L-Rhamnose (6-deoxy-L-mannose) is a rare monosaccharide exiting in nature but not easily accessible. It is described as a high value molecule for the food industry, pharmaceuticals, or nutraceuticals due to its unique physiological effects. Regarding a complex and expending market and the existing issues for its actual production (old and non green chemistry, sourcing, purity, etc.) , the methodology (processes) for the next decades must be challenged through (i) an intelligent sourcing, (ii) more up-to-date processing technologies (especially for purification) and (iii) new enzymatic proposals (cocktails, designed enzymes, etc.) for boosting L-Rha production (yield, performance, etc.). RAh is an interdisplinary public partnership that combines approaches in microbiology, biochemistry, physico-chemistry, and process engineering. Its overarching to significantly increase the level of knowledge and proficiency of new sustainable processes for large-scale production of L-Rhamnose. RAh project is directly relevant to the axis H.7 “Bioeconomy, from biomass to uses: chemistry, materials, processes and systemic approaches” of the transversal domain “Ecological and environmental transition” as well as the themes LS_07 “Environmental technology and bioengineering” and LS_11 “Biomass production and utilization, biofuels”. This project will therefore directly support the development and competitiveness of French industry in a strategic domain, integrating important constraints such as carbon, water footprints and the ethical use of natural resources, according to the axis H.7. Adopting an approach based on circular economy and sustainability will also help to support the cross-sectorial approaches and monitor future developments regarding societal and environmental challenges on a 2030 horizon.

The Nipah and Hendra viruses (NiV & HeV) (Henipavirus genus) are zoonotic pathogens responsible for severe encephalitis in humans. Their natural reservoir are fruit bats. Inter-human transmission of NiV and circulation of Henipaviruses in bat species from a growing number of countries, constitute a serious threat to human health. Although an efficient vaccine against HeV in horses is available, neither vaccines nor therapeutic treatments are available in humans against Henipaviruses. The high pathogenicity, wide host range and interspecies transmission of NiV & HeV led to their classification as BSL4 pathogens and potential bio-terrorism agents. The prevention and/or the containment of present and future epidemics will depend on our capacity to conceive effective strategies to combat these viruses. The long-term goal of this project is to shed light onto the molecular mechanisms of Henipavirus pathogenesis as a prerequisite for the rational design of future therapeutic approaches. Their V and W proteins are key players in the evasion of the host innate immune and inflammatory response. V and W share an N-terminal intrinsically disordered (ID) domain (NTD) and have distinct C-terminal domains. A region of HeV NTD was found to confer to V the ability to undergo a liquid to gel phase transition accompanied by the formation of amyloid-like fibrils. Congo red staining of transfected or infected cells suggests fibril formation also in cellula, and fluorescence microscopy showed that W forms condensates in the nuclei of transfected cells. Our hypothesis is that the phase-separated condensates formed by the V/W proteins may sequester key cell proteins involved in the cell innate immune and inflammatory response thereby contributing to the high pathogenicity of these viruses. Our aim is to further investigate the abilities of these proteins to phase separate and fibrillate and to shed light onto their functional implications. We will combine in vitro (Partner 1) and in cellula (Partners 2 & 3) studies. In vitro studies will use purified wt and mutated proteins and will enable deciphering the molecular and sequence determinants governing the ability of V/W proteins to phase separate and fibrillate. In cellula studies will imply V/W transient expression and infection experiments followed by quantitative cell imaging, interaction studies with cellular proteins of the innate immune pathway and measurements of IFN-stimulated genes & chemokines responses. V/W transient expression and infection studies will also use bat cells, which will enable unveiling possible differences between human and bat cells, thus potentially providing hints on the mechanisms by which bats efficiently control Henipavirus infection. We will also investigate the ability of well-known inhibitors of protein fibrillation to block Henipavirus V and W fibrillation and possibly hamper the adverse effects of V/W proteins on cell functions. Partner 1 is an expert in the structure-function relationships of ID regions from paramyxoviral proteins, Partner 2 is an expert of Henipavirus infection, bat cells engineering & innate immune response, and Partner 3 developed innovative approaches to image and quantify viral amyloids in infected cells. The strength of this project lies in (i) its feasibility (availability of multiple preliminary data & tools already generated by the partners), (ii) the expertise and complementarity of the partners, (iii) its originality (very few studies have investigated the impact of phase separation by viral proteins on host functions and even fewer have described amyloidogenic viral proteins), (iv) its potential to unveil a new molecular mechanism underlying Henipavirus pathogenesis, (v) to shed light on the molecular basis by which bats control Henipavirus infection, (vi) to illuminate the relationships between intrinsic disorder, phase transitions and amyloid formation and (vii) to set the stage for the development of new antiviral strategies.

Assembly of the myriad of synapses enabling communication between neurons is a crucial process of the CNS development and dictates the generation, maintenance and functioning of neural circuitries. Moreover, the function and specificity of synapses make them the locus of expression of most neurological disorders. This project aims at better characterizing the molecular and cellular mechanisms underlying synapse formation, maturation and specification. At the level of individual axon/dendrite contacts, synaptogenesis is a complex process initiated by recognition of specific adhesion proteins and followed by the recruitment of scaffolding molecules and functional receptor-channels. Among cell-adhesion molecules, the neurexins (NRXN), neuroligins (NLGN) and leucine-rich repeat transmembrane proteins (LRRTM) are implicated in synapse formation, differentiation and functional validation. In the mammalian brain, specific recognition between isoforms and splice variants of these molecules dictate the formation of, mainly : i) excitatory synapses relying on presynaptic glutamate release in front of AMPARs and NMDARs, which are stabilized at the postsynapse by NLGN1, LRRTM2, and PDZ domain-containing scaffolding molecules such as PSD-95; ii) inhibitory synapses relying on presynaptic GABA release, which activates GABARs stabilized by NLGN2 and scaffolding molecules such as gephyrin. Pathological mutations in the NRXN and NLGN genes are related to autism, X-linked mental retardation and schizophrenia, supporting the need of studying how adhesion molecules modulate synapse formation and functioning. However, the mechanisms by which these molecules, besides maintaining together axonal and dendritic membranes, dynamically assemble functional pre- and postsynaptic elements are still unclear. The study of synaptogenesis is hindered by the limited spatial resolution of conventional microscopy and the lack of selective molecules able to promote or perturb synapse formation. This project is aimed at overcoming these limitations by developing i) new strategies for protein labeling with small fluorescent probes combined with super-resolution imaging, ii) new peptidic ligands designed after the 3D structures of the extra- and intracellular domains of the adhesion proteins and to be used as interaction modulators. This project should lead to a better understanding of the role of adhesion molecules in the assembly of synapses and to the rational design of new therapeutic agents alleviating neurodevelopmental disorders. To address this multi-disciplinary and ambitious project, we built a consortium of three teams with complementary expertise. The first two teams, of O. Thoumine and M. Sainlos from the same institute (IINS, Bordeaux), have been collaborating for several years and interact on a day-to-day basis. They will bring their respective expertise in the biology of synaptic adhesion molecules, cell culture systems, high-resolution imaging and electrophysiology, and in the design and production of peptides mimicking the interaction of adhesion molecules to their extra- and intracellular partners. The third team associates P. Marchot, a biochemist and pharmacologist, and Yves Bourne, a structural biologist, who have strong collaborative records and now work in the same lab (AFMB, Marseille). They will add their complementary expertise in documenting the structure-function relationships of various ligand-receptor and protein-protein complexes and their functional implications. Our program involves three main collaborative tasks: Task 1. Structure-function analysis of the extracellular NRXN/LRRTM complexes and design of peptidic effectors of the NRXN interactions with NLGN and LRRTM Task 2. Intracellular interactions of the NRXNs, NLGNs and LRTTMs with scaffolding molecules, characterization of novel peptidic effectors, and high-resolution microscopy of the complexes Task 3: Signaling and other functions mediated by the NRXNs, NLGNs and LRRTMs

Lactic acid bacteria (LAB)-infecting bacteriophages (phages, or bacterial viruses) use diverse host-binding mechanisms, yet the overall picture of the interactions between LAB phages and their host remains incomplete. Unraveling the molecular details of phage-LAB interactions is essential not only for decoding phage biology, but also for industrial and public health purposes since LAB are important micro-organisms in food fermentations and in the human gut microbiota. Phages infecting the LAB species Lactococcus lactis and Streptococcus thermophilus have enjoyed extensive scientific scrutiny since they may disrupt LAB-dependent processes in dairy plants with serious concomitant economic losses. In contrast, there is a significant knowledge gap on the interactions between plant-associated LAB and their phage, even though they may also significantly impact fermentation processes. This is true for fermented beverages as exemplified by the emblematic field of winemaking that heavily relies on the LAB species Oenococcus oeni. Recently, we have shown that representative phages that infect O. oeni possess host-binding devices of distinct composition and morphology, and being different from those of lactococcal and streptococcal phages, that likely employ novel host-binding mechanisms. Moreover, we have observed that wine polyphenolic compounds (PCs), which are abundant in the O. oeni ecological niche, can interfere with the phage infection process. These organic compounds, being sterically similar to cell surface saccharides recognized by phages, may occupy phage receptor-binding sites, thereby preventing host binding. Moreover, PCs could also induce modifications in the cell wall saccharide composition, which would also prevent phages from binding to their host. In this context, our overall aim is to unravel molecular interactions between the wine LAB O. oeni, their viral predators, and PCs. We will work on three representative oenophages, all of which infect the same strain but use different host-binding devices differently affected by wine PCs. We will leverage complementary approaches covering the fields of structural biology, biochemistry, and microbiology, to meet our stated aim. We will 1) determine structure-function relationships of distinct host-binding devices combining cryo-electron microscopy, X-ray crystallography, biophysical characterization of protein-ligand interactions, and host cell-binding assays, 2) explore host-binding capabilities of these phages and the impact of PCs combining phenotypic analyses (generation of bacteriophage-insensitive mutants, phage plaque assays, adsorption tests) and comparative genomics, and 3) map phage-specific host cell saccharide receptors and examine the potential effects of PCs on the synthesis of these receptors through the analysis of gene expression, cell wall biochemical composition, and chemical structure of surface polysaccharides. Investigating molecular interactions between the wine LAB O. oeni and its phages, as a model system of the interactions between plant-related LABs and their phages, will significantly advance current knowledge of phage biology and structure, while simultaneously defining the role and potential inhibitory action of plant PCs on phage infection. We will produce important knowledge of LAB-phage interactions with expected high gains for the wine industry, as well as other plant-fermented products. Of note, plant-based fermented products are currently one of the most innovative and dynamic food categories, in response to the increasing popularity of vegetarian and vegan diets in western countries. Lastly, addressing the role of plant PCs on phage-host interactions may also lead to a better understanding of gut microbiota dynamics and to the rational development of ‘green’ phage-based biocontrol strategies, thereby opening perspectives in the socio-economically important fields of human health and agriculture.

The skin lesions observed in psoriasis patients are characterised by chronic inflammation in which the overexpression of heparanase 1 (Hpa1) contributes, although its precise role remains to be demonstrated. Indeed, Hpa1 could participate in the inflammatory processes through its unique ability to degrade heparan sulphates in humans on the one hand or independently on the other hand. Heparanase 2 (Hpa2), a protein related to Hpa1, has anti-inflammatory properties and seems to be able to inhibit the enzymatic activity of Hpa1 by a mechanism that remains to be elucidated. The precise contribution of the imbalance between Hpa1 and Hpa2 expression and their activities in an inflammatory context has not yet been assessed. Hpa1 is naturally inhibited by heparin, which cannot be used as an anti-inflammatory strategy due to its anti-thrombotic properties. We have thus developed an oligosaccharide called lambda-CO derived from algal polysaccharides. The latter is an inhibitor of Hpa1 and also seems to have anti-inflammatory properties. In this context, the HeparInfSkin project aims i) to describe the mode of interaction between the two heparanases and their respective roles in the regulation of inflammation in macrophages, keratinocytes and skin fibroblasts; ii) to characterise the Hpa1-dependent and -independent mode of action of lambda-CO; iii) to evaluate the expression of the two heparanases in psoriasis patients; and finally iv) to validate the beneficial effects of lambda-CO in a relevant mouse model of psoriasis. For this purpose, different cell culture models of macrophages, epidermis and reconstructed skin will be set up from original mouse models, where the expression of the two heparanases will be specifically controlled. The HeparInfSkin project gathers the strengths of three French laboratories, with complementary and recognised expertise in their respective fields, combining the mastery of biochemistry, biotechnology, immunology, pharmacology, biophysics and structural biology approaches. This original and ambitious project, at the interface of fundamental and clinical research, will make it possible to characterise the contribution of heparanases during the different phases of the disease, particularly in the initiation, maintenance or resolution of cutaneous inflammatory processes and the mechanisms of action of lambda-CO, particularly its capacity to inhibit Hpa1 without interfering with the beneficial effects of Hpa2 on Hpa1. This project, which has no national competition, will certainly pave the way for new anti-inflammatory therapeutic strategies in humans based on the use of oligosaccharide of marine origin that modulates heparanase metabolism.