Pathogenic mycobacteria have evolved a remarkable ability to evade the immune system and to colonize and survive within the host. Among the key evasion strategies is the capacity of these bacteria to parasitize host macrophages, which represent the major effector cells against intracellular pathogens that can be used as long-term cellular reservoirs. In order to manipulate the macrophage functions to survive and establish infection, mycobacterial pathogens employ a vast panoply of virulence factors, including cell envelope lipids, playing a key role in the modulation of the host innate response. We recently characterized several lipids that are specific to Mycobacterium abscessus (Mabs), acting as virulence factors in macrophage subversion. Mabs is a rapidly growing mycobacterium increasingly acknowledged as a serious non-tuberculous mycobacterial pathogen. It can cause extra-pulmonary infections as well as severe pulmonary disease among otherwise healthy individuals and poses a serious threat to cystic fibrosis patients, where infection correlates with a decline in lung function. Mabs presents distinct smooth (S) and rough (R) colony morphotypes, determined by the presence (S) or absence (R) of cell wall surface associated glycopeptidolipids (GPL). The S variant is thought to be the colonising form and is capable of producing mature biofilms whilst the R variant is impaired in these abilities but forms serpentine cords, a feature which is associated with intense inflammation and hypervirulence. In addition to GPL, we recently characterized a unique lipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol, participating in phagosomal membrane damage and in intracellular survival of Mabs in various cellular and animal models. We also identified a yet uncharacterized unique genomic locus related to the GPL locus and absent in M. tuberculosis, encoding putative “GPL-like” lipids, whose disruption leads to strongly impaired intramacrophage growth. Collectively, these observations support the view that complex lipids are potent immuno-modulators, conditioning the pathophysiology and outcome of Mabs infection. We, thus, foresee Mabs as a new training ground for the discovery of new lipids to decipher the structural variability of the mycobacterial lipid repertoire. This will increase our knowledge regarding how lipids benefit mycobacteria to manipulate the host innate and adaptive responses and contribute to Mabs virulence and the pathophysiology of Mabs infections, which are different from M. tuberculosis infections. SUNLIVE is anticipated to 1) Establish the lipidome of the Mabs with a special emphasis on the structural determination of major cell wall lipids; 2) Describe the role of these complex lipids (particularly GDND, GPL, GPL-like) in physiology and immunomodulation and by addressing their biological functions in macrophages thanks to the generation of lipid mutants and purified lipids coupled to beads; 3) Evaluate the role of these lipids in the pathophysiology of Mabs infection in relevant and complementary animal models (zebrafish and “Kramnick” mice). This 3-year project builds on a large set of data already generated by the four teams and the implementation of an integrated approach employing a broad panel of complementary and cutting-edge techniques to characterize new virulence and physiopathological determinants of one of the most drug-resistant and difficult-to-treat mycobacterial species. From an applied perspective, the results obtained may lead to innovative strategies to prevent the development of the severe clinical forms, often fatal in cystic fibrosis patients, and also new insights into the evolution of environmental mycobacteria towards pathogenicity. We anticipate also that the identification of a typical lipid signature will open the path to innovative diagnostic applications against Mabs infection, fitting with the objectives of Axis 3.6 “Immunology, Infectiology and Inflammation”.

.Skeletal dysplasias with multiple dislocations (SDM) are severe disorders characterized by dislocations of large joints, scoliosis and pre and postnatal growth retardation. More than 10 recessive disorders have been described so far and the majority of them have been linked to pathogenic variants in genes encoding glycosyltransferases (“linkeropathies”), sulfotransferases, epimerases or sulfate transporters, all requested for the biosynthesis of the heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains attached to HS and/or CS proteoglycan (HSPG and CSPG) core protein. These findings support the recognition of a new group of inborn errors leading to defects in GAG biosynthesis. This process is tightly regulated in the Golgi, especially through ion homeostasis. In our cohort of patients with SDM, pathogenic variants have been also identified in genes encoding proteins with no known functions directly related to proteoglycan synthesis, such as calcium activated nucleotidase-1 (CANT-1), that were associated with defective proteoglycans synthesis. More recently, we identified homozygous mutations in SLC10A7, which encodes a member of the SLC family of uncharacterized transporters. Furthermore, we developed a Slc10a7-deficient mouse model that mimics the human bone phenotype. Preliminary results, interestingly, demonstrated a strong specific decrease in HS level in both patient fibroblasts and Slc10a7-/- mouse cartilages and a congenital disorder of glycosylation type II (CDG II) profile, i.e. resulting in the presence of truncated and abnormal N-glycan structures, in SLC10A7 deficient patients. The link of SLC10A7 with divalent ion homeostasis is not clear but suggested by yeast studies showing that SLC10A7 orthologs could act as negative regulator of cytosolic calcium homeostasis. This proposal is built on the hypothesis that mutations identified in genes encoding transporters or ion binding proteins, such as SLC10A7 or CANT-1 lead to impaired Golgi ion homeostasis then resulting in Golgi glycosylation and specific GAG synthesis defects. As such, it is ambitious to hypothesize that the restoration of Golgi ion homeostasis will lead to a normalization of the GAG biosynthesis process, and will open the field of novel therapies for SDM patients. In order to avoid a scattering of our efforts, we will strategically focus our work on SLC10A7, for which both patient fibroblasts and Slc10a7-/- mouse model are already available. The ambition is to develop a framework that will then serve as a model to study the functions of the other identified transporters. We will combine the synergistic complementary expertise of three teams) in skeletal dysplasia and ossification process 2) in Golgi glycosylation and Golgi homeostasis and 3) in GAG biosynthesis. This team complementarity will be essential in pursuing our three main objectives: WP1: Elucidation of the roles of SLC10A7 in Golgi glycosylation and ion homeostasis (Team 2) The contribution of SLC10A7 in Ca2+/ Mn2+ homeostasis will be fully studied. WP 2: Analysis of SLC10A7 deficiency specific consequences on endochondral and membranous ossification using Slc10a7-/- mouse model (Teams 1 and 3). Ion supplementation efficiency will be tested to reverse the GAG synthesis and glycosylation defects in Slc10a7-/- mice WP 3: Golgi glycosylation, toward new therapeutics (Teams 1, 2 and 3). It will include i) a structural analysis of glycoconjugates and a characterization of GAG synthesis defects in patient and mouse samples, ii) whole exome analysis on samples from SDM patients with still unknown molecular bases. The established framework will be applied to any relevant new gene.

Bioorthogonal ligation methods are extensively explored for the development of protein conjugates, which are of considerable importance in the therapeutic field and in biotechnology industries. Popular applications include i) the conjugation of toxins, imaging agents, or radiopharmaceuticals to monoclonal antibodies in cancer therapies, ii) the design of glycoconjugate vaccines where microbial oligosaccharides are coupled to a protein carrier, iii) the pegylation of therapeutic proteins to improve their serum half-lifes and therapeutic index. Direct protein modifications are generally performed onto amino group of the abundant lysine amino-acid with N-hydroxysuccinimide-activated esters, sulfonyl chlorides or iso(thio)cyanates. Alternatively, the relatively rare cysteine residues can also be modified for single-site functionalization through disulfide exchange and Michael addition with maleimides. In comparison, the remaining 18 amino-acid have been much less exploited. One of the most promising recent alternatives, is the click-like reactions specifically targeting the tyrosine (Y) residues. Chemical oxidation of phenyl-urazole anchors like 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) react with phenol side chain of Y through an ene-like reaction. The method proved relatively chemoselective for Y, allowing the specific modification of peptides and proteins. Nevertheless, the method suffers from serious drawbacks such as (i) the use of an oxidizing chemical to generate the highly reactive PTAD species, (ii) the rapid PTAD decomposition in the presence of water (iii) the use of a specific buffer to scavenge the formation of an isocyanate side-product from rapid PTAD decomposition, resulting in the unwanted modification of lysines. This cross-reactivity limit the scope of the tyrosine-click reaction and the high PTAD reactivity prevents the site-specific targeting of a selected Y. The electrochemically promoted tyrosine-click-chemistry for protein labelling (e-CLICK) project will considerably broaden the scope of the click-Y by providing the reactive PTAD specie on demand, in different buffers, and without the needs of an oxidizing chemical. This ambitious goal will be reached by using a more promising strategy in terms of chemo-selectivity and protein modification. We propose here to develop the first electrochemically driven probes for Y-specific protein conjugations. The application of an appropriate electrochemical potential will allow us to activate the PTAD dormant specie in situ, on demand, without oxidizing the sensitive amino-acids from the protein or the ligands linked to the PTAD. Our preliminary results prove the feasibility on biologically relevant peptides and proteins. High coupling yields were obtained with a complete Y-selectivity and without compromising protein functionality. The possibility to activate a dormant urazole specie in situ will also offer the unique opportunity for site-specific Y electro-labeling (ss-e-click). In the currently described click-Y approaches, the most accessible Y are labeled preferentially but the distribution of the anchored urazoles at the protein surface is broad and aspecific. We strongly believe that ss-e-click will allow the selective labelling of Y residues at the entrance of protein binding sites. To reach this ambitious goal, we plan to attach the urazole anchor to a specific ligand of the targeted protein through an appropriate flexible linker. Y selectivity should be achieved after competitive binding followed by electrooxidation. We forsee that the e-CLICK and ss-e-CLICK methodology will nicely complement the arsenal of current biorthogonal ligations, allowing site-specificity in the design of covalent conjugate inhibitors, molecular probes for protein target identification, or the design of therapeutically relevant bio-conjugates.

Antibodies (Abs) exert their function by binding to distinct Fc receptors and complement. The affinity of these interactions is traditionally ascribed to the Ab isotype. At least for the IgGs this view is evolving. N-linked glycans are increasingly proposed to influence binding to the classical Fc-gamma receptors (Fc?R) and allow for binding to non-classical type II receptors. This proposal will study the functional significance of Fc-glycosylation in potentiating or regulating the chronic inflammatory response in Multiple Sclerosis (MS) and its animal models. MS is a chronic disabling disease during which inflammatory lesions in the central nervous system cause local tissue damage, including demyelination and neuronal (axonal) loss. The genetic susceptibility points to a causal role for the immune response, but the triggers that provoke T and B cells to attack myelin, the factors that cause disease relapses, and the anatomical localisation and composition of the inflammatory lesions in relation to the heterogeneous clinical expression of MS remain active fields of investigation. Antibodies contribute to MS. Their presence in the cerebrospinal fluid is a diagnostic hallmark. Active lesions contain antibodies and reveal complement activation. Autoantibodies recognising potassium channel KIR4.1, Neurofascin, as well as glycolipids have been identified. Within MS lesions antibodies specific for Myelin oligodendrocyte glycoprotein (MOG) can be detected. The pathogenicity of MOG Abs is raising attention as their titers are increased in juvenile MS, but also optic neuritis, and in a subset of patients with Neuromyelitis Optica (NMO). The crystallisable fragment (Fc) of Abs binds to distinct Fc receptors (FcRs) and complement. For human IgGs this tightly regulated process relies on the structural heterogeneity of the Ig-Fc domain that arises from differences among the four subclasses (IgG1, IgG2, IgG3 and IgG4), and the complex bi-antennary N-linked glycan attached to the conserved Asn297. The glycosylation of the Fc-domain is proposed to create two structural conformations, an “open” conformation that favors binding to type I FcR that include the activating receptors Fc?RIA (CD64), Fc?RIIA (CD32a), Fc?RIIC, Fc?RIIIA (CD16a) and Fc?RIIIB, as well as the inhibitory receptor Fc?RIIB (CD32b). Accordingly, the absence of sialic acids and low levels of galactosylation confer important pro-inflammatory properties to IgG by facilitating their binding to activating Fc?Rs. In addition, the absence of core-fucose improves the affinity to the Fc?IIIA, thereby enhancing antibody-dependent cellular cytotoxicity. In contrast, a “closed” conformation favours binding to type II FcRs that comprise the C-type lectin receptor CD209 (DC-SIGN) and CD23. These sialylated Abs are endowed with a potential anti-inflammatory effect. The overarching aim of this research project is to assess whether this diversity of Fc-effector functions impacts on the severity of chronic inflammatory diseases of the central nervous system. Our strategy is to study this impact functionally using a translational approach. In MS patients we will analyse the N-glycome and the Fc-glycome of the antibody response. In animal models of MS we will study the pathogenicity of Fc-glycovariants.

Norovirus (NoV) and rotavirus (RV) are re-emerging enteric viruses polluting the environment and causing foodborne gastroenteritis outbreaks. These viruses are shed at high concentrations and very resistant in the environment, thus contaminating surface waters. Oysters are a high-risk food since they can concentrate virus by filter-feeding and express glycan motifs similar to histo-blood group antigens (HBGA), which are attachment factors for NoVs and RVs in their human host. Since distinct viral strains attach to different glycan motifs, the HBGA polymorphism in the human population dictates sensitivity to infection. The presence of some of the HBGA motifs in the oyster tissues could likewise contribute to the selective accumulation of virus strains. Similar ligands are also found at the surface of some bacteria, including bacteria present in seawater. Our main hypothesis is that glycan recognition by both NoVs and RVAs is a key determinant of their environmental behavior, selecting viral strains and affecting their diffusion, infectivity, stability and persistence. Depending on a given strain’s HBGA specificity the outcome might vastly differ. Thus, attachment of the virus particle to bacteria may increase (or decrease) the virus persistence. It may also facilitate (or impair) bioaccumulation in oyster and it might either facilitate or impair infection of human cells. Project GOyAVE has 3 objectives: characterizing virus glycan ligands of oysters and of bacteria; deciphering the role of these environmental HBGA in NoV and RV infection of human intestinal cells; and assessing their role in the persistence of infectious virus in oysters or on bacterial cells. Using advanced methods such as nanoparticles enabling controlled multivalent presentation of the carbohydrate-binding domains of virus strains, state of the art MS and NMR methods dedicated to glycome analysis, in situ hybridization for localization of bioaccumulated viruses, human enteroids to detect infectious norovirus, gut-on-a chip microfluidics enabling improved realistic virus culture and NGS analysis of bioaccumulated virus, GOyAVE will provide a global approach encompassing the molecular analysis levels, the cellular and tissular levels, up to the environment levels. For the three tasks, all identified bottlenecks have a solution proposed based on the complementarity and experience of the three partners in their respective fields. The impact of GOyAVE will benefit to fundamental science by improving the knowledge of NoV and RV biology and to applied food science as it will provide a better understanding of persistence of infectious human enteric viruses in oysters. By combining different approaches, GOyAVE will allow the development of several tools meeting the social and economic demand for safer food.