Allergic inflammatory disorders of the airway like asthma and atopic asthma are complex, often long-term diseases that generate large public health and socioeconomic footprints especially in developed countries like US, UK and Australia. In 2009, approximately 8.2%, 24.6 million people in United States were affected by asthma. Currently 235 million people are affected by asthma worldwide and about 90% of those have allergic (atopic) asthma. An important factor in patients with allergic respiratory tract diseases is sensitization to fungi. Other risk factors for asthma include inhaled allergens that irritate the airways. Up to 70% of mold allergic patients have skin test reactivity to Alternaria. Alta1, an allergen produced by A. alternata also produces a prolonged and intense IgE mediated reaction in sensitized patients. Therefore A. alternata is not only a risk factor in development of asthma but also can lead to exacerbation of severe and potentially lethal asthma than any other fungus. Despite the well-documented clinical importance of Alternaria in allergic airway diseases, little knowledge exists about the role of individual fungal genes and gene products in theses pathological states besides a small repertoire of allergens and proteolytic enzymes. Moreover, the importance of small, secreted molecules of fungal origin has not been explored whatsoever in regards to immune responses triggered by Alternaria. This study addresses the hypothesis that Alternaria derived small molecule's have immune modulatory properties. A major thrust of this project was to assess the role of Alternaria secondary metabolites that are synthesized by genes called polyketide synthases (PKS) in immune responses of lung epithelial cells. / Master of Science

Au cours de ma thèse je me suis intéressée aux relations hôte-pathogène entre Drosophila melanogaster et Pseudomonas aeruginosa PA14. RhlR, un facteur de transcription bactérien permet à la bactérie d’échapper à la phagocytose. Mon projet de thèse consistait à identifier comment RhlR exerce cette fonction. Mes résultats suggèrent que RhlR exercerait également une fonction indépendante du quorum sensing. Un crible de mutants PA14 nous a permis d’isoler trois gènes importants pour la virulence de la bactérie et possiblement reliés à RhlR: xcpR, vfR et sltB1. L’utilisation de mutants de drosophile tep4, m’a permis de montrer que le rôle d’échappement à la phagocytose se ferait au niveau de la détection de la bactérie. Par ailleurs, mes résultats suggèrent aussi l’intervention d’un composé volatil qui permettrait de synchroniser la virulence de la bactérie. Dans une dernière partie, j’ai étudié les effets d’une co-infection entre un virus entérique et PA14. / During my PhD, I studied the host-pathogen interactions between Drosophila melanogaster and Pseudomonas aeruginosa PA14. We previously identified RhlR as a bacterial transcription factor that allows the bacteria to circumvent phagocytosis. My main PhD project was to study and identify how RhlR exerts this function. My first results suggested that RhlR plays also a role independently its the quorum sensing. A screen of PA14 mutants allowed me to identify three genes involved in PA14 virulence and possibility in RhlR function: xcpR, vfR and sltB1. By using tep4 fly mutants, I have shown that RhlR’s role against phagocytosis is most likely required at the level of PA14 detection. Beside this, my results indicated that possibly a volatile compound is involved to synchronize PA14 virulence. In the last part, I studied the effects of a co-infection between an enteric virus and PA14.

Les microARNs, régulateurs post-transcriptionnels de l’expression des gènes eucaryotes, sont impliqués dans la défense contre les pathogènes. Afin de favoriser leur multiplication, les virus et les bactéries ont développé des stratégies pour altérer la voie des miRNAs. Dans ce travail, nous avons montré que Helicobacter pylori, une bactérie responsable chez l’Homme de pathologies gastriques sévères, telles que l’ulcère ou le cancer, réprime un cluster de microARNs spécifique des cellules souches embryonnaires dans une lignée épithéliale gastrique. En utilisant une technique de séquençage à haut débit, nous avons identifié miR-372 comme le miRNA le plus exprimé dans cette lignée gastrique. Avec miR-373, miR-372 permet la prolifération cellulaire réprimant l’expression d’un inhibiteur du cycle cellulaire, the LArge Tumor Suppressor 2 (LATS2). Au cours de l’infection par H. pylori, l’expression de miR-372&373 est réprimée, provoquant une accumulation de LATS2 et un arrêt du cycle cellulaire. De manière importante, la répression de ces miRNAs est dépendante de la translocation de l’effecteur bactérien CagA dans la cellule hôte. Ces données constituent un nouvel exemple d’interaction hôte-pathogène impliquant les miRNAs et ont identifié le couple LATS2/miR-372&373 comme un mécanisme inattendu dans l’arrêt du cycle cellulaire observé au cours de l’infection. Ce mécanisme pourrait refléter l’inhibition de l’auto-renouvellement de l’épithélium gastrique, processus impliqué dans la défense contre les infections bactériennes. / MicroRNAs, post-transcriptionnal regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies to suppress miRNA functions with the aim to establish a sustainable infection. In this work, we report that Helicobacter pylori, a bacterium responsible for severe human gastric inflammatory diseases and cancers, down-regulates an embryonic-specific microRNAs cluster in a gastric epithelial cell line. We reveal by using a deep sequencing approach that hsa-miR-372 is the most abundant miRNA expressed in this gastric cell line where, together with hsa-miR-373, it promotes cell proliferation by silencing the expression of a cell cycle inhibitor, the LArge Tumor Suppressor 2 (LATS2). Upon H. pylori infection, miR-372&373 synthesis is inhibited, leading to the derepression of LATS2 and thus, to a cell cycle arrest at the G1/S transition. Importantly, this down-regulation of a specific cell cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells. These data constitute a novel example of host-pathogen interplay involving microRNAs and unveil the couple LATS2/miR-372&373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells which may be relevant of inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections.

Paired receptor families, consisting of multiple genetically and structurally similar but functionally opposite activating and inhibitory cell surface receptors, are among the fine tuners of the immune regulation. Recent studies on the evolutionary origin of these receptor families have suggested links to pathogen driven diversification, according to which activating receptors continuously evolve in order to counterbalance pathogens that try to subvert the immune response by stimulating the inhibitory receptor through their virulence factors. This thesis is about the CD200R paired receptor family. This family consists of an inhibitory receptor CD200R which is expressed on various leukocytes and delivers inhibitory signals upon engagement with its ligand CD200. In this study, the possibility that the activating members of the family evolved under pathogen pressure was investigated. Genomic DNA from twenty two different mice strains was screened for the presence of members of CD200R family. The number of activating receptors varied, CD200RLe and CD200RLc were found to be mutually exclusive and three strains possessed previously unknown members of CD200R family. In addition, the possibility that CD200R family members and other paired receptors interacted directly with bacteria was tested with a new assay but only the interaction of PIR-A1 with <em)S. aureus was found as previously reported. The rabbit CD200R family has been characterized and ligand receptor interaction between rabbit CD200 and rabbit CD200R has been demonstrated. However, no interaction between rabbit CD200R and a candidate viral CD200 homologue, the M141R protein of myxoma viruses, could be shown. This finding suggested a CD200R independent role for M141R molecule and possibly other homologues in pox viruses. Finally, two novel antibodies (OX131 and OX132) were characterized together with formerly generated antibodies against mouse CD200R family. The binding specificities and their effects on the CD200-CD200R interaction have been shown. This will help usage of these antibodies in various studies on the functionality and distribution of these receptors.

La coévolution ainsi que les différentes interactions entre hôte et pathogène contribuent à former la diversité génétique de ces deux organismes. Dans le cadre de cette thèse, nous nous sommes intéressés à l’étude de la variabilité génétique de 1760 gènes immunitaires choisis suivant des critères définis, pour essayer d’expliquer pourquoi il existe une variation individuelle face aux infections. L’objectif principal de ce projet était alors de caractériser et d'analyser de nouveaux gènes et polymorphismes immunitaires pouvant expliquer le contrôle ou la susceptibilité à certaines infections. Deux études pilotes nous ont permis de développer le pipeline de détection de polymorphismes. Pour la première, le polymorphisme des 3 gènes CD28, CTLA4, et ICOS a été caractérisé. Dans la deuxième, nous avons caractérisé le polymorphisme de 10 gènes impliqués dans la réponse immunitaire contre M. tuberculosis. Ces gènes ne sont pas très polymorphes et trois d’entre eux sont très conservés. Ces deux études nous ont aidés à préparer l’analyse à grande échelle avec les mises au point et l’amélioration du pipeline. Nous avons sélectionné 1760 gènes en se basant sur des critères définis. La variabilité génétique a été étudiée dans les populations humaines par une analyse minutieuse in silico de données de séquençage d’exomes générées par différents projets et consortiums pour plus de 700 individus représentant 20 populations à travers le monde. 30 gènes les plus polymorphes ont été ainsi identifiés. Ces gènes pourront être entièrement caractérisés et les données produites pourraient être comparées avec des données de résistance/sensibilité de certaines maladies infectieuses. / Host-pathogen co-evolution and interactions contribute in shaping the genetic diversity of both organisms. The objective of this thesis is to define the genetic basis of variability in disease resistance/susceptibility through the development of large-scale in silico screens to identify novel gene candidates implicated in host-pathogen interactions (such as tuberculosis).A pilot study was conducted on CD28, CTLA4, and ICOS to investigate their polymorphism. As a first step in our study based on data available in the literature, we selected a set of ten genes relevant for the immune response against M. tuberculosis. Seven of these genes were moderately polymorphic, while three of them were highly conserved. This analysis was used to prepare and setup the large scale analysis using the same developed pipeline for polymorphism detection and allele reconstruction. For our in silico, we used sequence data from several projects and consortiums to isolate most polymorphic human genes amongst a list of over 1760 candidates selected based on already established relevance for infections and on evolutionary considerations. A first screen of 64 individuals from eight different populations from several regions of the world was performed and most variable genes were selected for further extensive analyses on a larger panel (715 individuals). 30 most polymorphic genes were thus identified. The extent of polymorphism and the allelic worldwide variants of each of these 30 genes are ready to be fully characterized. The data generated could be compared against infectious disease resistance/susceptibility data to identify potentially relevant gene variation.

La macroautophagie, appelée ici autophagie, est un processus de dégradation lysosomale qui joue un rôle clé dans l'immunité en dégradant des micro-organismes intracellulaires mais également en activant des réponses immunitaires. Cependant, de nombreux virus ont développé des stratégies pour inhiber, utiliser voire détourner l'autophagie à leur propre bénéfice. L'objectif de cette thèse a été d'analyser la place de l'autophagie dans l'infection par le virus de la rougeole (VR). Ce travail démontre que les souches atténuées du VR utilisent plusieurs voies moléculaires successives d'induction d'autophagie dans les cellules infectées. En effet, elles sont capables d'induire une première vague d'autophagie très précoce mais transitoire par l'engagement de l'une des isoformes de leur récepteur d'entrée CD46. Après un bref retour à l'état basal, une deuxième vague d'autophagie est induite par l'interaction de la protéine virale non structurale C avec la protéine cellulaire autophagique IRGM. La formation de syncytia conduit à une troisième voie d'induction d'autophagie qui permet de maintenir l'autophagie mise en place. Cette autophagie soutenue est exploitée par le VR afin de limiter la mort des cellules infectées, ce qui promeut la production de particules virales. Les souches virulentes du VR, incapables de lier CD46, et utilisant comme récepteur d'entrée la protéine CD150, n'induisent que l'autophagie tardive qui est également utilisée pour favoriser la production de particules virales infectieuses. Ce travail de thèse montre donc que l'induction d'une autophagie soutenue lors de l'infection par le VR promeut l'infection, principalement en limitant la mort cellulaire / Macroautophagy, thereafter referred to as autophagy, is a lysosomal degradation which plays a key role in immunity by directly degrading intracellular pathogens but also by favouring innate and adaptive immune responses. However, several viruses have evolved strategies to inhibit, exploit or even hijack autophagy for their own benefit. The aim of this thesis was to analyse the role of autophagy in the course of measles virus (MeV) infection. This work demonstrates that attenuated strains of MeV induce successive autophagy signalling in infected cells, via distinct and uncoupled molecular pathways. First, attenuated MeV strains are able to induce a first early and transient wave of autophagy through the engagement of one of the isoform of their cellular receptor CD46. Soon after infection, a new autophagy signalling is initiated by the interaction of the non-structural MeV protein C with the cellular autophagic protein IRGM. Strikingly, this second autophagy signalling can be sustained overtime within infected cells via a third autophagy input resulting from cell-cell fusion and the formation of syncytia. Sustained autophagy is exploited by MeV to limit the death of infected cells and to improve infectious viral particle formation. Interestingly, virulent strains of MeV, which do not use CD46 as a cellular receptor but use CD150, are unable to induce the early autophagy wave, whereas they induce and exploit the late and sustained autophagy. Thus, this work demonstrates that the induction of a sustained autophagy during MeV infection promotes infectivity, mostly by limiting cell death. Overall, this work describes an unusual and complex interplay between autophagy and MeV

Further functional investigation revealed that knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells, concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium Conversely, overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta 1 (IFN-beta1). In this connection, IFN-beta1-mediated 2', 5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover, GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p= 0.019) as compared with their counterpart pre-treatment liver biopsies. / Hepatitis B virus (HBV) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although considerable progress has been made over the past decade, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. / In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via IFN-beta1-OAS-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection. / In this study, we applied a two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach to globally analyze the host early response to HBV by using an inducible HBV-producing cell line HepAD38. Twenty-three proteins were identified as differentially expressed, with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, as well as in HBV-infected human liver biopsies by immunohistochemistry. / Ma, Yan. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

L’émergence et la dissémination récentes de clones hypervirulents et multi-résistants de Klebsiella pneumoniae ont renouvelé l'intérêt général envers Klebsiella. Cependant, notre connaissance de la pathogénèse de Klebsiella au niveau moléculaire et cellulaire reste faible. Dans ce travail, nous avons mené deux axes de recherche focalisés sur la pathogénèse de Klebsiella. Le premier projet visait à caractériser un mutant de capsule de K. pneumoniae incapable de produire une toxine colibactine fonctionnelle. La colibactine est un métabolite secondaire génotoxique produit principalement par des souches commensales et extraintestinales pathogènes d’Escherichia coli, mais également par des souches de K. pneumoniae. Elle induit des cassures double brin conduisant à la formation de tumeurs dans des cancers colorectaux et contribue à une virulence accrue de la bactérie. Cependant la structure, les voies de biosynthèse, la sécrétion et le mode d’action de la colibactine restent à définir. Le laboratoire avait préalablement observé qu’un mutant de capsule de K. pneumoniae n’était pas capable de produire une colibactine fonctionnelle, suggérant un rôle de la capsule dans ce processus. Nous avons ensuite démontré qu’en fait la capsule n’est pas impliquée dans la fonction de la colibactine, et que l’incapacité du mutant de la capsule à produire une génotoxicité est due à une mutation avec un fort effet dominant négatif dans la protéine ClbD, une enzyme essentielle de la voie de synthèse de la colibactine. Nous caractérisons actuellement cette mutation pour comprendre comment elle affecte la structure et la fonction de ClbD. Le second projet étudiait le rôle de la capsule dans la pathogénèse de K. rhinoscleromatis. K. rhinoscleromatis est une sous-espèce de K. pneumoniae, responsable du rhinosclérome, une maladie chronique granulomateuse des voies aériennes supérieures spécifiquement humaine, et caractérisée par la formation de macrophages spumeux atypiques appelés cellules de Mikulicz. Or les mécanismes physiopathologiques de cette pathologie sont peu connus. A l’aide d’un modèle murin, nous avons observé qu’un mutant de capsule de K. rhinoscleromatis est atténué in vivo, mais aussi que les cellules de Mikulicz sont recrutées lors d’une infection avec un inoculum élevé du mutant de capsule de K. rhinoscleromatis. Ces données nous indiquent 1) que la capsule est un facteur de virulence de K. rhinoscleromatis qui n’est pas impliqué dans la formation et le recrutement des cellules de Mikulicz, et 2) que des facteurs spécifiques de K. rhinoscleromatis contrôlant la formation de cellules de Mikulicz existent et restent à identifier. Les nouvelles données concernant la pathogénèse de Klebsiella apportées par notre travail représentent une contribution significative dans la connaissance du rhinosclérome et du rôle d’une enzyme impliquée dans la synthèse de la colibactine, tout en ouvrant de nouveaux axes de recherche sur la pathogénèse de K. pneumoniae et K. rhinoscleromatis / The recent emergence and global expansion of hypervirulent and multidrug-resistant clones of K. pneumoniae have increased general interest in Klebsiella. However, knowledge of Klebsiella pathogenesis at the molecular and cellular level is still scant. We pursued two lines of research focused on Klebsiella pathogenesis. The first aimed to characterize a K. pneumoniae capsule mutant unable to produce a functional colibactin. Colibactin is a genotoxic secondary metabolite produced mainly by commensals and extraintestinal pathogenic E. coli strains, but also by some K. pneumoniae strains. It induces double-strand DNA breaks leading to tumor formation in colorectal cancer and contributes to increased virulence. However, its structure, biosynthesis, secretion and mode of action have yet to be fully defined. Previous work from our laboratory showed that a K. pneumoniae capsule mutant was unable to produce a functional colibactin, suggesting a role for capsule in this process. We report herein that capsule does not in fact have a role in the colibactin effect and that the inability of the capsule mutant to induce DNA damage is due to a strong dominant negative mutation in ClbD, an essential enzyme of the colibactin biosynthetic pathway. We are currently characterizing this mutation to understand how it deeply affects ClbD structure and function. The second project explored the role of capsule (CPS) in K. rhinoscleromatis pathogenesis. K. rhinoscleromatis is a K. pneumoniae subspecies responsible for rhinoscleroma, a human specific chronic granulomatous disease of the upper airways characterized by the formation of atypical foamy macrophages called Mikulicz cells. However, little is known about the pathophysiological mechanisms underlying this disease. Using our mouse model, we report that a K. rhinoscleromatis CPS mutant is attenuated in vivo but also that Mikulicz cells are observed upon infection with higher doses of K. rhinoscleromatis CPS mutant. Altogether, our data indicate that 1) CPS is a virulence factor of K. rhinoscleromatis, which is not involved in the specific appearance of Mikulicz cells and that 2) the K. rhinoscleromatis specific factors controlling the appearance of Mikulicz cells remain to be identified. The new insights brought to Klebsiella pathogenicity by this work represent a significant contribution to the understanding of rhinoscleroma pathogenesis and of the role of an enzyme implicated in colibactin biosynthesis. This opens new lines of research on K. pneumoniae and K. rhinoscleromatis pathogenesis

Infections with the bacterial pathogen Chlamydia trachomatis are a critical public health problem. Chlamydia remains the number one cause of preventable blindness worldwide and the leading cause of bacterial sexually transmitted infections in the United States. In humans, repeat and persistent infections with Chlamydia result in severe inflammation. Inflammation in the conjunctiva can result in blindness, while inflammation in the genital tract can result in pelvic inflammatory disease, ectopic pregnancy or infertility. In order to curb the increasing incidence of Chlamydia infections worldwide it will be necessary to develop a protective vaccine that affords long-term protection and prevents pathologies. To better inform vaccine development we must understand the mechanisms that drive long-term immunity in the genital tract and elucidate critical interactions between Chlamydia and host cells to uncover potential mechanisms of immune evasion.

Parasites of phylum Apicomplexa cause devastating diseases on a global scale. Toxoplasma gondii, the etiological agent of toxoplasmosis, and Plasmodium falciparum, the most virulent agent of human malaria, have the most substantial effects on human health and are the most widely studied. The success of these parasites is due in part to a sophisticated molecular arsenal that supports a variety of novel biological processes including a unique form of host cell invasion. Accessing the protective environment of the host cell is paramount to parasite survival and is mediated through an active invasion process: the parasite propels itself through a circumferential ring known as the moving junction (MJ) formed between its apical tip and the host cell membrane. The MJ ring is comprised of a parasite surface protein (AMA1) that engages a protein secreted by the parasite into the host cell and presented on the host cell surface (RON2). Thus, through an intriguing mechanism the parasite provides both receptor and ligand to enable host cell invasion. Prior to the studies described herein, the characterization of the AMA1-RON2 association was limited to low-resolution experiments that provided little insight into the functional and architectural details of this crucial binary complex. Towards elucidating the mechanism of AMA1-RON2 dependent invasion, I first structurally characterized T. gondii AMA1 bound to the corresponding binding region of RON2; analysis of the AMA1-RON2 interface along with biophysical data revealed an intimate association likely capable of withstanding the shearing forces generated as the parasite dives through the constricted MJ ring. To investigate the role of the AMA1-RON2 complex across genera, species and life-cycle stages, I next characterized the AMA1-RON2 complex from a distantly related genus within Apicomplexa (Plasmodium) and from a divergent pairing within T. gondii. By combining structural, biophysical and biological data, I was able to generate a detailed model describing the role of AMA1 and RON2 in MJ dependent invasion, which is currently supporting efforts to develop novel vaccines and cross-reactive small molecule therapeutics. / Graduate / 0487 / tonkin.ml@gmail.com