorische Antwort, während m-ICcl2 trotz guter Adhärenz nur schwach von H. hepaticus stimuliert wurde. Es wurde gezeigt, dass LPS und Flagelline von H. hepaticus nur eine geringe immunstimulatorische Wirkung besitzen, während Lipoproteine und vermutlich auch Peptidoglykan die wichtigsten PAMPs von H. hepaticus darstellen. Durch die Analyse der durch H. hepaticus ausgelösten globalen Genregulation in J774 und NCTC Klon 1469 wurde nachgewiesen, dass H. hepaticus nicht primär über NF-κB, sondern über MAP-Kinasen eine proinflammatorische Antwort auslöst. Außerdem wurde gezeigt, dass H. hepaticus untypisch für extrazelluläre Bakterien eher eine Wirtsantwort auslöst, welche der durch intrazelluläre Bakterien ähnelt. In diesen Modellen führten HHGI1-negative Stämme oder Mutanten der HHGI1-Insel zu einer leicht verringerten proinflammatorischen Antwort. Dies spiegelte sich auch in der transkriptionellen Regulation von Schlüsselfaktoren der angeborenen Immunantwort wie TLR2, IL-12, NOD2 oder Tollip wieder. In m-ICcl2-Zellen führte eine Koinkubation mit lebenden H. hepaticus oder Lysaten zu einer verringerten durch E. coli-LPS ausgelösten Induktion von MIP-2. Darauf basierend wurde gezeigt, dass LPS von H. hepaticus einen wesentlichen Faktor für diese Inhibierung der proinflammatorischen Antwort darstellt, nicht jedoch die HHGI-1-Insel oder andere vermutete Virulenzfaktoren. Zumindest auf mRNA-Ebene wurde durch H. hepaticus auch die Induktion anderer Cytokine wie TNF-α oder MIP-1α gehemmt. Eine primäre Koinkubation von m-ICcl2 mit E. coli-LPS führte zu einer Toleranzinduktion gegenüber einer zweiten Stimulation. Diese Toleranzinduktion wurde durch eine Inkubation mit H. hepaticus ebenfalls gehemmt. Die Hemmung der proinflammatorischen Antwort durch H. hepaticus-LPS konnte auch in NCTC Klon 1469 und unter serumfreien Bedingungen für die durch S. typhimurium- Flagellin induzierte IL-8 Sekretion in der humanen Kolonkarzinomzelllinie Caco2 nachgewiesen werden. Damit war diese Hemmung weder zellspezifisch noch spezifisch für die TLR4-abhängige Stimulation. Basierend auf dieser Arbeit wurde ein Modell für die Entstehung einer chronischen Entzündung im Intestinaltrakt entwickelt, welches Erklärungsansätze für die Entwicklung einer chronisch entzündlichen Darmerkrankung im Menschen liefern könnte
H. hepaticus is the prototype species of the enterohepatic group of Helicobacter species and leads to a persistent infection in mice. It is able to cause a chronic inflammation of the intestinal tract in immuno-deficient mice that resembles the common human inflammatory bowel diseases Crohn’s disease und ulcerative colitis. Therefore H. hepaticus is widely used as a model organism to study the possible immunological causes underlying the development of inflammatory bowel disease in the animal model. H. hepaticus can also lead to diseases in the liver and biliary tract of susceptible mouse strains (e.g. Balb/c, C3H/An) such as hepatitis and even liver cancer. But in most studies H. hepaticus was mainly used to trigger these diseases, while only little attention was paid to the bacterial determinants of pathogenesis. In this work the complete genome sequence of the H. hepaticus reference strain ATCC 51449 was determined and annotated in cooperation with GeneData, MWG Biotech and the Massachusetts Institute of Technology (MIT). The genome has a size of 1,799,146 bp and codes for 1,875 proteins. Generally the average similarity of the genome is about equal to the sequenced genomes of H. pylori and C. jejuni. But H. hepaticus misses most of the virulence factors of H. pylori such as adhesins (e.g. SabA, BabA, or AlpA), the vacuolating cytotoxin VacA and most genes of the cag pathogenicity island. On the other hand it possesses many orthologs of C. jejuni virulence factors like the cytolethal distending toxin CDT or the adhesion factor Peb1. In addition to several smaller genomic islands, the genome of H. hepaticus contains a large 71 kb genomic island, which we called HHGI1. It presumably codes for a type IV secretion system and several additional virulence factors. By a microarray-based genome comparison study, we could show that this island was missing in seven out of 13 isolates. While only mice that harbored an island positive strain showed signs of liver disease, not a single mouse with an island negative strain showed any pathological changes of the liver or the biliary tract. In cooperation with the MIT, it was shown in an animal model that two island negative strains led to a reduced colonization and weaker inflammation of the liver compared to the island positive reference strain ATCC 51449. By the genome comparisons, it was also shown that despite low sequence variability the genome contents of different H. hepaticus isolates can differ widely and that smaller islands are either present or absent in different strains. In the framework of these investigations, several isogenic H. hepaticus mutants in the HHGI1 island were constructed. These mutants showed in comparison to the wild type a deficiency to activate macrophages, but the exact mechanism could not be identified so far. The isogenic mutants are currently further tested in animal models for their disease-eliciting potential. Because no well-characterized cell culture model was yet available for the in vitro examination of H. hepaticus-associated pathogenesis, such models were established in this dissertation. Therefore the murine intestinal epithelial cell line m-ICcl2 which resembles the primary habitat of H. hepaticus in the mouse caecum, the murine hepatocyte cell line NCTC clone 1469 which imitates one possible secondary habitat (mouse liver canaliculi) and the murine macrophage cell line J774 were used. While a wide range of ligands for pattern recognition receptors were able to stimulate NCTC clone 1469 and J774, m-ICcl2 cells only reacted substantially to the TLR4 ligand E. coli LPS. Accordingly, infection with H. hepaticus led to a strong proinflammatory response in J774 und NCTC clone 1469, while m-ICcl2 cells were only weakly stimulated by H. hepaticus despite good adherence. It was shown that H. hepaticus LPS and flagellins are only weak stimulators of the innate immune system while, as the results suggested, the proinflammatory response was mainly induced by lipoproteins and probably also by peptidoglycans of H. hepaticus. By analyzing the global gene regulation in J774 and NCTC clone 1469 after coincubation with H. hepaticus, it was established that the proinflammatory response is not mainly dependent on NF-κB but on MAP kinases. Also, the global response of the cells resembled more those induced by intracellular than extracellular pathogens. In these model systems, HHGI1-negative strains or mutants in the HHGI1 island led to a weaker proinflammatory response than HHGI1-containing strains. This was also in concordance with a different regulation pattern of different factors like TLR2, IL- 12, NOD2 or Tollip. In m-ICcl2 cells, after coincubation with live H. hepaticus or lysates, a reduced secretion of MIP-2 after stimulation with E. coli LPS was observed. It was shown that H. hepaticus LPS is one important factor for this inhibition of LPS-induced MIP-2 secretion, but not the HHGI-1 island nor other presumed H. hepaticus virulence factors. On the mRNA level, the induction of other cytokines like TNF-α or MIP-1α was also reduced by H. hepaticus. We could show, that, as described in other cells before, a primary coincubation with E. coli LPS leads to a tolerance against a second stimulation round. This tolerance development was inhibited by H. hepaticus. Inhibition of the proinflammatory response by H. hepaticus LPS was also obtained with NCTC clone 1469 cells. When using the human intestinal epithelial carcinoma cell line Caco2, S. typhimurium flagellin triggered IL-8 secretion was almost completely reduced under serum-free conditions, while no inhibition was found under serum-containing conditions. Therefore, this inhibitory effect was neither cell-specific nor specific for induction via TLR4. Based on this work, a model for the development of a chronic inflammation of the intestinal tract could be established, which may offer possible explanations for the development of inflammatory bowel diseases in humans

Tese de mestrado, Biologia (Biologia Evolutiva e do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2015
Diferentes estudos desenvolvidos pelo nosso grupo envolveram colonização com Helicobacter hepaticus (Hh) reproduzindo um tipo de interação hospedeiro-microorganismo. Dados anteriores aos que são apresentados neste projeto reportaram que após a colonização com Hh em ratos adultos saudáveis B6 WT é desencadeada uma resposta robusta de imunoglobulina (Ig) específica contra Hh, acompanhada por uma redução da densidade bacteriana (dados não pulicados). Também foi observado que é necessária uma resposta de células B dependente de células T para controlar a densidade de Hh e que a citoquina responsável pela persistência de Hh, IL-10, é essencial para modular esta resposta. Curiosamente, aquando da colonização com esta bactéria em ratos neonatos B6 WT, a resposta de Igs específicas não se verificou e estes animais apresentaram densidades elevadas de Hh. Por fim, a tolerância a Hh – mediada por IL-10 e sustentada por células T regulatórias CD25+ – foi mantida mesmo quando os animais atingiram a idade adulta (dados não publicados). Tendo em conta as evidências referidas acima, o grande foco deste trabalho residiu no contraste entre a colonização com Hh no período neonatal e no adulto. Neste contexto, decidimos explorar o papel da presença de uma microbiota complexa na resposta de tolerância a Hh. Assim, a partir de animais GF (desprovidos de qualquer microorganismo) B6 foram produzidos animais Ad.mcol – colonização ig com Hh de cultura em ratos com 8 semanas e analisados 9 semanas pós-colonização – e animais Nb.mcol – nascidos a partir de mães mono-colonizadas com Hh e analisados com 9 semanas de idade. Semelhante ao que tinha sido demonstrado para animais SPF Nb.col (dados não publicados), não foi detetada IgA anti-Hh nas fezes de ratos Nb.mcol provando que a tolerância a Hh é independente da microbiota. Para além disso, demonstrámos que a Ig específica contra Hh se liga diretamente à sua superfície. Isto foi inferido através da incubação de extratos fecais de ratos Ad.col e Nb.col, usando como controlo negativo animais SPF, com Hh crescida em cultura. Posteriormente, a análise por citometria de fluxo confirmou que o anticorpo produzido especificamente contra Hh se liga efetivamente à sua superfície, o que ainda não tinha sido demonstrado até ao momento. A concentração de IgA detetada através de ELISA envolve a incubação de amostras de fezes com um lisado de Hh, sendo apenas determinada a IgA específica que a reconhece e se liga aos seus antigénios. Pelo contrário, uma vez que incubámos amostras de fezes com uma suspensão de Hh crescida em cultura no ensaio descrito acima, a integridade estrutural da parede celular bacteriana não foi comprometida. Assim, foi possível replicar a interação entre o sistema imunitário do hospedeiro e as bactérias num contexto in vivo, sendo considerado como um modelo plausível do que acontece ao nível fisiológico quando a IgA encontra estas bactérias intactas dentro do hospedeiro: a IgA reconhece os antigénios que se encontram na superfície de Hh presente no lúmen do intestino e a sua afinidade promove uma ligação específica; estes microorganismos ficam revestidos com IgA e impedidos de atravessar a barreira intestinal, mecanismo que ainda não é claro. Neste trabalho, também foram identificados os nichos intestinais ocupados por esta bactéria determinando a densidade de Hh em animais Rag2-/- (imunodeficentes), Ad.col e Nb.col. Os nossos dados após a análise da densidade de Hh por qPCR mostraram que a Hh está presente em maior densidade no caecum (em geral, 104 cópias de 16S de Hh por 16S total), de seguida no íleo e cólon (Rag2-/- = ~102 cópias de 16S de Hh/16S total; B6 Ad.col = ~1 cópia de 16S de Hh/16S total; B6 Nb.col = ~101-102 cópias de 16S de Hh/16S total) e em menor número no jejuno (Rag2-/- Ad.col e B6 Nb.col = ~10 cópias de 16S de Hh/16S total; B6 Ad.col < 1 cópia de 16S de Hh/16S total). Os animais Rag2-/- Ad.col exibiram ainda uma elevada densidade de Hh no recto comparativamente aos outros grupos (104 cópias de 16S de Hh/16S total). O facto de Hh ter sido isolada originalmente a partir do cólon e também do fígado de ratos com hepatite crónica ativa, tumores hepáticos e IBD, motivou a investigação da sua capacidade de atravessar a barreira epitelial intestinal sob as nossas condições experimentais. Dados obtidos anteriormente no nosso grupo sugeriram que Hh é capaz de atravessar o epitélio intestinal em ratos B6 Ad.col, indicado pela presença de IgM (primeira Ig a ser produzida por células B maduras) e IgG (produzido mediante hipermutação somática após estimulação) específicos para Hh no seu soro. Por outro lado, tínhamos evidências de que a densidade de Hh nas fezes em ratos Rag2-/- Ad.col desprovidos de células B e T maduras (mutantes em que a função da enzima que participa na recombinação V(D)J está comprometida, tanto no locus de Ig como no receptor das células T) é maior do que em ratos B6 Ad.col. Assim, averiguámos se 1) a ausência do sistema imunitário adaptativo e 2) o período de colonização, alteram o tráfico de Hh para fora do epitélio intestinal. Para perceber se esta bactéria tem a capacidade de atravessar o epitélio nestas condições experimentais – em animais Rag2-/- Ad.col, B6 Ad.col e B6 Nb.col – a densidade de Hh em vários órgãos internos analisada por qPCR. A capacidade de Hh atravessar a barreira intestinal permaneceu indeterminada; embora tenha sido verificado um maior número de Hh nos MLN de ratos Rag2-/- Ad.col relativamente aos outros grupos, esta bactéria não foi detetada no fígado e baço em nenhum dos grupos analisados. Por outro lado, foram detetadas aproximadamente 10 cópias de 16S de Hh por ng de DNA do hospedeiro na GB de ratos B6 Nb.col, mas não em Rag2-/- ou B6 Ad.col. A cultura de Hh a partir dos diferentes órgãos internos referidos revelou-se necessária para confirmar ou esclarecer estes resultados. Finalmente, decidimos comparar o perfil inflamatório destes dois grupos numa situação de disrupção da barreira intestinal. Também investigámos se a colonização com Hh promove a progressão da resposta inflamatória no intestino, comparando estes grupos com animais SPF. Assim, o efeito da colonização com Hh numa situação de patologia intestinal foi analisado. Para replicar esta situação foi utilizado um modelo de colite induzida por DSS, avaliando a severidade da inflamação pela perda de peso corporal e pelos níveis de Lcn-2 nas fezes (ELISA) dos animais analisados (n=5 por grupo). Globalmente, uma relativa reprodutibilidade foi verificada entre as duas experiências realizadas independentemente. Como esperado, foi observada uma diminuição gradual do peso a partir do 4º dia do tratamento com DSS em ambas as experiências. No geral, a colonização com Hh não afetou o peso dos animais analisados até ao dia 9 (1 dia após ter sido removido DSS). Na primeira experiência, foi observada uma perda de peso constante até ao 11º dia em todos os grupos; porém, posteriormente, houve uma grande variação entre os ratos B6 Nb.col provocada por 2 animais, que apresentaram uma perda de peso mais drástica. Ainda assim, todos os animais voltaram a ganhar peso sensivelmente a partir dos 12º-13º dias, após o DSS ter sido removido, acabando por recuperar o peso inicial. Na segunda experiência, também foram detectadas algumas diferenças posteriormente ao 10º dia, embora num menor número de dias. Contudo, os níveis fecais de Lcn-2 não se correlacionaram com o do peso corporal: níveis elevados de Lcn-2 vs recuperação do peso inicial. A regeneração da mucosa intestinal bem como a manutenção da tolerância a Hh serão alvo de estudos futuros.
Helicobacter hepaticus (Hh)-newborn colonization in B6 WT mice generates a tolerant response characterized by: undetectable faecal and serum Hh-specific IgA, high bacterial loads and maintenance of tolerance until adult life. In this study, monocolonization with this pathobiont revealed that this response is microbiota-independent. Moreover, we demonstrated that the specific Ig produced against Hh binds specifically to its surface. The niches inhabited by this microbe within the mouse GI tract were identified: higher Hh loads were found in the caecum followed by colon and ileum and, at lower number, in jejunum. In immunodeficient mice a high Hh load was found within the rectum. Hh ability to traverse the intestinal epithelial barrier remained uncertain as our data was unclear in this respect. Although a higher Hh load was estimated within MLN from immunodeficient Ad.col mice, it was not detected within the liver and spleen from any of the groups analysed. Still, we estimated around 10 copies of Hh 16S per ng of host DNA only within the GB from B6 Nb.col mice. Culture of Hh from the various internal organs is required to confirm these results. Finally, we investigated the effect of Hh colonization in the context of intestinal pathology. DSS-induced colitis was used to model intestinal disease, evaluating the severity of the inflammation. Generally, Hh colonization did not affect animal weight; however, the levels of faecal Lcn-2 did not correlate with body weight in both experiments: high Lcn-2 levels vs recovery of the initial weight. Mucosal healing and maintenance of tolerance to Hh will be subject for further work.