Legionella pneumophila est une bactérie aquatique qui prolifère en se répliquant à l’intérieur de cellules amibiennes. Elle peut persister dans ces environnements en vivant en communauté sous forme de biofilms. L’inhalation par l’Homme d’eau contaminée, vaporisée par les réseaux d’eau chaude ou les tours aéro‐réfrigérantes, peut mener à l’infection des macrophages pulmonaires qui se traduit par une grave pneumonie appelée légionellose. Le di‐GMP cyclique (diGMPc) est impliqué, chez diverses espèces bactériennes, dans la transition entre les modes de vie mobiles et sessiles, et chez certains pathogènes, dans la régulation de la virulence. Mon travail de thèse vise à démontrer l’implication des voies de signalisation à diGMPc dans le contrôle de la virulence et de la formation de biofilms par L. pneumophila. Cette implication a été étudiée grâce à l’inactivation systématique de chacun des gènes codant les protéines du métabolisme du diGMPc chez la souche L. pneumophila Lens. Notre étude a révélé que trois de ces protéines, Lpl0780, Lpl0922 et Lpl1118, sont spécifiquement requises pour le contrôle de la virulence et, plus particulièrement, pour la survie précoce lors de l’infection de cellules‐hôtes via l’orchestration de la sécrétion de facteurs de virulence dans la cellule‐hôte. Lpl1118 participerait également à la biogénèse de la vacuole de réplication. Cinq autres de ces protéines sont impliquées dans la régulation de la formation et de l’architecture des biofilms. L’une d’elles est, plus particulièrement, requise pour la formation de biofilms en présence d’oxyde nitrique. Ces résultats contribuent à une meilleure compréhension de l’organisation complexe et spécifique des voies de signalisation à diGMPc chez L. pneumophila et pourraient permettre d’envisager une lutte plus efficace contre ce pathogène / Legionella pneumophila is a bacterium that proliferates in fresh water environments through the replication within amoebas. These bacteria can persist in these environments through biofilm formation. The inhalation of aerosolized contaminated water through hot water systems or cooling towers can induce the infection of human lungs, leading to a severe pneumonia called legionellosis. Cyclic di‐GMP (c‐di‐GMP) in involved, in various bacterial species, in the motility‐to‐sessility transition, and in some pathogens, in virulence control. My work aims to demonstrate the involvement of signaling pathways that use c‐di‐GMP in virulence control and biofilm formation of L. pneumophila. This involvement was investigated by systematically inactivating each gene encoding a c‐di‐GMP‐metabolizing enzyme in L. pneumophila Lens strain. Our work revealed that 3 of these proteins, Lpl0780, Lpl0922 and Lpl1118 are specifically involved in virulence control and, particularly, in the early survival during host cell infection through the orchestration of virulence factors secretion within host cell. Lpl1118 is particularly required for replicative vacuole biogenesis. Five other proteins, participate in the formation and architecture of biofilms. One of them is more specifically involved in biofilm formation in the presence of nitric oxide. These results help to better understand the complexity and the specificity of c‐di‐GMP signaling pathways in L. pneumophila and should allow the exploration of more effective ways to fight this pathogen

La leptospirose est une zoonose causée par les bactéries du genre Leptospira. Elle touche près de 1 million d'individus par an dans le monde entier et sévit à l'état endémique dans les pays au climat tropical tel que La Réunion. Les manifestations habituelles sont variables d'un individu à l'autre et englobent une simple fièvre jusqu'aux défaillances poly-viscérales avec mortalité dans 5 à 10% des cas. Sa physiopathologie est encore mal comprise, en particulier la part que joue une réponse immunitaire inappropriée dans la genèse des manifestations graves qui surviennent en quelques heures, et avant la mise en place d'une réponse immunitaire adaptative propre à éliminer le microorganisme. Si l'échappement de la bactérie au système du complément est bien documenté, le rôle des acteurs cellulaires du système immunitaire inné reste à étayer. Notre étude avait donc pour objectif d'explorer l'immunopathologie de la leptospirose humaine dans la phase initiale de l’infection. Notre méthodologie s'est appuyée principalement sur des analyses quantitatives et qualitatives des acteurs cellulaires du système immunitaire inné à partir de prélèvements sanguins en phase précoce de la maladie, et comparaison avec la phase de convalescence et des sujets contrôles. Dans un premier temps nous avons montré qu'une population particulière de lymphocytes T impliquée dans la réponse immune innée, les lymphocytes Tγδ, avaient un taux abaissé et que cette baisse était corrélée à la charge bactérienne ainsi qu'à l’intensité de l'atteinte hépatique classiquement retrouvée lors de la leptospirose. Dans un deuxième temps, nous avons analysé les polynucléaires neutrophiles circulants dont le taux augmente d’autant plus que la maladie est sévère, mais sans pour autant présenter de modification de certains marqueurs d’activation ou de recrutement tissulaire (CD15, CD11b, CD182). Une des principales chimiokines des neutrophiles, l'interleukine 8, était à taux peu élevés. Les derniers travaux concernent les principales formes solubles issues des molécules membranaires impliquées dans le processus de recrutement/diapédèse leucocytaire. Nous retrouvons de manière isolée une forte élévation des formes solubles d'E-sélectine et ICAM-1 qui sont notamment exprimées par les cellules endothéliales. Ces augmentations n'étaient pas corrélées aux marqueurs de gravité de la maladie. La signification biologique de cette élévation n’est pas encore connue lors de la leptospirose. L'ensemble de nos données permet d’apporter des informations nouvelles sur des acteurs du système immunitaire inné présents dans le compartiment vasculaire lors de la leptospirose humaine. Cette réponse immunitaire semble inadaptée pour permettre une clairance du pathogène au stade de dissémination hématogène. / Leptospirosis is a bacterial zoonosis caused by Leptospira and affecting 1 million people each year worldwide and mainly in tropical areas such as Reunion Island. Usual presentations encompass flu-like syndrome to multiorgan failure with mortality rate between 5 to 10%. To date, pathophysiology in humans is poorly understood, notably the capacity of innateimmunity to mount a robust response to clear pathogen or to induce tissue damages and contributing to disease severity. Our study aimed at assessing the role of innate immune cells and molecules within the first days of leptospiral infection.Using blood samples, we performed quantitative and qualitative assessment of circulating innate immune cells from leptospirosis cases and healthy controls. The first study explored the levels of gamma-delta T-cells (γδT-cells), a subset of unconventional T cells with innate immune functions. Gamma-delta T cells were found deeply decreased and levels wereinversely correlated to bacterial burden and liver damage. The second study focused on membrane bound receptors indicative of activation and tissue migration ability of neutrophil polymorphonuclear cells: CD15, CD11b, and CD182. Although neutrophil rates were high in leptospirosis cases, the levels of studied receptors were either lower (CD15) or identical to healthy controls (CD11b, CD182). In addition, only low levels of interleukin-8, a key chemokine for neutrophils, was detected in patients. Lastly, we ascertained the plasmatic levels of several shed cell adhesion molecules notably expressed by endothelial cells. The levels of soluble E-selectin and ICAM-1 were significantly increased compared to controls, while P-selectin level was lower. We did not find any correlation with disease severity or organ failure. This finding indicates that endothelial cell may be activated but further experiments are warranted to explain the functional impact of our findings. Altogether, our results add to the field of knowledge of leptospirosis pathophysiology, and in particular the implication of key innate immune cells at the stage of plasmatic bacterial dissemination. Our findings will support the view that there is an inappropriate immune response to Leptospira.

Legionella pneumophila est une bactérie opportuniste qui émerge de l'environnement après multiplication dans des amibes et peut infecter accidentellement les macrophages alvéolaires humains, provoquant une pneumonie sévère, la légionellose. La capacité de L. pneumophila à survivre dans ses cellules hôtes est strictement dépendante du système de sécrétion de type 4 Dot/Icm, qui sécrète un large répertoire d'effecteurs dans le cytosol de l'hôte. Identifier la contribution individuelle de chaque protéine bactérienne sécrétée par le système Dot/Icm, dans le cycle infectieux de L. pneumophila reste un enjeu majeur pour comprendre les bases moléculaires de la virulence des légionelles. Mes travaux de thèse participent à cet objectif en caractérisant la voie cellulaire ciblée par la protéine kinase LegK2. Des tests d'interaction et de phosphorylation ont identifié le complexe nucléateur d'actine ARP2/3 comme cible de LegK2. Suite à l'adressage de LegK2 à la surface de la vacuole après sa translocation dans le cytosol de l'hôte, l'interaction LegK2-ARP2/3 inhibe la polymérisation d'actine sur le phagosome. Cette inhibition permet à Legionella de diminuer le trafic des endosomes tardifs et/ou des lysosomes vers le phagosome et favorise ainsi l'évasion du phagosome à la voie de dégradation endocytique. L'interaction LegK2-ARP2/3 met en évidence un mécanisme original de virulence dans lequel le remodelage local du cytosquelette d'actine de la cellule hôte permet à la bactérie de manipuler le trafic vésiculaire pour échapper aux défenses de l'hôte / Legionella pneumophila is an opportunistic bacterium that emerges from the environment after multiplication in protozoans and can accidentally infect human alveolar macrophages leading to a severe pneumonia, the legionellosis. The L. pneumophila ability to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Deciphering the individual contribution of each bacterial protein translocated by the Dot/Icm system in the L. pneumophila infectious cycle remains a major challenge to understand the molecular basis of Legionella virulence. My works contribute to this objective by characterizing the cellular pathway targeted by the protein kinase LegK2. Interaction and phosphorylation assays identified the actin nucleator ARP2/3 complex as the target of LegK2. Following the LegK2 addressing to the vacuole surface after its translocation into host cytosol, LegK2- ARP2/3 interplay inhibits the actin polymerization on the phagosome. This inhibition allows Legionella to decrease the late endosome/lysosome trafficking towards the phagosome and promotes the phagosome evasion from endocytic degradation pathway. LegK2-ARP2/3 interplay highlights an original mechanism of virulence wherein the local actin cytoskeleton remodeling of host cell allows bacteria to hijack the vesicles trafficking in order to escape host-cell defenses

Le champignon Fusarium graminearum est l'un des principaux agents responsables de la fusariose des épis, une maladie nécrosante des céréales associée à une contamination des grains et des aliments par des mycotoxines. De récentes observations suggèrent une évolution de l’agressivité des populations de ce pathogène, questionnant l’efficacité et la durabilité des moyens de luttes actuels. Mieux anticiper cette évolution nécessite une meilleure caractérisation de la diversité phénotypique et génotypique existante entre souches. Six nouveaux génomes de F. graminearum ont été séquencés et ont permis l’identification et la caractérisation de 243 000 variations génétiques. La majorité de ces variants (77%) est concentrée dans des îlots de polymorphisme, représentant 32% du génome et enrichis en probables effecteurs liés à la pathogénicité de F. graminearum. La construction d’une population recombinante, et son génotypage avec 1 300 marqueurs moléculaires, ont permis le développement de la première carte génétique à haute-densité de l’espèce. La corrélation entre le taux de recombinaison et le polymorphisme a mis en évidence une organisation « à deux-vitesses » du génome de cette espèce. Finalement, l’intégration de ces données dans une approche de génétique quantitative a permis l’identification d’un locus polymorphe, affectant le gène FgVeA, et responsable de 90% de la variation d’agressivité et de la production de mycotoxine observée. Les différents résultats obtenus durant ces travaux font l’objet d’une discussion générale sur le potentiel adaptatif et d’évolution de ce pathogène. / F. graminearum is one of the main causal agents of the fusarium head-blight (FHB), a cereal disease leading to head necrosis, in addition to grain and food/feed contamination by stable and toxic metabolites. Recent observations refer to an increase of pathogenicity, questioning efficiency and durability of current management practices. In order to anticipate this evolution, we must bring a deeper characterization of the currently existing diversity. Six new genomes of F. graminearum were sequenced, and 243,000 genetic variations have been identified and characterized. Seventy seven percent of the total number of the variants was located within 32% of the genome, delineating highly polymorphic islands. These islands are enriched with probable effectors linked to Fusarium’s pathogenicity. The construction and the genotyping on 1,300 molecular markers of a recombinant population have enabled the development of the first high-density genetic map of the species. The remarkable correlation between polymorphism and recombination rate highlighted the 'two-speed' genome organization of this pathogen. Finally, the integration of these data through a quantitative genetic approach allowed the discovery of one quantitative trait locus, likely to affect the gene FgVeA, and responsible for 90% of the observed variation of aggressiveness and mycotoxin production. These results are discussed in the light of F. graminearum’s adaptive potential and evolution.

De nombreuses bacteries pathogènes sont capables d'affecter les programmes transcriptionnels de la cellule hôte pendant l'infection. Cependant, les mécanismes contrôlant ce processus restent largement méconnus. En investigant les effets de la Listerai monocytogenes sur les modifications des histones de l'hôte, nous avons mis en évidence un nouveau mecanisme de régulation de transcription nécessaire pour la répression de certains gènes, pendant l'infection. Lors de l'infection par L. monocytogenes, le facteur de virulence sécrété, InlB, se lie au récepteur c-Met et active la signalisation par les intermédiaires PI3K et Akt. cette plateforme de signalisation est nécessaire pour la relocalisation de la deacetylase d'histone, SIRT2, au noyau et l'association à la chromatine.En caractérisant me mécanisme gouvernant la relocalisation nucléaire de SIRT2 lors de l'infection, nous avons démontrés que SIRT2 subit une modification post-traductionnelle. SIRT2 est déphosphorylée à un nouveau site de phosphorylation localisé à la partie terminale de la protéine. SIRT2 est recrutée au site de démarrage de la transcription des gènes réprimés lors de l'infection menant à la deacetylation de H3K18 et la répression transcriptionnelle. Nous avons mis en évidence que SIRT2 est détournée par L. monocytogenes et provoque une croissance des bactéries intracellulaires. Ces résultats démontrent un clef de SIRT2 en provoquant la deacetylation de H3K18 mors de l'infection et dévoilent un nouveau mécanisme imposée par les bactéries pathogènes dans le but de reprogrammer la cellule hôte. / Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.

<p>Two species of <i>Neisseria</i>, <i>N. meningitidis</i> and <i>N. gonorrhoeae</i>, are obligate human pathogens that cause meningitis and gonorrhea, respectively. Although generally asymptomatic, <i>N. meningitidis</i> can cause invasive meningococcal disease with high mortality rate. Due to emerging antibiotic resistance strains of <i>N. gonorrhoeae</i>, the Centers for Disease Control and Prevention (CDC) have designated it as an urgent threat to public health. Therefore, immediate interventions are required for fight against these Neisserial pathogens. Iron is an essential nutrient for all bacteria, including <i>Neisseria</i>. However, free iron is scarce in human, therefore, <i>Neisseria</i> have evolved to acquire iron from host proteins. These iron acquisition systems are immunogenic and important for infection and are promising therapeutic targets.</p> <p> In the host, lactoferrin sequesters free iron and limits iron availability to pathogens. However, <i>Neisseria</i> have evolved machinery to hijack iron directly from lactoferrin itself. Lactoferrin binding proteins, LbpA and LbpB, are outer membrane proteins that together orchestrate the acquisition of iron from lactoferrin. Additionally, LbpB serves an additional role in providing protection against host cationic antimicrobial peptides and innate immune response. Despite studies aimed at deciphering the roles of LbpA and LbpB, the molecular mechanisms underpinning iron acquisition and immune protection remain unknown. Here, we investigated the role of the lactoferrin binding proteins in iron acquisition and protection against cationic antimicrobial peptides. We obtained three-dimensional structures of <i>Neisseria</i> LbpA and LbpB in complex with lactoferrin using cryo-electron microscopy and X-ray crystallography. These structures show that both LbpA and LbpB bind to C-lobe of lactoferrin, albeit at distinct sites. Structural analyses show that while lactoferrin maintains its iron-bound closed conformation in the LbpB-lactoferrin complex, it undergoes a large conformational change from an iron-bound closed to an iron-free open conformation upon binding to LbpA. This observation suggest that LbpA alone can trigger the extraction of iron from lactoferrin. Our studies also provide an explanation for LbpB’s preference towards holo-lactoferrin over apo-lactoferrin and LbpA’s inability to distinguish between holo- and apo-lactoferrin. Furthermore, using mutagenesis and binding studies, we show that anionic loops in the C-lobe of LbpB contribute to binding the cationic antimicrobial peptide lactoferricin. Solution scattering studies of the LbpB-lactoferricin complex showed that LbpB undergoes a small conformational change upon peptide binding.</p> Together, our studies provide structural insights into the role of the lactoferrin binding proteins in iron acquisition and evasion of the host immune defenses. Moreover, this work lays the foundation for structure-based design of therapeutics against <i>Neisseria</i> targeting the lactoferrin binding proteins.

Adaptive evolution of Toll-like receptors in birds Hana Velová, PhD thesis 6 Abstract Toll-like receptors (TLRs) are one of the key and presumably also evolutionary most original components of animal immune system. As Pattern recognition receptors they form the first line of innate immune defence against various pathogens. The proper receptor binding of pathogenic ligands is crucial for their correct recognition and for subsequent triggering of an appropriate immune response. Because there exists a direct interaction between the receptor surface and the pathogenic ligand, host-pathogen coevolution on molecular level can be predicted. Thus, through variability of their ligands, TLRs are exposed to extensive selective pressures that may be detected on both genetic and protein levels. Surprisingly, the variability we revealed in birds is even higher than previously expected based on the reports from other vertebrates, mainly mammals. In my doctoral thesis I summarise the results of my contribution to the avian TLR research. We were the first who experimentally verify the absence of functional TLR5 in several avian species and duplication of TLR7 in others. We finally resolved the origin of duplication in TLR1 and in TLR2 family. An important part of my research project focused on the prediction of potentially...

The Gram-negative aerobic coccobacillus Bordetella pertussis is one of the few exclusively human pathogens and the main causative agent of the respiratory infectious disease called pertussis, or whooping cough. Despite global vaccination programs, pertussis remains an important public-health burden and still accounts for over 100,000 infant deaths and over a dozen of millions of whooping cough cases every year. Substantial effort is devoted to studies on the mechanisms of action of virulence factors of B. pertussis, but the biology of interactions of B. pertussis with its human host remains largely underexplored. Evolution, genetics and adaptation of B. pertussis to the complex environment of human nasopharynx and the mechanisms enabling B. pertussis to overcome host innate and adaptive mucosal immune defenses, remain poorly understood. In such situations, unbiased exploratory omics approaches represent valuable tools for uncovering of unknown aspects of host-pathogen interactions and open the path to detailed analysis of virulence-underlying processes by mechanistic studies. In this thesis, I am presenting the results of three omics projects on B. pertussis biology that involved high-throughput proteomics. In the inital phosphoprotemics project, we analyzed the kinase signaling pathways hijacked...

Interferon-inducible PYHIN protein family includes the DNA-binding proteins, AIM2 and IFI16, which form ASC-caspase 1 dependent inflammasomes, important in immunity against cytosolic bacteria, DNA viruses and HIV. The role of other members of this family in the recognition of DNA and/or regulation of immune responses is unclear. We identified an immune regulatory function of p205, another member of the PYHIN family, in the transcriptional control of immune genes. Knockdown of p205 in macrophages revealed that inflammasome activation due to dsDNA and ligands that engage the NLRP3 inflammasome were severely compromised. Detailed mechanistic analysis showed that loss of p205 was associated with a decrease in Asc mRNA and protein levels. p205 knockdown resulted in reduced RNA Polymerase II-mediated endogenous Asc gene transcription and mRNA processing, suggesting a co-transcriptional control of Asc gene expression. Ectopically expressed p205 induced expression of an Asc gene-luciferase reporter and collaborated with other transcription factors, such as c/EBPβ, p65/RelA, to further enhance expression. p205 knockdown also affected the expression of the immune genes Cd86, Cox2, Cxcl2, Il1α, Il10, Il12α, Il6 and Ifnα in LPS-stimulated macrophages. Together these findings suggest that p205 regulates inflammation through control of Asc gene expression, and other immune genes. Fungal infections activate both caspase 1-dependent and -independent inflammasomes. In an independent study, we show that Paracoccidioides brasiliensis fungal infection also induces caspase 8-dependent non-canonical inflammasome. Caspase 8-dependent IL-1β processing required dectin-1, Syk and Asc. Rip3-/- Casp8-/- mice infected with P. brasiliensis displayed increased fungal load and showed worse disease progression compared to wild type and Rip3-/- mice. These results revealed the importance of caspase 8 in activating and regulating inflammasome responses during fungal infection in vivo.

<p dir="ltr">Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), and human immunodeficiency virus-1 (HIV) are major public health concerns, individually and in combination. The status of the host immune system, previous <i>Mtb</i> infection and HIV-mediated T cell exhaustion, can have significant impacts on immune dynamics during reinfection. Individuals with asymptomatic latent TB infection (LTBI) may be protected against <i>Mtb </i>reinfection, as demonstrated by animal and <i>in vitro </i>studies. However, the underlying dynamics and protective mechanisms of LTBI are poorly understood. In HIV, long-term infection in children and associated T cell exhaustion leads to weakened immune responses to HIV reinfection. The complexity of these infections, particularly in the context of the heightened vulnerability of HIV+ individuals to TB, underscores the need for novel investigative approaches to study host-pathogen and pathogen-pathogen interactions. To this, we have developed an agent-based model (ABM) as a mechanistic computational tool to simulate the immune response to <i>Mtb </i>and HIV, separately and during coinfection. Our ABM integrates clinical and experimental data; simulates immune cell dynamics between macrophages, CD4+ and CD8+ T cells; and produces emergent granuloma-like structures – a critical response to <i>Mtb</i>. This <i>in silico</i> approach allows us to efficiently explore host-pathogen interactions and their clinical implications. By unraveling the complex interplay of immune cell activation, T cell exhaustion, and pathogen dynamics, our model offers insights that could guide the development of targeted therapies. By quantifying the multifaceted nature of these diseases and their interactions, we highlight the potential of computational approaches in understanding and treating complex diseases, individually and in combination.</p>