Caractérisation moléculaire de la modulation spatio-temporelle des fonctions du phagosome

La phagocytose est un processus par lequel des cellules spécialisées du système immunitaire comme les macrophages ingèrent des microorganismes envahisseurs afin de les détruire. Les microbes phagocytés se retrouvent dans un compartiment intracellulaire nommé le phagosome, qui acquiert graduellement de nombreuses molécules lui permettant de se transformer en phagolysosome possédant la capacité de tuer et dégrader son contenu. L’utilisation de la protéomique a permis de mettre en évidence la présence de microdomaines (aussi nommés radeaux lipidiques ou radeaux membranaires) sur les phagosomes des macrophages. Notre équipe a démontré que ces radeaux exercent des fonctions cruciales au niveau de la membrane du phagosome. D’abord nous avons observé que la survie du parasite intracellulaire L. donovani est possible dans un phagosome dépourvu de radeaux lipidiques. Parallèlement nous avons constaté qu’un mutant de L. donovani n’exprimant pas de LPG à sa surface(LPG-) est rapidement tué dans un phagosome arborant des radeaux membranaires. Pour comprendre le mécanisme de perturbation des microdomaines du phagosome par la molécule LPG, nous avons provoqué la phagocytose de mutants LPG- du parasite et comparé par microscopie les différences avec le parasite de type sauvage. Nous avons ainsi démontré que le LPG de L. donovani est nécessaire et suffisant au parasite pour empêcher la maturation normale du phagosome. Nous avons également découvert que la molécule LPG permet d’empêcher la formation des radeaux lipidiques sur le phagosome et peut aussi désorganiser les radeaux lipidiques préexistants. Enfin, nous avons montré que l’action de LPG est proportionnelle au nombre d’unités répétitives de sucres (Gal(β1,4)-Manα1-PO4) qui composent cette molécule. Nos travaux ont démontré pour la première fois le rôle important de ces sous-domaines membranaires dans la maturation du phagosome. De plus, nos conclusions seront des pistes à suivre au cours des études cliniques ayant pour but d’enrayer la leishmaniose. Le second objectif de ce travail consistait à effectuer la caractérisation des radeaux lipidiques par une analyse protéomique et lipidomique à l’aide de la spectrométrie de masse. Nous avons ainsi entrepris l’identification systématique des protéines présentes dans les radeaux membranaires des phagosomes et ce, à trois moments clés de leurmaturation. Le traitement des phagosomes purifiés avec un détergent nous a permis d’isoler les «Detergent Resistent Membranes» (DRMs) des phagosomes, qui sont l’équivalent biochimique des radeaux membranaires. Nous avons ainsi établi une liste de 921 protéines associées au phagosome, dont 352 sont présentes dans les DRMs. Les protéines du phagosome sont partagées presque également entre trois tendances cinétiques (augmentation, diminution et présence transitoire). Cependant, une analyse plus spécifique des protéines des DRMs démontre qu’une majorité d’entre elles augmentent en fonction de la maturation. Cette observation ainsi que certains de nos résultats montrent que les radeaux lipidiques des phagosomes précoces sont soit très peu nombreux, soit pauvres en protéines, et qu’ils sont recrutés au cours de la maturation du phagosome. Nous avons aussi analysé les phospholipides du phagosome et constaté que la proportion entre chaque classe varie lors de la maturation. De plus, en regardant spécifiquement les différentes espèces de phospholipides nous avons constaté que ce ne sont pas uniquement les espèces majoritaires de la cellule qui dominent la composition de la membrane du phagosome. L’ensemble de nos résultats a permis de mettre en évidence plusieurs fonctions potentielles des radeaux lipidiques, lesquelles sont essentielles à la biogenèse des phagolysosomes (signalisation, fusion membranaire, action microbicide, transport transmembranaire, remodelage de l’actine). De plus, la cinétique d’acquisition des protéines de radeaux lipidiques indique que ceux-ci exerceraient leurs fonctions principalement au niveau des phagosomes ayant atteint un certain niveau de maturation. L’augmentation du nombre de protéines des radeaux membranaires qui s’effectue durant la maturation du phagosome s’accompagne d’une modulation des phospholipides, ce qui laisse penser que les radeaux membranaires se forment graduellement sur le phagosome et que ce ne sont pas seulement les protéines qui sont importées.Macrophages are specialized cells of the immune system which mediate destruction and killing of invading micro-organisms. They do so by engulfing them by a process called phagocytosis. Microbes are then captured in an intracellular compartment, the phagosome, which gradually acquire molecules able to attack and degrade its cargo. Use of proteomics let us demonstrate the presence of flotillin-1 enriched microdomains (also called lipid rafts or membrane rafts) on the phagosomes. Our team demonstrated the crucial importance of these rafts in the phagocytosis process. Indeed, survival of L. donovani correlates with its presence in a ‘raftless’ phagosome while a mutated L. donovani without LPG is rapidly killed in a phagosome containing lipid rafts. To understand the membrane raft destabilisation mechanism mediated the LPG molecule, we induced phagocytosis of parasites devoid of LPG (LPG-) and compared it to the wild type parasite by microscopy. We first demonstrated that LPG alone is necessary to prevent normal maturation of the phagosome. Additionally, we discovered that the LPG molecule not only inhibits lipid rafts formation on the phagosome but also disorganise pre-existing lipid rafts. This effect of LPG is proportional to the number of repetitive sugar units (Gal( 1,4)-Man 1-PO4) which compose this molecule. Our work demonstrated for the first time an important role of the membrane rafts in the phagosome maturation. Moreover, our conclusions will give new interesting leads for clinical studies on leishmaniosis. The second goal of this work was to characterise them with proteomics and lipidomics tools. To do this, we undertook the systematic identification of proteins present on both subdomains of the phagosome (lipid rafts versus the rest of the phagosomal membrane). To achieve this, we purified phagosomes, from which we isolated lipid rafts by floating Triton X-100 insoluble membranes (DRMs for Detergent Insoluble Membranes). After that, we identified proteins by mass spectrometry

Études chimiques et immunologiques des capsules polysaccharidiques de Streptococcus suis

Streptococcus suis est l’un des plus importants pathogènes bactériens du porc, causant des pertes économiques substantielles à l’industrie porcine. De plus, c’est un agent zoonotique représentant de sérieux risques pour la santé humaine. Cette bactérie cause diverses pathologies, dont la méningite, la mort subite et le choc septique sont les plus fréquentes. De plus, il demeure qu’à ce jour, aucun vaccin efficace n’est disponible pour prévenir les infections à S. suis. Étant encapsulé, S. suis est classifié en 35 sérotypes définis par l’antigénicité de sa capsule polysaccharidique (CPS). Malgré cela, seule la structure de la CPS du sérotype 2 est connue à présent. Immunologiquement parlant, les CPS purifiées sont généralement des antigènes T-indépendant de par leur nature, faisant d’elles de très pauvres immunogènes. Ceci a déjà été démontré pour la CPS du sérotype 2. Paradoxalement, les anticorps anti-CPS sont fortement opsonisants et protecteurs, ce qui les rend désirables. Ainsi, les objectifs de cette thèse sont, d’abord, de poursuivre la caractérisation chimique des structures des CPS de différents sérotypes de S. suis, puis d’étudier les propriétés immunostimulatrices et immunogènes de ces nouvelles CPS. De plus, les travaux de cette thèse ont tenté d’améliorer l’immunogénicité de la CPS du sérotype 2 pour pouvoir l’utiliser comme antigène vaccinal. Dans un premier temps, un recensement de la littérature a permis de dresser un portrait à jour et mondial des infections chez le porc et chez l’homme, et d’identifier les sérotypes les plus importants. À l’aide de ces données épidémiologiques, les CPS des sérotypes choisis ont été purifiées, ce qui a permis de déterminer la structure pour huit CPS additionnelles (1, 1/2, 3, 7, 8, 9, 14 et 18). D’autre part, ces nouvelles connaissances sur la structure nous ont permis d’évaluer l’antigénicité et l’immunogénicité de ces nouvelles CPS. Ainsi, nous avons pu observer pour la première fois que la CPS du sérotype 3 de S. suis est hautement immunogène en produisant une réponse opsonisante d’IgG et d’IgM par un mécanisme T-indépendant. De plus, nous avons développé pour la première fois un vaccin glycoconjugué constitué de CPS du sérotype 2 couplé au toxoïde tétanique afin d’obtenir une réponse anticorps T-dépendante protectrice. Une preuve de concept chez le porc a permis de démontrer la protection conférée par notre vaccin glycoconjugué contre S. suis, et plus largement le potentiel des vaccins glycoconjugués pour combattre les infections bactériennes invasives en médecine vétérinaire. Enfin, l’étude de l’immunogénicité du vaccin glycoconjugué chez la souris a permis d’évaluer le rôle du choix du modèle animal et des adjuvants sur la réponse anti-CPS, en plus de produire trois nouveaux anticorps monoclonaux qui ont permis notamment d’étudier les mécanismes humoraux impliqués dans l’élimination de S. suis par opsonophagocytose. L’ensemble de ces études va permettre de souligner l’importance des anticorps anti-CPS dans la protection face aux infections bactériennes invasives.Streptococcus suis is one of the most important porcine bacterial pathogens and is responsible for substantial economic losses to the swine industry. Moreover, it is also a zoonotic agent representing serious risks to human health. This bacterium causes a variety of clinical signs, of which meningitis, sudden death, and septic shock are the most frequent. Furthermore, no efficient vaccine is available to protect against infections caused by S. suis. Being encapsulated, S. suis is classified into 35 serotypes based on the antigenicity of their capsular polysaccharide (CPS). However, only the structure of the serotype 2 CPS is known to date. Immunologically, purified CPSss generally behave as T-independent antigens, making them very poor immunogens. Indeed, this has been previously demonstrated to be the case for the serotype 2 CPS. Paradoxically, anti-CPS antibodies are strongly opsonizing and protective, making them attractive targets for vaccine development. Therefore, the objectives of this thesis are, firstly, to further characterize the chemical composition and structure of the S. suis CPSs from different serotypes, and secondly, to study the immunostimulatory and immunogenic properties of these additional CPSs. Moreover, this thesis will attempt to increase the serotype 2 CPS immunogenicity for potential use it as a vaccine antigen. In a first step, a review of the literature provided an updated and global view of S. suis infections in swine and humans and identified the most important serotypes. With these epidemiological data in hand, we then purified the CPSs of selected serotypes, which allowed us to determine the structures for eight additional CPSs (1, 1/2, 3, 7, 8, 9, 14, and 18). The knowledge obtained regarding the structure of theses CPSs allowed us to then evaluate their antigenicity and immunogenicity. We demonstrated for the first time that serotype 3 is highly immunogenic S. suis CPS, as it induces an opsonizing response composed of IgG and IgM by a T-independent mechanism. In addition, we have developped for the first time a glycoconjugate vaccine composed of serotype 2 CPS conjugated to tetanus toxoid in order to obtain a protective T-dependent antibody response. A proof of concept performed with pigs demonstrated protection conferred by our glycoconjugate vaccine against S. suis, and more importantly the potential of glycoconjugate vaccines in the fight against invasive bacterial infections in veterinary medicine. Finally, immunogenicity studies performed in mice with this glycoconjugate vaccine allowed us to evaluate the effect of the animal model and adjuvant choice on the anti-CPS response. It also allowed us to produce three new monoclonal antibodies that enabled us to study, among other aspects, the humoral mechanisms involved in S. suis clearance by opsonophagocytosis. Consequently, the work conducted during this thesis will serve to highlight the importance of anti-CPS antibodies in the protection against invasive bacterial diseases

Non-normal Recurrent Neural Network (nnRNN): learning long time dependencies while improving expressivity with transient dynamics

A recent strategy to circumvent the exploding and vanishing gradient problem in RNNs, and to allow the stable propagation of signals over long time scales, is to constrain recurrent connectivity matrices to be orthogonal or unitary. This ensures eigenvalues with unit norm and thus stable dynamics and training. However this comes at the cost of reduced expressivity due to the limited variety of orthogonal transformations. We propose a novel connectivity structure based on the Schur decomposition and a splitting of the Schur form into normal and non-normal parts. This allows to parametrize matrices with unit-norm eigenspectra without orthogonality constraints on eigenbases. The resulting architecture ensures access to a larger space of spectrally constrained matrices, of which orthogonal matrices are a subset. This crucial difference retains the stability advantages and training speed of orthogonal RNNs while enhancing expressivity, especially on tasks that require computations over ongoing input sequences

S-Layer From Lactobacillus brevis Modulates Antigen-Presenting Cell Functions via the Mincle-Syk-Card9 Axis

C-type lectin receptors (CLRs) are pattern recognition receptors that are crucial in the innate immune response. The gastrointestinal tract contributes significantly to the maintenance of immune homeostasis; it is the shelter for billions of microorganisms including many genera of Lactobacillus sp. Previously, it was shown that host-CLR interactions with gut microbiota play a crucial role in this context. The Macrophage-inducible C-type lectin (Mincle) is a Syk-coupled CLR that contributes to sensing of mucosa-associated commensals. In this study, we identified Mincle as a receptor for the Surface (S)-layer of the probiotic bacteria Lactobacillus brevis modulating GM-CSF bone marrow-derived cells (BMDCs) functions. We found that the S-layer/Mincle interaction led to a balanced cytokine response in BMDCs by triggering the release of both pro- and anti-inflammatory cytokines. In contrast, BMDCs derived from Mincle−/−, CARD9−/− or conditional Syk−/− mice failed to maintain this balance, thus leading to an increased production of the pro-inflammatory cytokines TNF and IL-6, whereas the levels of the anti-inflammatory cytokines IL-10 and TGF-β were markedly decreased. Importantly, this was accompanied by an altered CD4+ T cell priming capacity of Mincle−/− BMDCs resulting in an increased CD4+ T cell IFN-γ production upon stimulation with L. brevis S-layer. Our results contribute to the understanding of how commensal bacteria regulate antigen-presenting cell (APC) functions and highlight the importance of the Mincle/Syk/Card9 axis in APCs as a key factor in host-microbiota interactions.Fil: Prado Acosta, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Goyette Desjardins, Guillaume. University of Veterinary Medicine; AustriaFil: Scheffel, Jörg. Humboldt-Universität zu Berlin; AlemaniaFil: Dudeck, Anne. Otto-von-Guericke-Universität Magdeburg; AlemaniaFil: Ruland, Jürgen. Universitat Technical Zu Munich; AlemaniaFil: Lepenies, Bernd. University of Veterinary Medicine; Austri

Glycoengineered outer membrane vesicles: A novel platform for bacterial vaccines

The World Health Organization has indicated that we are entering into a post-antibiotic era in which infections that were routinely and successfully treated with antibiotics can now be lethal due to the global dissemination of multidrug resistant strains. Conjugate vaccines are an effective way to create a long-lasting immune response against bacteria. However, these vaccines present many drawbacks such as slow development, high price, and batch-to-batch inconsistencies. Alternate approaches for vaccine development are urgently needed. Here we present a new vaccine consisting of glycoengineered outer membrane vesicles (geOMVs). This platform exploits the fact that the initial steps in the biosynthesis of most bacterial glycans are similar. Therefore, it is possible to easily engineer non-pathogenic Escherichia coli lab strains to produce geOMVs displaying the glycan of the pathogen of interest. In this work we demonstrate the versatility of this platform by showing the efficacy of geOMVs as vaccines against Streptococcus pneumoniae in mice, and against Campylobacter jejuni in chicken. This cost-effective platform could be employed to generate vaccines to prevent infections caused by a wide variety of microbial agents in human and animals

Integrated immunovirological profiling validates plasma SARS-CoV-2 RNA as an early predictor of COVID-19 mortality.

peer reviewedDespite advances in COVID-19 management, identifying patients evolving toward death remains challenging. To identify early predictors of mortality within 60 days of symptom onset (DSO), we performed immunovirological assessments on plasma from 279 individuals. On samples collected at DSO11 in a discovery cohort, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA), low receptor binding domain–specific immunoglobulin G and antibody-dependent cellular cytotoxicity, and elevated cytokines and tissue injury markers were strongly associated with mortality, including in patients on mechanical ventilation. A three-variable model of vRNA, with predefined adjustment by age and sex, robustly identified patients with fatal outcome (adjusted hazard ratio for log-transformed vRNA = 3.5). This model remained robust in independent validation and confirmation cohorts. Since plasma vRNA’s predictive accuracy was maintained at earlier time points, its quantitation can help us understand disease heterogeneity and identify patients who may benefit from new therapies

Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-κB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-κB and Sp1 to bind to their binding sites. Knock-down of either NF-κB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-κB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis