The mucosal immune system encompasses a wide array of interactions that work in concert to protect an individual from harmful agents while retaining tolerance to molecules, microbes, and self-antigens that present no danger. The upheaval in the regulation-response balance is a critical aspect in both infectious and immune-mediated disease. To understand this balance and methods of its restoration, iterative and integrative modeling cycles on the pathogenesis of disease are necessary. In this thesis, I present three studies highlighting phases of a systems immunology cycle. Firstly, the thesis provides a description of the construction of a computational ordinary differential equation based model on the host-pathogen-microbiota interactions during Clostridium difficile infection and the use of this model for the development of the hypothesis that host-antimicrobial peptide production may correlate with increased disease severity and promote increased recurrence. Secondly, it provides insight into the necessity of trans-disciplinary analysis for the understanding of novel molecular targets in disease through the immunometabolic regulation of CD4+ T cell by NLRX1 in inflammatory bowel disease. Third, it provides the assessment of novel therapeutics in disease through the evaluation of LANCL2 activation in influenza virus infection. In total, the computational and experimental strategies used in this dissertation are critical foundational pieces in the framework of precision medicine initiatives that can assist in the diagnosis, understanding, and treatment of disease. / Ph. D. / Many diseases are a result of altered patterns of interaction between the body, bacteria, viruses or nutrients. When these patterns are altered, inflammation occurs. If not controlled, the inflammation can cause pain, damage to the affected area, and other specific symptoms depending on the type of disease. This dissertation details the use of alternative methods of treating disease and analyzing disease in the context of Clostridium difficile infection, inflammatory bowel disease and influenza infection. It provides insight into the development of computational models with equations to capture the response patterns. It assesses the connections between immunology and metabolism that can lead to inflammation. And, it identifies a new therapeutic target for influenza infection. Together, these three phases are important pieces toward a future with improved understanding of disease and treatments that can be specific and customized for every individual.

The gastrointestinal mucosal immune system has the daunting task of maintaining immune homeostasis by eliminating potentially harmful microorganisms and limiting tissue injury while inducing tolerogenic responses to luminal antigens including innocuous food, commensal bacteria and self-antigens. This carefully orchestrated system depends on elaborate down-regulating mechanisms that mediate and maintain a state of tolerance under normal conditions. Changes in such delicate balance are linked to the development of gastrointestinal pathology as well as systemic disease states. Despite the rapid increase in our appreciation of the gastrointestinal immune system, there is still a major disconnect between the description of how mucosal immune responses are organized and controlled and an insufficient mechanistic understanding of how such responses shape and influence disease outcome and pathogenesis. By using model enteric microorganisms Helicobacter pylori and Clostridium difficile, this dissertation presents a systematic effort to generate novel mechanistic hypothesis based on computational predictions and experimentally elucidate the mechanisms of action underlying mucosal immune responses and pathology in the gut. In this thesis I present i) an overview on mucosal immunology and the need to develop novel therapeutics that limit the pathogenic effects of invading bacteria while maintaining their protective functions, ii) the role of miRNAs in the modulation of immune responses to enteric pathogens, iii) the mechanisms by which Helicobacter pylori is able to limit effector inflammatory responses required for bacterial clearance thus favoring tolerance over immunity, iv) intracellular mechanisms of immune evasion that contribute to bacterial persistence and chronic infection. The knowledge generated throughout this dissertation exemplifies how a combination of computational modeling, immunoinformatics and experimental immunology holds enormous potential for discovering unforeseen targets and developing novel vaccines and cures for infectious, allergic and immune-mediated diseases. / Ph. D.

Clostridioides difficile is a bacterial pathogen that causes pseudomembranous colitis and the majority of antibiotic-associated diarrheal cases. Broad-spectrum antibiotic usage disrupts the normal gut microbiota and thereby compromises colonization resistance, the main defense against C. difficile infection. Treatment options are limited to vancomycin, fidaxomicin, and the fecal microbiota transplant. Addressing the scarcity of these therapeutics, we documented two explorations in C. difficile drug discovery: (i) evaluation of antibacterial and toxin-suppressing activity of (+)-puupehenone and similar derivatives, and (ii) clarification of a discrepancy in the hypothesized mechanism of auranofin against C. difficile. A better understanding of how C. difficile colonizes and thrives in the gut can greatly benefit therapeutic development. Interestingly, C. difficile can scavenge nutrients such as amino acids and possibly even purines during infection. Amino acids including proline and glycine act as substrates for Stickland metabolism, a bioenergetics scheme that partially relies on enzymes containing selenium in the form of selenocysteine (e.g., D-proline reductase and glycine reductase). Purines such as xanthine and uric acid can be degraded by bacterial molybdenum hydroxylases harboring an uncharacterized form of selenium, though the role of these enzymes in C. difficile physiology is poorly understood. Selenium likely plays a key role in the scavenging of these nutrients during C. difficile infection. Our investigation of these selenium-dependent enzymes revealed two new findings in C. difficile biology: (i) a link between proline-dependent growth and D-proline reductase, characterized as an energy "addiction," and (ii) a previously uncharacterized selenium-dependent pathway involved in the catabolism of xanthine and uric acid. Overall, these physiological analyses of C. difficile provide promising candidates for therapeutics and key information regarding the organism's nutrient preferences.

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The spore-forming opportunistic enteropathogen Clostridioides difficile (C. difficile) has emerged as the most common cause of nosocomial antibiotic-associated diarrhea. Disruption of the healthy gut microbiota upon antibiotic therapy is the major risk factor for developing an infection. The symptoms can range from mild diarrhea to life threatening disease, such as colitis and tissue necrosis. The severity of infection is mediated by the expression of several virulence factors, including two major exotoxins. Many factors regulating C. difficile pathogenicity are unknown. In recent years, it appeared that small non-coding RNAs (sRNAs) and RNA-binding proteins (RBPs) are involved in post-transcriptional regulation of nearly every facet of bacterial life. Most research has been conducted in Gram-negative species, generating a big knowledge gap in Gram-positives such as C. difficile. Despite ongoing efforts to unravel the functions of selected sRNAs and the RNA chaperone Hfq, we have only scratched the surface of studying post-transcriptional control in this pathogen. In this study, the complexomic approach gradient profiling by sequencing (Grad-seq) was applied to draft the landscape of cellular RNA and protein complexes in C. difficile. The sedimentation profiles of 3,541 transcripts (~88% of annotated transcripts) and 1,867 proteins (~50% of annotated proteins) were captured. Comparative analysis of the profiles reproduced major ribonucleoprotein particles (RNPs) and protein-protein interactions (PPIs). Based on several examples, the potential use of Grad-seq to imply protein function and discover unknown regulatory RNAs was demonstrated. With an established pull-down approach, the proteins KhpA and KhpB were identified as common interaction partners of similar behaving sRNAs. By applying crosslinking immunoprecipitation followed by RNA-sequencing (CLIP seq), a pervasive targetome of KhpB was revealed, comprising over one-third of all annotated transcripts, including sRNAs, tRNAs and untranslated regions. The scope of RNA-binding suggests that KhpB is a second globally acting RBP in C. difficile, next to Hfq. The combination of transcriptome studies and phenotypic characterization suggests that KhpA and KhpB have several physiological functions including the regulation of toxins and metabolic pathways associated with pathogenesis. Overall, this work provides a comprehensive dataset for the discovery of PPIs and RNA-protein interactions in C. difficile. Further, this study establishes the foundation for studying two novel players in post-transcriptional regulation in this important human pathogen. / Das sporulierende, opportunistische Enteropathogen Clostridioides difficile (C. difficile) ist die häufigste Ursache nosokomialer Antibiotika-assoziierter Diarrhö. Eine Störung des gesunden Darmmikrobioms durch eine Antibiotikatherapie ist der größte Risikofaktor für eine Infektion. Die Symptome können von milder Diarrhö bis hin zu lebensgefährlicher Kolitis oder Gewebsnekrosen variieren. Die Schwere der Infektion wird durch die Expression verschiedener Virulenzfaktoren beeinflusst, welche unter anderem auch die zwei krankheitsauslösenden Exotoxine Toxin A und Toxin B umfassen. Viele der Faktoren, die zur Pathogenität von C. difficile beitragen sind jedoch noch unbekannt. In den letzten Jahren hat sich herausgestellt, dass kleine nichtcodierende RNAs (sRNAs) und RNA-bindende Proteine an der posttranskriptionalen Regulation fast aller Aspekte des bakteriellen Lebens beteiligt sind. Der Großteil der Forschung wurde an Gram-negativen Organismen durchgeführt. Dies führte zu der Entstehung einer großen Wissenslücke in Gram-positiven Organismen wie C. difficile. Trotz fortwährender Bemühungen, die Funktionen ausgewählter sRNAs und dem RNA-Chaperon Hfq zu verstehen, kratzen wir immer noch an der Oberfläche der Erforschung posttranskriptionaler Regulation in diesem Erreger. In dieser Studie wurde mittels der Methode Grad-seq (gradient profiling by sequencing) eine Landkarte zellulärer RNA und Proteinkomplexe in C. difficile erstellt. Dies führte zur erfolgreichen Erfassung der Sedimentationsprofile von 3541 Transkripten (~88% aller annotierten Transkripte) und 1867 Proteinen (~50% aller annotierten Proteine). Mit einer komparativen Analyse der Verteilung von RNAs und Proteinen konnten wichtige Ribonukleoproteine und Protein-Protein Interaktionen reproduziert werden. Basierend auf mehreren Beispielen wie der Vorhersage von Proteinfunktion oder der Identifizierung unbekannter regulatorischer RNAs, konnte die potentielle Nutzung des Datensatzes demonstriert werden. Mittels eines etablierten Pulldown-Assays wurden die zwei Proteine KhpA und KhpB als Interaktionspartner einer Gruppe von sRNAs mit ähnlichem Sedimentationsverhalten identifiziert. Die Durchführung eines CLIP-seq (crosslinking immunoprecipitation followed by RNA seq) Experimentes führte zur Ermittlung eines umfangreichen Targetoms des Proteins KhpB, welches mehr als ein Drittel aller annotierten Transkripte umfasst. Dieses inkludiert neben mRNAs auch sRNAs, tRNAs und untranslatierte Regionen. Der Umfang der RNA-Interaktionen suggeriert KhpB als zweites global agierendes, RNA-bindendes Protein in C. difficile neben Hfq. Die Kombination von Transkriptom-Analysen und phänotypischer Charakterisierung deutet darauf hin, dass KhpA und KhpB verschiedene physiologische Funktionen in C. difficile haben. Diese umfassen unter anderem die Regulation von Toxinen und Stoffwechselwegen, welche mit der Pathogenese assoziiert werden können. Zusammengefasst stellt diese Studie einen umfangreichen Datensatz zur Analyse von Protein-Protein und RNA-Protein Interaktionen in C. difficile zur Verfügung. Des Weiteren setzt diese Arbeit die Grundlage zur Erforschung zweier neuartiger Akteure der posttranskriptionalen Regulation eines wichtigen humanen Krankheitserregers.

Clostridium difficile est une bactérie qui, avec l’appauvrissement de la flore intestinale causée par des antibiotiques, produit deux toxines qui altèrent la paroi intestinale et provoquent ainsi des diarrhées pouvant entraîner la mort. Les premiers traitements à base de vancomycine, ont fait apparaître des souches résistantes. Depuis 2012, Santé Canada a approuvé un seul nouvel antibiotique, la fidaxomicine ou tiacumicine B. Ce traitement est plus efficace pour lutter contre C. difficile que la vancomycine. Le macrocycle de la tiacumicine B, un macrolactone, est fabriqué par une bactérie du sol et les enzymes qui agissent dans la voie de biosynthèse sont connues mais leur ordre d’action ne l’est pas encore. Pour cela, il est nécessaire de connaître les substrats de chacune de ces enzymes. Afin de mieux comprendre la biosynthèse de la tiacumicine B, ce mémoire présente la détermination de la structure de TiaP1, un cytochrome P450 qui hydroxyle le carbone 18 du macrocycle. La structure de TiaP2, qui hydroxyle le carbone 20, étant déterminée, l’objectif était de prédire l’identité de son substrat à l’aide de l’arrimage moléculaire. Les résultats appuient l’hypothèse que TiaP2 agirait avant TiaP1 sur le macrocycle. D’après les simulations d’arrimage moléculaire, nous prédisons que la glycosyltransférase TiaG1 (non étudiée ici) ajouterait un résidu de sucre au macrolactone avant que TiaP2 n’hydroxyle le carbone 20. / Clostridium difficile is a bacterium that, with the depletion of gut flora during antibiotic treatment, produces two toxins that alter the intestinal wall causing potentially deadly diarrhea. The first treatments were based on vancomycin and resistance by C. difficile developed rapidly. In 2012, Health Canada approved a new antibiotic, fidaxomicin, which is also named tiacumicin B. This treatment is the most effective treatment against C. difficile. The macrocycle of tiacumicin B, a macrolactone, is produced by a soil bacterium and enzymes that act in the biosynthetic pathway are known. However, their order of action is not established. To know their order of action, it is necessary to know the substrates. This thesis presents the determination of TiaP1 structure, a cytochrome P450 that hydroxylates carbon 18 of the macrocycle of tiacumicin B. The structure of TiaP2, a P450 hydroxylating carbon 20, was already determined, and we attempted to predict its substrate by using a molecular docking approach. Our results confirm that TiaP2 hydroxylates before TiaP1. According to molecular docking results, we predict that the glycosyltransferase TiaG1 (not studied here) adds a sugar on the macrolactone of tiacumicin B before TiaP2 hydroxylates the carbon 20.

Bakgrunn: Clostridium difficile(CD)er antatt å være den utløsende årsak for 20-30 % av tilfeller med antibiotikaassosiert diaré. CD er den vanligste formen for helsetjenesteervervet diaré, og forår-saker økt sykelighet og dødelighet, samt økte kostnader for helsetjenesten. Desiste tiår er det rappor-tert om endringeri epidemiologien, forårsaket av en ny CD stamme, ribotype 027. Den spres lettere, lager mer alvorlig sykdom og tilbakefall. I Norge har denne stammen til nå ikke vært noe problem. Forekomsten kan reduseres ved en tydelig antibiotikapolitikk, og etterlevelse av anbefalte smitte-verntiltakfor å forebygge CD. Hensikt: Finne ut hvilke smitteverntiltak helsepersonell i sykehuset velger ved håndtering av pasien-ter med CD, og hvor de henter kunnskap om slike smitteverntiltak fra. Metode: En retrospektiv tverrsnittsstudie med spørreskjema til helsepersonell ble gjennomførtvåren og høsten 2011 i Sørlandet sykehus HF (SSHF), sør i Norge. Resultat: 168 helsepersonell deltok i undersøkelsen, fordelt på 59 leger og 109 pleiere. Svarprosen-ten var 94. Antibiotikarestriksjoner sier 94 % av medisinerne og 38 % av kirurgene atde har i sine avdelinger(x²=10.756, p&lt;0.001). Stetoskop brukt i isolat med CD pasienter sier 25 % legerat de spritdesinfiserer og tar med, mens 6 % pleiere sier det samme (x²=22.273, p&lt;0.001). 73 % av leger og 64 % pleiere sier at de både desinfiserer og vasker hendene etter kontakt med CD pasient (x²=6.451, p=0.011). Pasientinformasjon om viktigheten av håndhygiene sier 8 % leger og 36 % pleiere at de alltid gir (p&lt;0.001). Desinfeksjonsmiddel til bruk etter en CD pasient, sier 48 % medi-sinske pleiere og 43 % kirurgiske pleiere at de velgerVirkon (feil). Infeksjonskontroll-programmet (IKP) som kunnskapskilde brukes av 14 % leger og 46 % pleiere, (p&lt;0.001). Kunnskap om smitte-verntiltak ved CD sier 63 % legerog 72 % pleiere at de får fra kolleger. Konklusjon: Helsepersonell har generell kunnskap om smitteverntiltak, men mangler spesifikk kunnskap om smitteverntiltak ved CD. For å møte utfordringen med nye mer spredningspotente CD stammer, må etterlevelsen av smitteverntiltak øke. IKPalene, synes ikke å være tilstrekkelig for å holde helsepersonelloppdatert. Den manglende spesifikke kunnskapen om smitteverntiltak ved CD kan utsette pasienter i sykehuset for smitterisiko / Background: Clostridium difficile(CD) causes 20 %–30 % of all nosocomial infectious diarrhea, resulting in significant morbidity and increasing healthcare costs. Good antibiotic stewardship reduc-es the incidence of CD, andcompliance with infection control measures limits its spread. In recent years, ribotype 027, a new strain of CD, caused several disease outbreaks. Ribotype 027 spreads more easily and increases disease severity and relapse. Thus far, ribotype 027 has caused few prob-lems in Norway. Objective: This thesis aimed to determine whether hospital-based healthcare professionals comply with recommended infection control measures for CD prevention and identify how they learn about such measures. Method: A retrospective cross-sectional survey and questionnaire was performed among healthcare professionals at Sørlandet Hospital, Norway,during the spring and fall of 2011. Results: Survey participants included 168 health professionals (59 physicians and 109 nurses). The response rate was 94 %. Medical doctors (94 %) and surgeons (38 %) said that their clinics impose antibiotic restrictions (x ² = 10.756, p&lt; 0.001). After contact with a CD patient, physicians and nurs-es (73 % and 64 %, respectively) said they disinfect and wash their hands (x ²= 6.451, p&lt; 0.011). Notably, only 8% of physicians and 36 % of nurses always give patients information about the im-portance of hand hygiene (p&lt; 0.001). Even 25 % of physicians and 6 % of nurses reported using ethanol (does not eliminated CD spores) to disinfect stethoscopes before leaving a CD isolation room (x ² = 22.273, p&lt; 0.001). Medical and surgical nurses (48 % and 43 %, respectively) incorrectly used Virkon as a disinfectant in the CD patient’s room. Physicians and nurses (63 % and 72 %, respective-ly) mainly obtain knowledge about infection control measures from colleagues, compared to physi-cians and nurses (14 % and 46 % , respectively) who gain such knowledge from the hospital’s infec-tion control program (p&lt; 0.001). Conclusion: Healthcare professionals have some knowledge about infection control measures, but lack knowledge specific to limiting the spread of CD. Increased compliance with infection control measures is crucial to meeting the challenge of new and more potent strainsof CD. Guidelines alone are likely insufficient to keep healthcare professionals up to date. The lack of specific knowledge about infection control measures for CD may expose hospitalized patients to CD infection / <p>ISBN 978-91-86739-69-0</p>

Objective To describe the use of Lactobacillus by hospitalized patients and to examine its relationship with various Clostridium difficile infection (CDI) related outcomes. Methods The characteristics of Lactobacillus users and non-users and the initiation of Lactobacillus with respect to initiation of antibiotic therapy and CDI treatment were described using national hospital discharge database. The relationships between Lactobacillus use and post-CDI length of stay, mortality, switch of CDI therapy, and readmission were analyzed. Results Lactobacillus users and non-users were different in most characteristics. Metronidazole and fluoroquinolones were the most frequently used antibiotics by Lactobacillus users. They were mainly CDI cases, used multiple antibiotics, extremely ill, and started Lactobacillus five or more days after initiation of antibiotics or CDI treatment. Lactobacillus use was associated with increased length of stay and switching of CDI therapy. Conclusions The true association between Lactobacillus use and CDI remains unclear. This study provides foundation for future research.

Clostridium difficile est responsable de 15 à 25% des cas de diarrhées post-antibiotiques (ATB) et de plus de 95% des cas de colite pseudomembraneuse. Depuis 2003 et suite à l'émergence du clone 027, les ICD sont devenues plus fréquentes et plus sévères. Compte tenu des conséquences, il a été décidé d'explorer en détail et prospectivement cette maladie au Groupement Hospitalier Edouard Herriot L'inclusion des patients a débuté fin février 2011 et devrait s'arrêter fin février 2014. Dans une méta-analyse, nous avons montré que l'ICD communautaire est associée à l'exposition aux mêmes ATB qu'une ICD nosocomiale. Une analyse de la littérature, en utilisant la grille ORION comme outil, nous a permis de synthétiser les connaissances sur la prévention et le contrôle d'ICD en milieu hospitalier. Par la suite sur la base d'une étude rétrospective, le sexe, la CRP et l'exposition aux fluoroquinolones ont été identifiés comme associés à une ICD sévère chez les patients hospitalisés en réanimation. Entre 2011 et 2013, 430 patients ont été inclus dans notre cohorte. L'analyse des données de la prise en charge thérapeutique de 118 cas d'ICD a montré un niveau insuffisant de la connaissance des recommandations actuelles concernant le traitement de cette infection. L'analyse pronostique a montré un taux de mortalité de 19,5% dans les 30 jours qui suivent le diagnostic. L'ICD était indiquée comme une cause principale ou contributive de décès dans quinze cas (65,7% des décédés). Les analyses multivariées ont montré que les facteurs associés au décès sont différents entre les patients avec une ICD et les patients présentant une diarrhée non liée au Clostridium difficile / Clostridium difficile is responsible for almost all cases of pseudomembranous colitis and for 15%-25% of cases of post-antibiotic (ATB) diarrhea. Since 2003 and the emergence of 027 strain, CDI epidemiology is changing, with evidence of rising incidence and severity. In response to the alarming situation we decided to conduct a prospective study at Eduard Herriot Hospital to explore in details this infection. Patient’s inclusion has started in February 2011 and will end in February 2014. In a meta-analysis we found that the risk profiles for antimicrobial classes as risk factors for community-acquired CDI are similar to those described for nosocomial CDI. We used the ORION statement (Outbreak Reports and Intervention Studies Of Nosocomial infection) to synthesize knowledge of interventions to reduce and to control CDI in hospitals. Then in a retrospective study, we found that male gender, rising serum C-reactive protein level, and previous exposure to fluoroquinolones were independently associated with severe CDI in ICU. Between 2011 and 2013, 430 patients were included in our prospective cohort study. Data analysis of 118 cases of CDI showed an inefficient knowledge of current recommendations of CDI treatment. The crude mortality rate within 30 days after CDI diagnosis was 19.5%, with 15 deaths (65.7% of deceased patients) related to CDI. In a multivariate cox regression model, gender, serum albumin, antidiarrheal medications, cephalosporins, peritonitis and septic shock were independently associated with mortality in CDI patients. When diarrhea was not related to C. difficile, mortality was rather associated with cancer and high WBC level

No description available.