Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.

No description available.

Neutralisierende Antikörper sind entscheidend in der Eindämmung der Virusinfektion, indem sie den Eintritt in die Wirtszelle hemmen bzw. die Aktivierung der Komplementkaskade initiieren. Distinkte wirtseigene Oberflächenrezeptoren für die Fc-Domäne von IgG (FcyR) sind für die Kommunikation von humoraler und zellulärer Immunantwort verantwortlich. Auch Mitglieder der Herpesviren kodieren für Fc-bindende Proteine, die Kandidaten für immunevasive Funktionen darstellen könnten. Der Nachweis der HCMV-kodierten Fc-bindenden Proteine gp34 und gp68 als Bestandteil der Virushülle lies auf eine immunevasive Funktion hinsichtlich neutralisierendem IgG und Komplement-vermittelter Virolyse schließen, wie für den HSV-1-kodierten FcyR gE beschrieben. Weder für gp34 noch für gp68 konnte in vitro ein hemmender Effekt auf Neutralisation und Virolyse beobachtet werden. In unserem Labor wurde jedoch gezeigt, dass gp34 und gp68 selektiv die IgG-abhängige Aktivierung zellulärer FcyR inhibieren. Die glykosylierungsunabhängige Ligandenbindung von gp34 und gp68 wies auf unterschiedliche Interaktionsmechanismen zwischen den zellulären und den viralen FcyR hin. Mithilfe eines mutierten Fc-Fragments konnte für gp68 eindeutig eine mit HSV-1 gE überlappende Bindestelle an IgG identifiziert werden. Die für die Ligandenbindung erforderlichen Aminosäuren 71-292 von gp68 binden Fc in einer 2:1 Stöchiometrie, wobei die N-, nicht aber die O-Glykosylierung des vFcyRs essentiell sind. Darüber hinaus formt gp34 auf infizierten Zellen und auf der Virushülle kovalente Homooligomere. gp34-Cysteinpunktmutanten auf Basis der für die Bindung notwendigen Aminosäuren 24-140 lassen vermuten, dass die Oligomerisierung Voraussetzung für die Fc-Bindung ist. Im Gegensatz zu gp68 scheint der Mechanismus der Fc-Bindung von gp34 einzigartig unter den bekannten Fcy-Rezeptoren zu sein. Diese Ergebnisse lassen vermuten, dass trotz redundanter Expression der HCMV-FcyR der Bindungs- und Wirkungsmechanismus selektiv ist. / Neutralizing IgGs play a key role in diminishing virus infectivity by inhibiting the entry into host cells. Additionally, IgG-bound particles may be inactivated by virolysis through the activation of complement. Surface receptors specific for the Fc domain of IgG represent host proteins, connecting humoral and cellular immune responses. Also members of the herpes virus family code for proteins with Fc binding properties, implying functions that could intervene with antibody-dependent effector mechanisms. The presence of gp34 and gp68 on the virion membrane raised the question whether they are able to inhibit neutralising IgG and complement-mediated virolysis. The HSV-1-encoded FcyR gE was described to affect neutralisation and virolysis. Despite extensive analysis, there were no implications found that gp34 or gp68 interfere with neutralising IgG or virolysis in vitro. However, our lab could demonstrate that gp34 and gp68 selectively inhibit the IgG-dependent activation of the different host FcyRs. In contrast to the cFcyRs Fc recognition by gp34 and gp68 occurs independently of N-linked glycosylation of IgG, which points to a different binding mechanism among host and viral FcyRs. By taking advantage of a mutated Fc fragment, overlapping binding regions of the HSV-1 gE and gp68 were identified. For gp68 the amino acids 71-292 including the N-glycans are strictly required for Fc binding in a 2:1 stoichiometry. Interestingly, gp34 forms covalently linked homo-oligomers in infected cells and on the virion. Based on the minimal binding domain comprising the amino acids 24-140 of gp34, targeted cysteine exchange mutants revealed that oligomer formation by gp34 is absolutely required for Fc binding. In contrast to gp68, the Fc binding characteristics of gp34 appears to be unique among the known FcyRs. These findings allow us to postulate that even if the HCMV-encoded FcyRs are redundantly expressed the mechanistic details and binding properties are selective.

Streptococcus pneumoniae ist ein gram-positives Bakterium und ein Krankheitserreger des Menschen. Ein Charakteristikum des Bakteriums ist, dass es Cholin-Reste in seine dicke Zellwand einbaut. Das Ziel meiner Doktorarbeit war herauszufinden, inwiefern diese Cholin-Reste zur Virulenz des Bakteriums während experimenteller Sepsis und Meningitis beitragen. Dabei konnte ich feststellen, das cholinierte Wildtyp-Bakterien hoch virulent waren, ungestört im Wirt wachsen konnten und letztendlich zu einer massiven Überaktivierung des Wirts-Immunsystems (gemessen anhand der Zytokine IL-1beta, IL-6, IL-12, TNFalpha) sowie zum Tode der Versuchtiere führten. Im Gegensatz dazu waren cholin-freie Bakterien nicht in der Lage eine permanente Infektion im Wirt zu etablieren. So wuchsen sie nur anfangs und wurden vom Wirts-Immunsystem kontrolliert und beseitigt, sodass alle Tiere überlebten. Die Injektion von cholin-freien und cholinierten, hoch aufgereinigten Zellwänden, führte zu der Schlussfolgerung, dass Cholin in der Zellwand ein Immunevasionsmechanismus der Bakterien sein muss. Ausserdem waren cholin-freie Bakterien in der Lage einen protektiven, serotyp-spezifischen Immunschutz im Wirt zu induzieren. / Streptococcus pneumonia is a major human pathogen. Since it is a gram positive bacterium it is characterized by the production of a thick cell wall. Being auxotrophic for choline, the pneumococcus attaches this aminoalcohol to its teichoic acids thus decorating its surface with choline-residues. The aim of this work was to investigate the role that these choline residues play in the virulence of the bacterium. By using an isogenic choline-containing and choline-free pair of S. pneumoniae I was able to demonstrate that surface bound choline is essential for the virulence of the bacterium in animal models of experimental sepsis and meningitis. In either model choline-containing bacteria were able to persistently grow within the host system, continuously stimulate the production of proinflammatory cytokines (IL-1beta, IL-6, IL-12, TNFalpha) and eventually led to the death of all infected animals within 24h. In contrast, choline-free bacteria showed only transient growth within the host and induced only moderate and limited expression of cytokines. Consequently, the bacterium was virtually avirulent and all animals survived. Intracisternal application of highly purified cholinated and choline-free cell wall preparations, induced a comparable activation of the immune system. These findings led to the conclusion that choline residues contribute to an immunevasion strategy that allows the bacteria to grow despite an ongoing immune response. Although being avirulent choline-free bacteria were able to induce serotype specific immunity in the animals.

Um die Wirkung herpesviral-kodierter FcgammaRezeptoren auf wirtskodierte zelluläre FcgammaRezeptoren und IgG-vermittelten Effektorfunktionen untersuchen zu können, einen methodisch neuen Ansatz wurde entwickelt, der die Detektion FcgammaR-aktivierender Antikörper ermöglicht. Dieses neuartige Assay beinhaltet die Kokultivierung virusinfizierter Zellen, die mit virusspezifischen IgG-Antikörpern opsoniert sind, mit FcgammaR-zeta BW5147-Transfektanten als Reporterzellen. Diese stabilen Transfektanten exprimieren chimäre Rezeptoren, die aus der extrazellulären Domäne der zellulären FcgammaRezeptoren bestehen, welche mit der TM und intrazellulären Domäne der murinen CD3zeta-Kette fusioniert wurden. Die Aktivierung der CD3zeta-Kette führt zu einer IgG-dosisabhängigen mIL-2 Sekretion, die im ELISA gemessen werden kann. Die FcgammaR-spezifische immune IgG könnte eine wichtige biologische Rolle in der antiviralen Immunabwehr spielen. Herpesviren exprimieren auf der Oberfläche infizierter Zellen viral-kodierte Fc-bindende Glykoproteine. Um zu bestimmen, ob virale FcgammaRezeptoren die IgG-abhängige Aktivierung von wirtskodierten FcgammaRezeptoren beeinflussen können, wurde das oben beschriebene Assay angewandt. Es wurde festgestellt, dass der HCMV-kodierte FcgammaR gp68 die Aktivierung und die nachfolgende Signalkaskade von CD16>CD32=CD64 inhibiert, während der HCMV-kodierte FcgammaR gp34 die Aktivierung von CD16>CD64>CD32 inhibiert. In klarem Kontrast dazu wirkt der HSV-kodierte FcgammaR gE, der CD16 Aktivierung vermindert, CD32 hingegen nur sehr schwach und CD64 gar nicht beeinflußt. Der MCMV-kodierte FcgammaR m138/fcr-1 vermindert die Aktivierung des murinenCD16. Zusammenfassend betrachtet zeigen die ermittelten Daten, dass es sich bei den herpesviral-kodierten FcgammaRezeptoren um hierarchische und redundante Antagonisten der wirtskodierten zellulären FcgammaRezeptoren handelt. Herpesviral-kodierte FcgammaRezeptoren wirken somit der Aktivierung des Immunsystems entgegen. / To study the possible interference of the herpesviral vFcgammaRs with the host FcgammaRs and IgG-mediated effector functions, a new methodological approach to detect FcgammaR activating antibodies was developed. The novel assay comprises the co-cultivation of virus infected cells upon opsonization with immune IgG antibodies and the stably transfected FcgammaR-zeta BW5147 transfectants as responder cells. The transfectants express chimeric receptors bearing the extracellular domain of the host FcgammaRs fused to the transmembrane and tail domains of the murine CD3zeta chain. Triggering the CD3zeta chain is sufficient to elicit IL-2 secretion in a dose dependent manner which is measured in an ELISA. The setup of the new assay provides a defined effector cell population bearing one Fcgamma receptor on the surface, which becomes activated in the presence of immune IgG antibodies bound to the native viral antigens displayed on the surface of infected cells. The assay system allows us to detect and quantify Fc gamma receptor-activating immune IgG in an FcgammaR-specific way, which is thought to have an important biological function in antiviral defense. Several alpha- and beta- herpesviruses express on the surface of infected cells virally encoded Fc binding glycoproteins. The assay described above was applied to determine if the viral FcgammaRs are able to impair IgG-mediated activation of host FcgammaRs. In a systematic approach, the effect on each host FcgammaR by each of the herpesviral FcgammaR was investigated. It was found that HCMV FcgammaR gp68 affects activation and downstream signaling of CD16 > CD32 = CD64, while gp34 attenuates CD16 > CD64 > CD32. In clear contrast, HSV gE impairs CD16 activation and weakly CD32, but has no effect on CD64. Furthemore, MCMV m138/fcr-1 diminishes activation of mouse CD16. Taken together, this data uncover herpesviral FcgammaRs as hierarchical and redundant antagonists precluding host FcgammaRs from triggering immune responses.

Le virus de l’hépatite B (HBV) est l’agent étiologique de l’hépatite B. Ce virus est responsable d’hépatite chronique B, de cirrhose et de cancer du foie au niveau mondial. L’absence d’activation de la voie Interféron (IFN) suite à l’infection par HBV est encore mal comprise. Récemment, le senseur cellulaire cytosolic GMP-AMP synthase (cGAS) a été décrit comme un senseur efficace de DNA double brin possédant également une activité antivirale envers des virus à ADN et à ARN. Le but de mes travaux de thèse a été de contribuer à la compréhension des relations existants entre le HBV et cGAS, à des stades précoces et tardifs de l’infection HBV en utilisant des expériences de perte- et gain- de function ainsi que du profilage génomique des génes apparentés à cGAS dans un modéle cellulaire permissif au HBV. Mes travaux ont démontré (1) que cGAS exerce une forte activité antivirale envers le HBV incluant une réduction de la forme nucléaire du génome, le cccDNA; (2) alors que le rcDNA génomique nu est reconnu par la voie cGAS/STING et induit une réponse IFN efficace, la nucléocapside virale protège le DNA génomique viral et l’empêche d’être détecté par la réponse immunitaire innée; et (3) que l’infection par HBV diminue l’expression des acteurs de la voie cGAS-STING et des gènes impliqués dans la réponse immunitaire innée in vitro et in vivo. Ce dernier point met en lumière le rôle de cGAS dans un nouveau mécanisme d’échappement du HBV au système immunitaire inné dans les cellules hépatocytaires et dans ce mécanisme. / Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic GMP-AMP synthase (cGAS) was identified as a DNA sensor. In this PhD work, we aimed to investigate the functional role of cGAS in sensing of HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss- and gain-of-function experiments combined with cGAS effector gene expression profiling in an HBV infection-susceptible cell culture model. Collectively, our data show that (1) the cGAS-STING pathway exhibits robust antiviral activity against HBV infection including reduction of viral cccDNA levels; (2) naked HBV genomic rcDNA is sensed in a cGAS-dependent manner whereas packaging of the viral genome during infection abolishes host cell recognition of viral nucleic acids; (3) HBV infection down-regulates the cGAS/STING pathway actors as well as innate immune effector gene expression in vitro and vivo. Overall, this work led to describing new aspects of the complex interaction between HBV and the DNA sensor cGAS in hepatocytes.

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No. of bitstreams: 2 Tese - Andréia de Souza Gonçalves - 2017.pdf: 2952330 bytes, checksum: 1e8c4975cba85c509fcbc41ceea34245 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Study Rationale: Oral potentially malignant disorders (OPMDs) consist of morphologically altered tissues, which present a greater risk of malignant transformation than normal tissue. The most frequently found OPMDs are leukoplakia (OL) and actinic cheilitis (AC). It is now known that mutated or genetically altered cells have developed immunomodulatory strategies which allow them to escape antitumor immune response. The immune escape mechanisms used by mutated cells include the expressions of HLA-G, HLA-E and PD-L1 molecules and the IL-10 and TGF-β cytokines. Objective: To evaluate tissue and salivary expressions of HLA-G, HLA-E and PD-L1 molecules and IL-10 and TGF-β1, -β2 e -β3 cytokines in OPMDs, and relate such immunomodulatory mediators with antitumor immune response and potential for malignant transformation of lesions. Methods: Samples of patients with OL (n= 80) were submitted to immunohistochemistry and ELISA techniques to evaluate tissue and salivary expressions of the HLA-G, HLA-E, PD-L1, IL-10, TGF-β1, TGF-β2 and TGF-β3. In addition, samples of patients with QA (n= 30) were submitted to immunohistochemistry technique to evaluation tissue expression of HLA-G, HLA-E, PD-L1 and IL-10. Control group (n= 20) consisted of saliva and tissue of healthy individuals. The immunostained tissue samples were measured using a semi-quantitative method in association with staining intensity. The expression of these molecules and cytokines were related with the malignant potential of the lesions (epithelial dysplasia grading, proliferation- Ki-67 and apoptosis index- mutated p53). Moreover, the association with the density of granzyme B (GB+) cells and regulatory T cells FOXP3+ was investigated. The statistical tests used were: Fisher’s exact or Pearson Chi-Squared, Mann-Whitney and Kruskal- Wallis tests. The significance level was set at P < 0.05. Results: Fifteen samples showed severe dysplasia, twenty moderate, thirty-two mild and thirteen non-dysplasia. Forty samples (50%) of OL presented combined high Ki-67/p53. Irrespective of the severity of epithelial dysplasia and proliferation/apoptosis index in OL, an overexpression of HLA-G, -E, PD-L1, IL-10, TGF-β2 and -β3 was found in OL when compared with control (P < 0.05). The number of GB+ and FOXP3+ cells in OL was similar to control. Salivary concentration of sHLA-G, IL-10 and TGF-β did not allow distinction between OL patients and healthy individuals (P > 0.05). As regards to AC, we showed that this lesion had an increase in expression of HLA-G, HLA-E, IL-10 and PD-L1 when compared to control; however this increase was statistically significant only for PD-L1 (P= 0.04). Conclusion: The OL showed a reduced cytotoxic immune response (low number of GB+ cells) associated with a high expression of immunomodulatory mediators; however, this expression was independent of epithelial dysplasia grading, proliferation and apoptosis index. Regarding AC we also showed an increase in expression of HLA-G, -E, IL-10 and PD-L1 when compared to the control. Thus, our findings suggest that this lesion present an immunosuppressive microenvironment which favors the escape of mutated keratinocytes in any stage dysplastic, proliferation or apoptosis which this disease finds itself. / Justificativa do estudo: As desordens potencialmente malignas (DPMs) de boca consistem em tecidos morfologicamente alterados, os quais apresentam maior risco de transformação maligna que o tecido normal. Dentre as DPMs de boca, as mais frequentes são a leucoplasia (LE) e a queilite actínica (QA). Atualmente, sabe-se que células mutadas ou geneticamente alteradas são capazes de desenvolver estratégias imunomodulatórias que lhes permitem a evasão à resposta imune antitumoral. Dentre esses mecanismos, pode-se citar a expressão das moléculas HLA-G, HLA-E e PD-L1 e das citocinas IL-10 e TGF-β. Objetivo: Avaliar a expressão tecidual e salivar das moléculas HLA-G, HLA-E e PD-L1 e das citocinas IL-10 e TGF-β1, -β2 e -β3 em DPMs de boca e relacionar tais mediadores imunomodulatórios com a resposta imune antitumoral e com o potencial de transformação maligna dessas lesões. Metodologia: Amostras de pacientes acometidos pela LE (n= 80) foram submetidas às técnicas da imunoistoquímica e ELISA para avaliação da expressão tecidual e salivar, respectivamente, das proteínas supracitadas. Adicionalmente, amostras de pacientes acometidos pela QA (n= 30) foram submetidas à técnica da imunoistoquímica para avaliação da expressão tecidual das proteínas HLA-G, HLA-E, PD-L1 e IL-10. O grupo controle (n= 20) consistiu de tecido e saliva de indivíduos saudáveis. As amostras teciduais imunomarcadas foram mensuradas por um método semi-quantitativo associado à intensidade de marcação. A expressão dos mediadores imunomodulatórios foi associada com o potencial de malignização das lesões (gradação de displasia epitelial, índice de proliferação celular- Ki-67 e apoptose- p53 mutado). Além disso, a associação com a densidade de células granzima B+ (GB+) e células T regulatórias FOXP3+ foi investigada. Os testes estatísticos utilizados foram: teste exato de Fisher ou qui-quadrado de Pearson, Mann-Whitney e Kruskal- Wallis. O nível de significância foi estabelecido em P < 0,05. Resultados: No que se refere a LE, o presente estudo demonstrou que 15 amostras apresentaram displasia severa, 20 moderada, 32 leve e 13 sem displasia. Quarenta amostras (50%) apresentram o combinado alto Ki-67/p53. Independente da severidade da displasia epitelial e do índice de proliferação celular/ apoptose, observou-se uma expressão aumentada de HLA-G, HLA-E, PD-L1, IL-10, TGF-β2 e -β3 quando comporado ao controle (P < 0.05). O número de células GB+ e FOXP3+ nas LE foi similar ao controle. Os níveis salivares de HLA-G solúvel (HLA-Gs), IL-10 e TGF-β não nos possibilitou uma diferenciação entre os pacientes com LE e indivíduos saudáveis (P > 0,05). Em relação aos resultados obtidos para QA, evidenciou-se que essa lesão teve um aumento na expressão de HLA-G, HLA-E, IL-10 e PD-L1 em comparação ao controle, todavia esse aumento só foi estatisticamente significativo para o PD-L1 (P= 0,04). Conclusão: As LEs apresentaram uma reduzida resposta imunológica citotóxica (baixo número de células GB+) associada a uma elevada expressão de mediadores imunomodulatórios; todavia essa expressão não possui relação com a gradação de displasia epitelial e índice de proliferação celular e apoptose. Em relação à QA, nós também evidenciamos uma maior expressão de HLA-G, HLA-E, IL-10 e PD-L1 se comparado ao controle. Desta forma, nossos achados sugerem que tais lesões apresentam um microambiente imunossupressor que favorece a evasão de queratinócitos mutados em qualquer estágio de alteração displásica, proliferação ou apoptose que essa doença se encontre.

Le domaine TIR (Toll/interleukin (IL)-1 receptor) est une composante essentielle du système immunitaire inné, celui-ci est présent dans les récepteurs TLR (Toll-like receptor) et les protéines adaptatrices associées comme MyD88 et TIRAP. La détection de pathogènes déclenche l'interaction entre les domaines TIR permettant ainsi l'initiation et la propagation de la signalisation par les TLRs. Aussi, de nombreux pathogènes produisent des effecteurs contenant un domaine TIR tels que BtpA et BtpB chez Brucella abortus, TirS chez Staphylococcus aureus ou TcpC chez l'uropathogènique Escherichia coli. Tous ces effecteurs bloquent la signalisation des TLRs et sont capables de perturber les voies de signalisation de l'immunité innée pendant l'infection. Cependant les mécanismes moléculaires impliqués restent la plupart du temps non caractérisés et dans certains cas controversés. Dans le but de mieux comprendre le fonctionnement de ce type d'effecteurs bactériens, j'ai caractérisé chez Pseudomonas aeruginosa PA7 un nouvel effecteur contenant un domaine TIR que nous avons renommé PumA pour Pseudomonas UBAP1 Modulator A. En parallele, j'ai aussi participé à des projets de caractérisation de deux autres effecteurs avec un TIR domain : BtpB et TirS. Ainsi, PumA est un facteur essentiel pour la virulence de P. aeruginosa PA7 et son domaine TIR est essentiel pour interaction avec deux protéines adaptatrice, TIRAP et MyD88. Durant l'infection de cellules épithéliales pulmonaires par P. aeruginosa PA7, PumA est responsable du contrôle de la translocation du facteur de transcription NF-κB dans le noyau. De plus, la production de PumA dans une souche de P. aeruginosa non-TIR confère à cette bactérie de nouvelles propriétés d'immuno-modulation. PumA cible aussi UBAP1, une protéine du complexe de tri endosomal requis pour le transport, ESCRT-I (endosomal sorting complex require for transport I) qui a été récemment montré pour moduler l'activation de récepteur de cytokine. Nos résultats montrent que UBAP1 peut s'associer avec TIRAP et MyD88, provoquant le mouvement de MyD88 à la membrane cytoplasmique, suggérant une nouvelle voie cellulaire commune entre UBAP1 et les TLRs, et révélant UBAP1 comme nouvelle cible pour des effecteurs bactériens dans le cadre du contrôle des réponses immunitaires de l'hôte / In higher eukaryotes, the innate immune system provides the first line of defense against invading pathogens. The Toll/interleukin-1 receptor (TIR) domain is an essential component of the innate immune system. This domain is present in Toll-like receptors (TLRs) and associated adaptor proteins such as MyD88 and TIRAP. Pathogen detection requires interaction between the TIR domains, which initiates and triggers propagation of TLR signaling. However, many pathogens produce a TIR domain-containing protein such as BtpA and BtpB in Brucella abortus, TirS in Staphylococcus aureus or TcpC in the uropathogenic strain Escherichia coli. These effectors block TLR signaling and are able to disrupt innate immune response during infection. However, the molecular mechanisms involved remain mostly uncharacterized and in some cases controversial. The objective of this thesis was to study bacterial effectors containing a TIR domain particularly at the molecular level. For this, we focused on Pseudomonas aeruginosa PA7, an atypical multi-drug resistant strain that contains an effector with a TIR domain that we named PumA, for Pseudomonas UBAP1 Modulator A. In addition, during these four years of thesis work I also participated in the characterization of two other effectors with a TIR domain: BtpB in B. abortus and TirS in S. aureus.We found that PumA is essential for virulence of P. aeruginosa PA7 and its TIR domain is the key element for interaction with two adaptor proteins MyD88 and TIRAP. During infection of lung epithelial cells by P. aeruginosa PA7, PumA is responsible for controlling the translocation of NF-?B into the nucleus indicative of activation of this transcription factor. In addition, production of PumA by a TIR-deficient strain of P. aeruginosa confers to this bacterium a new immuno-modulation property. Furthermore, PumA targets ubiquitin-associated protein 1 (UBAP1), a protein of the endosomal sorting complex required for transport I (ESCRT-I) which has recently been shown to modulate cytokine receptor activation. Our results also show that UBAP1 can associated with TIRAP and MyD88, causing movement of MyD88 to the cytoplasmic membrane and suggesting a new cellular pathway between UBAP1 and TLRs. In summary, our data reveal UBAP1 as a novel target for bacterial effectors implicated in control of host immune responses

Mon projet a porté sur la caractérisation du mécanisme moléculaire des enzymes d'arenavirus (une 3'-5' exoribonucléase et une endonuclease) impliquées dans l'inhibition de la réponse innée IFN de type I et dans le vole de coiffe respectivement, et le développement d'une stratégie thérapeutique basée sur leur structures. Premièrement, j'ai résolu deux structures cristallographiques à haute résolution du domaine exoribonucléases du virus Mopeia (NP-exo MOPV) -un homologue du virus Lassa pathogène- en complexe avec deux ions différents. Ensuite, j'ai effectué une caractérisation fonctionnelle de l’activité exoribonucléase 3'-5' codée par ce domaine. Une corrélation entre la structure et la fonction de NP-exo MOPV démontre que; L’activité exoribonucléase 3'-5' est conservée chez les arenavirus pathogènes ainsi que chez les non-pathogènes. J'ai démontré pour la première fois que l'exoribonucléase est capable d'exciser un ARN misapparié, suggérant ainsi une potentielle activité de correction d'erreur par cette enzyme. Avec la structure de NP-exo MOPV, j'ai développé une stratégie in silico pour identifier des inhibiteurs potentiels spécifiques contre son activité et un inhibiteur a était identifié.En parallèle, nous avons résolu deux structures cristallographiques du domaine de l'endonuclease du virus de la LCMV en complexe avec deux ions catalytiques et deux composés appartenant a la famille des diketo. En résumé, ce travail éclaircit le rôle des exoribonucléases de la famille d'Arenaviridae allant de l’évasion de l'immunité innée à son implication directe dans la réplication. Il ouvre également la voie au développement des inhibiteurs contre ces nucléases. / My PhD work focused on the characterization of the molecular mechanism of two arenavirus enzymes - a 3'-5' exoribonuclease and an endonuclease - implicated in type I IFN suppression and mRNA cap-snatching respectively and the design of a structure based-drug strategy against them. First I solved two high resolution crystal structures of the exoribonuclease domain of Mopeia virus (NP-exo MOPV) -a non pathogenic homologue of the highly pathogenic Lassa virus- in complex with different metal ions. Next I performed an in depth functional characterization of the 3'-5' exoribonuclease activity encoded by this domain. By correlating the structure and function of NP-exo MOPV, I showed that; the 3'-5' exoribonuclease activity is conserved in pathogenic as well as in non-pathogenic arenaviruses. Also, I showed for the first time that this enzyme is able to excise a mismatched RNA suggesting that, arenaviruses might posses a mechanism to limit error incorporation by the RdR polymerase during replication. Using the crystal structure of NP-exo MOPV I designed a structure-based strategy to identify potential inhibitors specific for the 3'-5' exoribonuclease activity and have identified a potential inhibitor.Alongside, we solved two crystal structures of the endonuclease domain of LCMV in complex with two catalytic ions and two compounds belonging to the diketo family.In conclusion, this work has a deep implication extending the role of the Arenaviridae exoribonuclease from innate immunity evasion to direct implication in replication. It also paves the way for the development of inhibitors against these arenavirus nucleases.

Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Protective immunity against pathogenic mycobacteria depends principally on cell-mediated immunity executed by efficient anti-infectious functions of type 1 T helper (Th1) subset of CD4+ T cells. The polarization of Th1 responses is orchestrated by IL-12 secreted by antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs). A hallmark of Th1 type CD4+ T cells is the production of IFN-γ that activates plethora of innate cell-mediated immunity. It is well known that cytokines such as IFN-γ, IL-12 and TNF-α are required for control of mycobacterial infection in humans as well as in mice. However, it remains unclear that why the immune response controls mycobacteria, but does not eradicate infection suggesting critical roles for series of survival strategies employed by pathogenic mycobacteria. In general, these evasion strategies include blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, induced secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. that ultimately suppress the secretion of IL-12 and IFN-γ from APCs and T cells respectively, culminating in a skewed Th1/Th2 balance towards unprotective Th2 responses. Th2 cells secrete IL-4, IL-5, IL-9, IL-10 and IL-13 but are deficient in clearing intracellular infections including pathogenic mycobacteria. This eventually leads to inhibition of host’s immuno-protective responses with concomitant increase in the vulnerability to chronic mycobacterial infection. In this intricate process, modulation of cyclooxygenase-2 (COX-2) levels, a key enzyme catalyzing the rate-limiting step in the inducible production of prostaglandin E2 (PGE2), by mycobacteria like Mycobacterium bovis BCG assumes critical importance in influencing the overall host immune response. PGE2, an immunosuppressive member of prostaglandin family, is known to restrain production of IL-12, as well as reactive oxygen intermediates. PGE2-mediated inhibition of IL-12R, diminishes IL-12 responsiveness of macrophages and dendritic cells. PGE2 also inhibits the secretion of IFN-γ, which is important in activating T cells and macrophages. In contrast, PGE2 promotes IL-10 production by macrophages, dendritic cells and Th1-to-Th2 shift of acquired immune responses by inhibiting IL-2 and enhancing IL-4 production. Albeit, mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways are generally believed to be involved, little is known about the signaling molecules playing significant roles upstream of MAPK and NF-κB pathways during mycobacteria triggered COX-2 expression. Further, information on early receptor proximal signaling mechanisms essential during mycobacteria mediated induction of COX-2 remains scanty. In this regard, signaling cascade triggered upon recognition of mycobacterial components by pattern recognition receptors (PRR) signify as critical event in overall regulation of cell fate decisions. PRR like Toll like receptor (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2) are two nonredundant recognition mechanisms of pathogenic mycobacteria. Several components of mycobacteria have been identified as being responsible for TLR2-dependent activation including 19-kDa lipoprotein, lipomannan etc.; while NOD2 recognizes mycobacterial peptidoglycans through its interaction with muramyl dipeptide (MDP). Interestingly, although mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), trehalose 6,6′-dimycolate (TDM; cord factor), PE/PPE family proteins etc., are released and traffic out of the mycobacterial phagosome platform into endocytic compartments. Importantly, these antigens could gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represents a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. Further, PIM2 is a known TLR2 agonist and reported to activate NF-κB, AP-1, and MAPK suggesting that mycobacterial envelope antigen PIM2 could modulate the inflammatory responses similar to mycobacteria bacilli. In this context, we explored the signaling events modulated by M. bovis BCG, and role for TLR2 and NOD2 in this intricate process, to trigger the expression of COX-2 in macrophages. Our studies demonstrated that M. bovis BCG triggered TLR2-dependent signaling leads to COX-2 expression and PGE2 secretion in vitro in macrophages and in vivo in mice. Further, the presence of PGE2 could be demonstrated in sera or CSF of tuberculosis patients. Similarly, mycobacterial TLR2 agonist PIM2 and NOD2 ligand MDP triggered COX-2 expression in macrophages. The induced COX-2 expression in macrophages either by M. bovis BCG or PIM2 or MDP was dependent on NF-κB activation, which was in turn mediated by iNOS/NO and Wnt-β-Catenin dependent participation of the members of Notch1-PI3K signaling cascade. Importantly, loss of iNOS activity either in iNOS null macrophages or by pharmacological intervention in wild type macrophages severely abrogated M. bovis BCG ability to trigger the generation of Notch1 intracellular domain (NICD) as well as activation of PI3K signaling cascade. On contrary, treatment of macrophages with SIN-1, an NO donor, resulted in a rapid increase in generation of NICD, activation of PI3K pathway as well as the expression of COX-2. Interestingly, pharmacological inhibition as well as siRNA mediated knockdown of Wnt-β-Catenin signaling compromised ability of M. bovis BCG to induce activation of Notch1-PI3K signaling and drive COX-2 expression. Concomitantly, activation of Wnt-β-Catenin signaling by LiCl triggered activation of Notch1 and PI3K pathway as well as COX-2 expression. Stable expression of NICD in RAW 264.7 macrophages resulted in augmented expression of COX-2. Further, signaling perturbation experiments suggested involvement of the cross-talk of Notch1 with PI3K signaling cascade. In this perspective, we propose TLR2 and NOD2 as two major receptors involved in mycobacteria mediated activation of Notch1PI3K signaling, and the activation of iNOS/NO and Wnt-β-Catenin signaling axis as obligatory early receptor proximal signaling events during mycobacteria induced COX-2 expression in macrophages. Functional characterization of mycobacterial antigens that are potent modulators of host immune responses to pathogens by virtue of induced expression of COX-2 assumes critical importance for deciphering pathogenesis of mycobacterial diseases as well as to identify novel therapeutic targets to combat the disease. In this context, a group of novel antigens carried by M. tuberculosis that are expressed upon infection of macrophages belong to PE and PPE family of proteins. Ten percent of the coding capacity of M. tuberculosis genome is devoted to the PE and PPE gene family members, exemplified by the presence of Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many members of the PE family exhibit multiple copies of polymorphic guanine-cytosine– rich sequences (PGRS) at the C-terminal end, which are designated as the PE_PGRS family of proteins. A number of PE/PPE proteins associate with the cell wall and are known to induce strong T & B cell responses in humans. However information related to effects of PE/PPE antigens on the maturation and functions of human dendritic cells and eventual modulation of T cell responses as well as underlying signaling events remains obscure. Our results demonstrated that two cell wall associated/secretory PE_PGRS proteins PE_PGRS 17, PE_PGRS 11 and PPE family protein PPE 34 recognize TLR2, induce maturation and activation of human dendritic cells and enhance the ability of dendritic cells to stimulate CD4+ T cells. In addition, tuberculosis patients were found to have a high frequency of T cells specific to PE_PGRS and PPE antigens. We further found that PE/PPE proteins-mediated activation of dendritic cells involves participation of ERK1/2, p38 MAPK and NF-κB signaling pathways. While, PE_PGRS antigens-matured dendritic cells secreted high amounts of inflammatory cytokine IL-12, PPE 34 triggered maturation of dendritic cells was associated with secretion of high amounts of anti-inflammatory cytokine IL-10 but not the Th1-polarizing cytokine IL-12. Consistent with these results, PPE 34-matured dendritic cells favored secretion of IL-4, IL-5 and IL-10 from CD4+ T cells and contributed to Th2 skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, PPE 34-skewed Th2 immune response involved induced expression of COX-2 in dendritic cells. Our results suggest that by inducing differential maturation and activation of human dendritic cells, PE/PPE proteins could potentially modulate the initiation of host immune responses against mycobacteria. Taken together, our observations clearly signify the potential role for TLR2 and NOD2 triggering by M. bovis BCG in activating receptor proximal Notch1-PI3K signaling during induced COX-2/PGE2 expression which represents a crucial immune subversion mechanism employed by mycobacteria in order to suppress or attenuate host immune responses. Further, differential maturation of human dendritic cells by PE_PGRS and PPE antigens as well as their ability to stimulate CD4+ T cells towards Th1 and Th2 phenotype respectively, improves our understanding about host-mycobacteria interactions and clearly paves a way towards the development of novel combinatorial therapeutics.