Eduard Baquero

Juliette Fedry

Pierre Legrand

Thomas Krey

Felix A Rey
email: felix.rey@pasteur.fr

Species-specific functional regions of the green alga gamete fusion protein HAP2 revealed by structural studies

The cellular fusion protein HAP2, which is structurally homologous to viral class II fusion proteins, drives gamete fusion across several eukaryotic kingdoms. Gamete fusion is a highly controlled process in eukaryotes, and is allowed only between same species gametes. In spite of a conserved architecture, HAP2 displays several species-specific functional regions that were not resolved in the available X-ray structure of the green alga Chlamydomonas reinhardtii HAP2 ectodomain. Here we present an X-ray structure resolving these regions, showing a target-membrane interaction surface made by three amphipathic helices in a horseshoe-shaped arrangement. HAP2 from green algae also features additional speciesspecific motifs inserted in regions that in viral class II proteins are critical for the fusogenic conformational change. Such insertions include a cystine-ladder-like module evocative of EGF-like motifs responsible for extracellular protein-protein interactions in animals, and a mucin-like region. These features suggest potential HAP2 interaction sites involved in gamete fusion control.

Introduction

Cell-cell fusion is ubiquitous across eukaryotes. It is required for fertilization, where two gametes of opposite type merge together to form a zygote [START_REF] Georgadaki | The molecular basis of fertilization (Review)[END_REF]. During the development of multicellular organisms, the formation of syncytial tissues also occurs via fusion of somatic cells [START_REF] Zito | United we stand: Adhesion and molecular mechanisms driving cell fusion across species[END_REF]. The molecular mechanism driving the merger of lipid bilayers has been studied extensively in the case of intracellular fusion events, such as the fusion of transport vesicles [START_REF] Jahn | SNAREs--engines for membrane fusion[END_REF][START_REF] Martens | Mechanisms of membrane fusion: disparate players and common principles[END_REF][START_REF] Sudhof | Membrane fusion: grappling with SNARE and SM proteins[END_REF][START_REF] Wickner | Membrane fusion[END_REF] or the homotypic fusion of intracellular organelles [START_REF] Farmer | Tying trafficking to fusion and fission at the mighty mitochondria[END_REF][START_REF] Hu | Fusion of the endoplasmic reticulum by membrane-bound GTPases[END_REF]. In contrast, relatively 2 little is known about extracellular fusion [START_REF] Hernandez | The hallmarks of cell-cell fusion[END_REF][START_REF] Sampath | Myoblast fusion confusion: the resolution begins[END_REF]. The only well-characterized extracellular fusion process is that of enveloped viruses, which fuse the viral lipid bilayer either with the plasma membrane or with an endosome from the luminal side [START_REF] Harrison | Viral membrane fusion[END_REF]. HAP2/GCS1 is the first bona fide gamete fusion protein identified. Discovered in flowering plants [START_REF] Johnson | Arabidopsis hapless mutations define essential gametophytic functions[END_REF][START_REF] Mori | GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization[END_REF], subsequent studies in the green alga Chlamydomonas reinhardtii and the malaria organism Plasmodium showed that it was essential for bilayer merger [START_REF] Liu | The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes[END_REF]. The presence of HAP2 in organisms across the main branches of eukaryotes [START_REF] Cole | Function of the Male-Gamete-Specific Fusion Protein HAP2 in a Seven-Sexed Ciliate[END_REF][START_REF] Ebchuqin | Evidence for participation of GCS1 in fertilization of the starlet sea anemone Nematostella vectensis: implication of a common mechanism of sperm-egg fusion in plants and animals[END_REF][START_REF] Kawai-Toyooka | Sex-specific posttranslational regulation of the gamete fusogen GCS1 in the isogamous volvocine alga Gonium pectorale[END_REF][START_REF] Steele | Evolutionary history of the HAP2/GCS1 gene and sexual reproduction in metazoans[END_REF] suggests that it is likely an ancestral gamete fusogen already present at the origin of eukaryotic life [START_REF] Wong | Is HAP2-GCS1 an ancestral gamete fusogen?[END_REF].

HAP2 is present exclusively in male gametes in most of the organisms where it was identified -or in minus type gametes in the case of isogamous organisms such as algae. The gamete fusion process has been studied in highest detail in C. reinhardtii, where HAP2 was shown to localize to the "mating structure", a projecting organelle at the plasma membrane located in between the two flagella of the minus gametes [START_REF] Pan | Protein transport and signal transduction during fertilization in chlamydomonas[END_REF].

We recently reported the x-ray structure of the C. reinhardtii HAP2 ectodomain. Although this structure lacked information about important functional regions, it revealed clear homology to class II viral fusion proteins [START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF], a notion supported by parallel functional studies on HAP2 from other eukaryotic phyla [START_REF] Angrisano | Targeting the Conserved Fusion Loop of HAP2 Inhibits the Transmission of Plasmodium berghei and falciparum[END_REF][START_REF] Pinello | Structure-Function Studies Link Class II Viral Fusogens with the Ancestral Gamete Fusion Protein HAP2[END_REF][START_REF] Valansi | Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens[END_REF]. The structural study also showed that the HAP2 ectodomain trimerizes upon insertion to membranes, adopting a hairpin arrangement typical of the postfusion form of the viral proteins, with the transmembrane (TM) proximal region and the fusion loops together at one end of the trimer (Figure 1).

In spite of being structurally unrelated, the three characterized structural classes of viral fusion proteins, termed class I, II and III [START_REF] Igonet | SnapShot: Viral and eukaryotic protein fusogens[END_REF], were shown to function via a similar mechanism [START_REF] Harrison | Viral membrane fusion[END_REF]. Anchored in the viral lipid envelope by a C-terminal TM segment, they have a bulky N-terminal ectodomain folded in a metastable conformation at the viral surface. Interactions with the target membrane trigger an exergonic conformational change during which the protein exposes a polypeptide segment, termed "fusion peptide" when it is N-terminal or "fusion loop" when it is internal. This segment inserts firmly into the outer leaflet of the target cell membrane while the protein adopts a transient, elongated trimeric conformation (Figure 1A). The trimer subunits then further re-organize into a hairpin that brings both membranes into close apposition (within 1 nm), thereby overcoming the repulsive force between the two membranes generated by the required dehydration of the outer leaflets to allow lipid contact, helping catalyze the membrane fusion reaction.

The reported HAP2 structure displayed the three characteristic β-sheet-rich domains of viral class II fusion proteins, organized around the central domain I. In the post-fusion form, domain I is a 10-stranded β-sandwich with the inner A 0 B 0 I 0 H 0 G 0 β-sheet packing against the outer J 0 C 0 D 0 E 0 F 0 β-sheet (Figure 1B). At the domain I "front" end, two of the connections between consecutive β-strands make very long excursions to form the elongated domain II: the D 0 E 0 connection spans domain II β-strands a' through e', while the H 0 I 0 connection makes β-strands f through l (Figure 1B andC). The fusion loops are located in the segment connecting β strands c and d, at the very tip of domain II. At the opposite end of domain Ithe "bottom" end -a flexible linker connects to domain III, which has an immunoglobulin superfamily fold and is in turn connected to the C-terminal viral TM anchor via a flexible segment called "stem". Although the structure of HAP2 in the pre-fusion form is not known, structural studies on pre-and post-fusion forms of viral class II fusion proteins [START_REF] Bressanelli | Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation[END_REF][START_REF] Gibbons | Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus[END_REF]Guardado-Calvo et al., 2017;[START_REF] Halldorsson | Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion[END_REF][START_REF] Modis | Structure of the dengue virus envelope protein after membrane fusion[END_REF] have shown that the transition mostly involves a rearrangement of the three domains with respect to each other, with domain III undergoing an important translation to relocate to the sides of the trimer (diagrammed in Figure 1A). Importantly, in all cases studied, the central domain I undergoes the most significant internal rearrangement, with β-strand A 0 switching from the inner to the outer β-sheet. This change is accompanied by formation of βstrand J 0 , which in the pre-fusion form is part of the linker between domains I and III, and by a significant rearrangement of the A 0 B 0 , C 0 D 0 and E 0 F 0 inter-strand connections (Figure 1). These studies suggest that these same regions are also likely to undergo structural changes during the HAP2 trimerization to adopt its post-fusion form.

Although they drive fusion in an overall similar way, the viral fusion proteins of the various classes have different ways of ensuring a stable enough insertion into the target membrane to withstand the molecular gymnastics diagrammed in Figure 1A. In the case of the influenza virus hemagglutinin (HA) (the prototype class I fusion protein), the fusion peptide displays a random coil conformation in the pre-fusion form [START_REF] Wilson | Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution[END_REF], but restructures upon exposure to a membrane into an α-helical hairpin projecting nine bulky non-polar side chains [START_REF] Harter | Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes occurs through the "fusion peptide[END_REF][START_REF] Lorieau | The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface[END_REF]. In contrast, for certain class II proteins such as that of the Rift Valley fever virus (RVFV, a bunyavirus), the fusion loop maintains the same conformation it has in the pre-fusion form. In this case, a pre-formed pocket accommodates the head groups of glycerophospholipids of the membrane's outer leaflet by making multiple hydrogen bonds (Guardado-Calvo et al., 2017). Combined with the insertion of only one or two bulky aromatic side chains of the fusion loop, these polar interactions provide stable enough anchoring to drive the fusion process.

Here, we report the X-ray structure of the C. reinhardtii HAP2 ectodomain derived from a different crystal form that resolves most of the regions that were missing in the previous structure. It shows that the fusion loops are folded as three amphipathic helices in a horseshoe-shaped arrangement. Mutagenesis altering the non-polar character of the exposed residues in the fusion helices disrupted the ability of the HAP2 ectodomain to insert into liposomes. This structure also showed the presence of an unanticipated cystine ladder-like motif reminiscent of the epidermal-growth factor (EGF)-like domains inserted in loop C 0 D 0 , packing against a mucin-like region in loop E 0 F 0 at the bottom end of domain I.

Results

Crystallization of the intact HAP2 ectodomain. The crystals used to determine the previously reported HAP2 X-ray structure grew only after limited proteolysis of the HAP2 ectodomain [START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF]. Here we screened for crystallization conditions of the unproteolysed HAP2 ectodomain in complex with HAP2-specific monoclonal antibodies (Mabs) obtained previously [START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF]. The complex with the single chain variable fragment (scFv) of one of these antibodies resulted in crystals in the hexagonal space group P6 3 22 diffracting to 2.6Å resolution (Figure S1). Structure determination by molecular replacement using the atomic model of HAP2, PDB code 5MF1, resulted in an electron density map of HAP2 displaying 1 protomer per asymmetric unit (i.e., with the trimer axis coincident with a crystallographic 3-fold axis) and 80% of the volume occupied by solvent (Table 1). Unexpectedly, this map did not reveal density for a bound scFv. It is possible that formation of the crystal lattice displaced the bound antibody, or that it is present but disordered in the large solvent volumes in between HAP2 molecules (Figure S1). In any event, the electron density allowed tracing the polypeptide chain for most of the HAP2 ectodomain, including loops that were not resolved in the available structure. In total, the new model contains 551 out of 573 residues (Figure 1B), with a break of about 15 residues in the fg loop in domain II -a loop projecting laterally, perpendicular to the trimer axis and roughly midway between top and bottom. Potential antibody binding to this loop could explain the absence of scFv density in the crystal, and will be the object of future experiments. Also missing was part of loop E 0 F 0 at the trimer bottom (Figure S1).

Crystallographic refinement led to a final model containing 4136 atoms in total, with free R factor of 24.3% at 2.6Å. Comparison of the new model with the previously determined structure resulted in a root mean square deviation (RMSD) of 0.528 Å for 528 aligned Cα atoms, indicating that they are very close, with minor changes at the tip of domain II, where the proteolytic treatment to obtain the crystals may have locally affected the protein conformation.

Potential protein-protein interaction modules in domain I

Mucin-like region (MLR). The new crystals revealed density for loops C 0 D 0 and E 0 F 0 (Figure 2). These two segments are highly variable in sequence and length in HAP2 orthologs across eukaryotes (Figures S2 andS3), and are particularly long in HAP2 from green algae. In C. reinhardtii HAP2, the longest is loop E 0 F 0 (residues 239-284), a 45-residue long segment rich in proline and serine/threonine residues (Figure S2A), which are typical of heavily Oglycosylated proteins such as the mucins [START_REF] Shogren | Role of glycosylation on the conformation and chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary mucin[END_REF] (Figure 2A, left panel and Figure S2A). MLRs, which are known to confer unique rheological properties on proteins [START_REF] Tabak | In defense of the oral cavity: structure, biosynthesis, and function of salivary mucins[END_REF], are present on viral proteins involved in attachment to cells, for instance in glycoprotein C of herpes simplex viruses [START_REF] Rajcani | The role of herpes simplex virus glycoproteins in the virus replication cycle[END_REF], the respiratory syncytial virus glycoprotein G [START_REF] Satake | Respiratory syncytial virus envelope glycoprotein (G) has a novel structure[END_REF][START_REF] Wertz | Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein[END_REF], or in the surface glycoprotein of Ebola virus [START_REF] Kirchdoerfer | Filovirus Structural Biology: The Molecules in the Machine[END_REF]. These viral proteins have in common the recognition and binding of glycosaminoglycans (GAGs) present at the surface of target cells, and the MLR was shown to modulate GAG binding [START_REF] Altgarde | Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction[END_REF]. In animals, MLRs are found on many proteins anchored at the cell surface, as well in secreted proteins, and were found to modulate cell recognition and adhesion via sugar/lectin interactions [START_REF] Tran | Mucin-type O-glycosylation during development[END_REF]. Although the new crystals did not entirely resolve the E 0 F 0 loop, they showed density for its N-terminal six amino acids (sequence PTAPSP, downstream of strand E 0 ) as well as its C-terminal 14 residues (271-APQFLSPPAPSTRE-285) preceding β-strand F 0 (Figure 2A), accounting in total for 40% of the loop. The latter the proline rich segment packs against the bottom of domain I, and also contacts loops A 0 B 0 and C 0 D 0 of the neighboring subunit. Of note, the downstream F 0 and G 0 β-strands in the previously reported structure were miss-assigned and were out of register by two residues, due to poor electron density in this region and the more limited resolution of those crystals. In addition, the density that had been interpreted as a potential O-glycosylation on residue Thr577 at the end of domain III, is clear in the new structure to correspond to an N-linked glycan attached to Asn578. These errors were corrected in the present structure.

Cystine ladder-like (CLL) motif. The 37-residue long C 0 D 0 loop is completely resolved in the new structure, and displays a CLL motif with three parallel disulfide bonds (Figure 2A, right panel). This motif is present only in HAP2 from green algae (Figure S3), across which it is highly conserved (Figure 2B). Authentic cystine ladder motifs have been observed in θdefensins [START_REF] Conibear | Structural characterization of the cyclic cystine ladder motif of theta-defensins[END_REF], which are 18-residue long cyclic peptides with antifungal, antibacterial, and antiviral activities first isolated from primates [START_REF] Tang | A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins[END_REF]. The CLL motif in the C 0 D 0 loop is however more reminiscent of EGF-like domains (Fig. 2D), which are extra-cellular modules identified in animals, found as single or as multiple copies connected sequentially [START_REF] Bork | Structure and distribution of modules in extracellular proteins[END_REF]. Protein-protein contacts involving EGF-like modules are associated with blood coagulation, complement activation, cell adhesion, or determination of embryonic cell fate during animal development [START_REF] Bork | Structure and distribution of modules in extracellular proteins[END_REF][START_REF] Carpenter | Epidermal growth factor[END_REF][START_REF] Stenflo | Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors[END_REF]. These domains are also around 40 aa long and contain six conserved cysteine residues forming three disulfide bonds. The arrangement of these bonds is intermediate between the CLL and the "cystine knot motif" observed in many bioactive peptides [START_REF] Daly | Bioactive cystine knot proteins[END_REF]. In HAP2, the CLL motif is inserted at the turn of a β-hairpin formed by domain I consecutive β-strands C 0 and D 0 , and so the N-and C-termini are next to each other (Figure 2D, left panel). In contrast, the "chain of beads" organization of sequential EGF-like domains directs their termini to opposite ends (Figure 2D). This different topology results in a change in the disulfide connectivity, with a 1-3, 2-4 and 5-6 bonding pattern in EGF-like modules and 1-2, 3-6 and 4-5 in the HAP2 CLL (Figure 2B). The HAP2 C 0 D 0 loop displays an electrostatically charged surface potential (Figure 2C) -a hallmark of EGF-like modules -although the CLL does not display the Ca 2+ binding motif present in a subset of the EGF-like domains [START_REF] Rao | The structure of a Ca(2+)-binding epidermal growth factor-like domain: its role in protein-protein interactions[END_REF].

The stem region.

Another region that had not been resolved previously is the stem segment that connects domain III to the C-terminal TM helix. The new structure resolves the N-terminal 12 residues of the stem, showing that it is directed toward the tip of domain II of the adjacent "left" subunit in the trimer (Figure 3). Compared to the two viral class II proteins for which the stem is resolved, RVFV Gc and rubella virus (RV) E1, the path of the stem to reach the fusion loops in the HAP2 post-fusion trimer is different. In spite of a similar positioning of domain III, the stem does not turn back to interact with domain II from the same protomer (in yellow in Figure 3) as in RVFV. HAP2 is also different to RV E1, as in this case not only the stem but also domain III interacts with the alternative adjacent subunit (to the "right" in the trimer, Figure 3, right panel). The HAP2 stem fills a surface groove in the adjacent protomer, formed between the bc loop and the aefg β-sheet of domain II. The polar contacts include a short segment of antiparallel main-chain β-interactions (strand m, residues 587-589, Figures 1B and1C) with the bc loop, inducing formation of strand s (residues 149-151) (Figure 3, inset). The construct used for crystallization ended at residue G592, followed by the affinity tag. The last residue ordered in the structure is N591 (Figure 1C), with the stem ending roughly at the level of the bdc β-sheet in the domain II tip. In full-length C. reinhardtii HAP2, there are 42 aa between this point and the TM helix, including an amphipathic membrane proximal external region (MPER). Comparison with the viral proteins (Figure 3) suggests that the distance remaining to reach the fusion loops would be spanned by about 10-12 residues, until about position 604. The sequence of the remaining C-terminal part (residues 605-633, Figure S4) is compatible with the MPER present in a number of viral fusion proteins [START_REF] Cai | Biochemistry and biophysics of HIV-1 gp41membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design[END_REF][START_REF] Guardado-Calvo | The Envelope Proteins of the Bunyavirales[END_REF][START_REF] Shogren | Role of glycosylation on the conformation and chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary mucin[END_REF]. The MPER has so far eluded structural characterization for any fusion protein in the context of the post-fusion form (the MPER is not represented, for clarity, in the diagram of Fig. 1A).

The HAP2 membrane insertion region

The packing between HAP2 trimers in the new crystals involves 2-fold symmetric head-tohead interactions along the 3-fold crystallographic axis (Figure S1). This "dimer of trimers" is likely to correspond to the "hexamer" observed in the size-exclusion chromatography (SEC) profile of the purified recombinant HAP2 ectodomain (Figure S1A, left panel). In this assembly, the non-polar residues at the tip of domain II are masked from solvent. This interaction between trimers results in the fusion loops being entirely resolved in the new structure. The segment between β-strands c and d of domain II, which is 40 residues long, projects two loops (cd1 and cd2 in Figure 3B) exposing non-polar side chains. In place of the single αF fusion helix observed in A. thaliana HAP2 and the three potential short fusion loops of T. cruzi HAP2 (Figure 4A), in C. reinhardtii the HAP2 membrane insertion surface is composed of helix αF1 in loop cd1 and αF2 in cd2, running roughly antiparallel to each other (Figure 5, left panels), and a small 3 10 helix (ηF) also in loop cd2 directly preceding αF2. The three "fusion helices" are arranged into a horseshoe shape, exposing the non-polar residues toward the target membrane (Figures 4 and5). αF1 is 9 aa long and exposes residues W173 and F177; ηF is only one turn and exposes F192 and W193, and αF2 is 7 aa long and exposes P196, L197, L200 and I201 (Figure 4). In addition to providing a firm hold of the protein onto the target membrane, the substantial membrane insertion surface is likely to generate a significant stress upon insertion into the outer leaflet of the opposing gamete membrane. The resulting lipid bilayer destabilization can potentially also play a role in facilitating the downstream membrane fusion process.

The HAP2 cd segment is long in comparison with the cd loop of the fusion protein of the well-studied flaviviruses (10 residues) [START_REF] Modis | Structure of the dengue virus envelope protein after membrane fusion[END_REF] or bunyaviruses like RVFV ( 15residues) (Guardado-Calvo et al., 2017), but not when comparing to other class II viral fusion proteins. For instance, RV E1 has a 48 aa long cd inter-strand connection [START_REF] Dubois | Functional and evolutionary insight from the crystal structure of rubella virus protein E1[END_REF], featuring an additional β strand (c') that augments the bdc β-sheet at the domain II tip into bdcc' and two fusion loops, cc' and c'd (Figure 3B). In HAP2, cd1 and cd2 are separated by the arginine residue (Figure 4A) making a strictly conserved salt bridge (R185-E126 in C. reinhardtii HAP2) with a glutamic acid in β-strand b [START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF]. This salt-bridge is a distinctive HAP2 feature among all class II proteins, linking the variable membrane insertion region to the highly conserved core of the protein [START_REF] Fedry | Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes[END_REF]. The R185 side chain not only makes a salt bridge but also donates multiple hydrogen bonds to backbone carbonyls of the highly conserved "ij loop" (Figure 4A), so named because it takes the place of the ij βhairpin of viral class II proteins. The HAP2 ij loop is the central part of the pfam 10699 motif [START_REF] Finn | Pfam: the protein families database[END_REF], a ~50 aa long stretch of conserved sequence that allowed the identification of HAP2 in numerous eukaryotic species.

Although the aa 184-190 stretch (i.e., including the conserved R185) in between cd1 and cd2 was ordered in the previous structure and formed an α-helix termed α0, it displays an extended conformation in the new structure (Figures 1B and 4A,left panel). This segment is also extended in Arabidopsis thaliana HAP2 (Figure 4A, middle panel) [START_REF] Fedry | Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes[END_REF].

The difference between the two C. reinhardtii HAP2 structures in this stretch could have been induced by the in situ proteolytic treatment used to grow the initial crystals. α0 helix folding/unfolding could also be a mechanism required to fully project the cd loop toward the target membrane during the fusogenic conformational change of HAP2. Indeed, the crystal structure of domain II of T. cruzi HAP2 does display an α0 helix in the same location and orientation (Figure 4A, right panel). In contrast to the fusion helices, α0 runs perpendicular to the target membrane. The only non-polar bulky residues present at the membrane insertion region are in the second turn of the T. cruzi HAP2 α0 helix, and are not fully exposed (Figure 4A, third panel), suggesting that this segment may have adopted an alternative conformation in the aqueous environment used for crystallization [START_REF] Fedry | Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes[END_REF].

The non-polar residues of the fusion helices are required for liposome binding.

Our previous results demonstrated that gamete fusion in vivo correlates with the ability of HAP2 to insert into liposomes in vitro [START_REF] Fedry | Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes[END_REF][START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF]. To confirm that the exposed residues of the fusion helices are indeed important for membrane insertion, we examined the behavior of the HAP2 ectodomain and of selected mutants in liposome coflotation in density gradients, as described in the STAR Methods section. All the mutants tested were similar to wild type in terms of expression yields and transit through the quality control system of the producer cells for secretion. The SEC profiles of the mutants were also similar to wild type, indicating that the introduced mutations did not induce any obvious misfolding, as expected for exposed residues such as those being tested.

As P196 induces an important kink causing the shift from ηF to αF2 in loop cd2, we tested the effect of the P196A mutation and found that there were no statistically significant differences with wild type (Figure 4C) in liposome co-flotation. The kink induced by P196 in the conformation of the fusion helices is thus not essential for insertion into membranes. In contrast, we found a more drastic effect when we substituted the non-polar residues by histidine, a polar aa. Indeed, the double mutants W173H/F177H (αF1), W192H/F193H (ηF) and the triple mutant L197H/L200H/I201H (αF2) displayed essentially no detectable liposome binding. The triple mutant L197Q/L200Q/I201Q had the same effect, suggesting that any polar side chain replacing the non-polar bulky side chains of these helices will interfere with membrane binding, as expected. This last result is also an important control, as the introduction of multiple neighboring histidines could induce local repulsion upon their protonation, although the membrane binding assays were done at pH 7.4 and the pKa of solvent-exposed histidine side chains is 6.8. Of note, in contrast to virus fusion driven by class II fusion proteins, which takes place in the acidic environment of the endosomes, Chlamydomonas gamete fusion occurs in the extracellular environment at neutral pH.

When the same bulky non-polar residues were replaced by alanine, which has a small nonpolar side chain, the effect on membrane insertion was less striking: the difference in binding of double mutant W192A/F193A in ηF was less significant with respect to wild type, and W173A/F177A (αF1) or L197A/L200A/I201A (αF2) displayed reduced binding although not as significant as the mutants with polar residues. It required a mutant with alanine substitution in both, αF1 and ηF, W176A/F177A/W192A/F193A, for a complete loss of binding comparable to the experiments involving substitution by polar residues (Figure 4C). These experiments therefore demonstrate that the bulky non-polar residues of the fusion loops indeed control the insertion of the HAP2 ectodomain into lipid bilayers. The differences observed when substituting them to alanine instead of polar residues suggest that certain positions are key anchoring sites requiring a bulky non-polar side chain, whereas others are necessary only to maintain the non-polar character of the surface. This is evidenced in particular by the significant difference in insertion to liposomes of the mutant W173A/F177A compared to W192A/F193A (Figure 4C, bottom panels).

Discussion

The X-ray structure of the C. reinhardtii HAP2 ectodomain described here has higher resolution and provides views of important functional regions that were not resolved in the previously reported structure. One crucial segment that was missing is the tip of domain II and its fusion loops, which must insert into the target membrane and are critical for the membrane fusion function of HAP2. The new structure shows that the domain II tip exposes a hydrophobic platform formed by three short helices projecting ten bulky non-polar residues that are used for membrane insertion (Figure 1A, steps ii to v). This region is involved in hydrophobic head-to-head interactions between two HAP2 trimers to form the crystals (Figure S1B), making a dimer of trimers that was also observed in solution (Figure S1A, left panel).

The arrangement of the membrane insertion surface in C. reinhardtii HAP2 is reminiscent of the α-helical hairpin of the influenza virus HA fusion peptide, which projects nine bulky nonpolar residues (Figure 5, second panel) into the target membrane (reviewed in [START_REF] Apellaniz | The three lives of viral fusion peptides[END_REF]). This structure forms only upon interaction of the HA fusion peptide with the outer leaflet of the membrane, whereas in HAP2 the helical arrangement appears to be pre-formed in the protein. An important difference is that in HA the two antiparallel amphipathic helices pack very tightly against each other, whereas in HAP2 they have a rather loose, horseshoeshaped arrangement. The HAP2 fusion loops are also very different to those of the viral class II proteins that have been characterized, such as the fusion proteins of flaviviruses -i.e, dengue virus E [START_REF] Klein | Structure of a dengue virus envelope protein late-stage fusion intermediate[END_REF][START_REF] Modis | Structure of the dengue virus envelope protein after membrane fusion[END_REF], of Gc from bunyaviruses such as RVFV (Guardado-Calvo et al., 2017) (Figure 5, right panels), and also to the RV E1 fusion loops (Figure 3B). Molecular dynamics analyses using RVFV Gc showed that insertion of the fusion loop involves a rearrangement of the membrane such that the bulky lipid head groups move apart to avoid clashes with the main chain of the inserted loop. The presence of lipids with very small polar heads were found to be required in the membrane to fill the void created in the aliphatic moiety at the site of insertion. This effect would be more drastic in the case of C. reinhardtii HAP2 as the inserted motif is much larger (compare panels in Figures 4A and5). We note that the tips of the three domains II in the HAP2 trimer leave an empty space at the center (Figure 4B), which we propose is important to accommodate the displaced bulky lipid head groups upon insertion of the fusion helices. In influenza virus HA2, the tight αhelical platform of the fusion peptide is loosely tethered via a flexible linker to the central, rigid trimeric α-helical coiled coil of the HA2 subunit of HA [START_REF] Bullough | Structure of influenza haemagglutinin at the pH of membrane fusion[END_REF][START_REF] Chen | N-and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triplestranded coiled coil[END_REF], providing flexibility and leaving space to accommodate the displaced lipid head groups. In HAP2, not only the lateral packing of the fusion helices appears to be loose (the minor effect of the P196A mutation in liposome binding shown in Figure 4C indeed suggest that this region displays conformational malleability), but their connection to the rigid bdc βsheet of the domain II tip also displays flexibility (Figure 4A) that is potentially important for membrane insertion. The relatively flexible connection to the bdc β-sheet could combine with a potential rocking about the strictly conserved R185-E125 salt bridge (Figure 4A). The malleability of this region is further exemplified by formation or not of a vertical α0 helix around the R185 position. We also note that sequence analysis of the HAP2 fusion loop region further suggests that the three fusion helices are not conserved in many algae (Figure S5), and some appear to have a single fusion helix, similar to HAP2 from the flowering plants [START_REF] Fedry | Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes[END_REF]. Future studies using molecular dynamics simulations by taking advantage of the currently available X-ray structures of three orthologs should be useful to better understand the HAP2 membrane insertion process.

A driver for the high variability observed in certain regions of HAP2 may be the requirement of barriers that avoid cross-species fertilization. In the viral class II proteins, the fusion loops are exposed only at the time of fusion, and until this moment the protein is maintained in a metastable conformation via interaction with itself (as diagrammed in Figure 1A, panel i) or most commonly with other proteins, such that that the fusion loops are masked [START_REF] Guardado-Calvo | The Envelope Proteins of the Bunyavirales[END_REF][START_REF] Kielian | Class II virus membrane fusion proteins[END_REF]. The high sequence variability of the HAP2 fusion loops could thus be driven by its necessary interaction with species-specific proteins that recognize and bind to this region. The controlled release of their grip on HAP2 could then be a way to trigger its fusogenic conformational change and exposure of the fusion helices (corresponding to panel ii in Figure 1A). In our in vitro experiments, in the absence of the putative interacting molecules normally present in the in vivo situation, the recombinant HAP2 ectodomain is triggered spontaneously to change conformation, with monomers trimerizing irreversibly in solution to form post-fusion trimers that interact head-to-head (Figure S1). The remaining monomers in our preparations remain competent for membrane insertion, by forming membrane-inserted post-fusion trimers when incubated with liposomes [START_REF] Fedry | Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes[END_REF][START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF]. Cellular fusion events are normally controlled so that they can take place fast but only at the required time and space. This is evidenced, for instance, in neurotransmitter release, where the fusogenic SNARE proteins are controlled by an array of other synaptic proteins that allow for very fast fusion of vesicles that are pre-bound at the cytosolic side of the plasma membrane. The rate-limiting steps involved in priming the fusion machinery have already taken place when a signal provided by calcium influx activates fusion [START_REF] Brunger | Molecular Mechanisms of Fast Neurotransmitter Release[END_REF]. In the case of HAP2-driven gamete fusion, our results now suggest that in addition to fusion loop masking, there may be additional controls at other steps -such as trimerization, which involves rearrangements in the bottom end of domain I. Our data further suggest additional potential means of identifying interaction partners of HAP2 in vivo, for instance by comparative co-immunoprecipitation using HAP2 wild-type and variants mutated at key sites at the domain I bottom. liposome binding experiments. PL completed the crystallographic refinement. TK, JF, EB and FAR conceived the research.

Declaration of interests:

The authors declare no financial interest. FAR is a consultant with Flagship Pioneering. [START_REF] Chao | Sequential conformational rearrangements in flavivirus membrane fusion[END_REF]. v) Fusion pore formation. Upon contact of the two membranes, fusion proceeds via an initial merger of the outer leaflets of the two membranes ("hemifusion", not shown) followed by formation of a fusion pore, as illustrated here.

B) The HAP2 ectodomain, crystallized in the post-fusion form (corresponding to panels iv and v in A). Left panel: the previously reported X-ray structure of a partially proteolysed C. reinhardtii HAP2 ectodomain to 3.3Å resolution. The foreground subunit is colored by domains as in A). The intervening α0 helix is highlighted in brown (brown arrow). Disordered In HAP2, the stem interacts with domain II of the "left" subunit in the trimer. In RVFV, it interacts with domain II of the same subunit (in yellow). In the RV E1 trimer, both domain III and the stem interact with the subunit to the right. For context, a fused membrane is schematized at the top, with hydrophilic and aliphatic moieties in light and dark grey, respectively. The inset (left panel) shows a more detailed view of the stem (magenta) inserted in the groove formed by the bc loop and aefg β-sheet of the adjacent protomer (grey). Dashed lines represent inter-chain hydrogen bonds between the stem and domain II. A. thaliana (center) and T. cruzi (right) [START_REF] Fedry | Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes[END_REF]. The helices in fusion loops 1 and 2 are colored light orange and yellow, respectively (as in Figure 1B). Broken orange lines mark a 2-residue disordered segment in the bc loop in T. cruzi HAP2 that could potentially also insert into the target membrane. The panels were aligned on the conserved salt bridge (indicated by a continuous horizontal line), showing that the membrane would be at a variable distance from the bdc core β-sheet (the horizonal dashed lines mark roughly the limits of the polar moiety of the membrane's outer leaflet). This figure highlights the potential malleability of the membrane insertion surface. density reaches approximately 7x10 6 cells/ml. At 5 days post-induction, the cells were pelleted and the soluble ectodomain was purified by affinity chromatography from the supernatant on a StrepTactin Superflow column. The strep tag was then removed by treatment with enterokinase and the protein was further purified by SEC on a Superdex200 column equilibrated with 10 mM bicine pH 9.3. As we reported previously [START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF], low ionic strength conditions at pH 9.3 reduce formation of HAP2 hexamers. As shown in Figure S1, hexamers appear to form by head-to-head interaction between two trimers via the hydrophobic fusion loops, thus interfering with HAP2 binding to membranes.

The yields of HAP2 ectodomain obtained from S2 cells was around 15 mg/l of cell culture.

The purified protein was concentrated to approximately 5 mg/ml.

We also generated an S2 stable cell line expressing antibody A20 scFv, which was expressed following the same procedures used for the HAP2 ectodomain. The complex between the HAP2 ectdomain and scFv A20 was obtained mixing both proteins in a molar ratio 1:2 and incubating overnight at 4˚C. The HAP2-scFv A20 complex was then separated from unbound scFv on a Superdex200 column. The fractions in Figure S1, right panel, corresponding to the complex were pooled and then concentrated to approximately 7 mg/ml.

Crystallization and Structure Determination

Crystals were grown using the sitting drop method at 293K by mixing HAP2-scFv A20 protein solution in a ratio 1:1 with reservoir solution containing 0.2M LiSO 4 , 0.1M Tris-HCl pH 8.5 and 25% w/v PEG 5000 MME. Small hexagonal plates obtained after 5 days were cryoprotected with reservoir solution containing 15% glycerol and plunged in liquid nitrogen.

Data collection was carried out at the SOLEIL synchrotron (St Aubin, France). Data were processed, scaled and reduced with XDS [START_REF] Kabsch | Automatic indexing of rotation diffraction patterns[END_REF] and AIMLESS [START_REF] Evans | How good are my data and what is the resolution?[END_REF]. Diffraction anisotropy was corrected with DEBYE and STARANISO programs using the STARANISO server (http://staraniso.globalphasing.org/). These programs perform an anisotropic cut-off of merged intensity data, a Bayesian estimation of structure amplitudes and apply an anisotropic correction to the data. The HAP2 structure was determined by molecular replacement using PHASER and the coordinates of the previous crHAP2e ectodomain (PDB accession code 5MF1) as search model. The model was corrected and completed using COOT [START_REF] Emsley | Features and development of Coot[END_REF] and the corrected anisotropic amplitudes were used for model refinement in BUSTER [START_REF] Bricogne | BUSTER version 2.9[END_REF]. The stereochemical properties of the model were validated with MolProbity [START_REF] Chen | MolProbity: all-atom structure validation for macromolecular crystallography[END_REF].

Figure 1 .

 1 Figure 1. The new structure reveals functional species-specific regions of HAP2. (A) Diagram illustrating the rearrangement of viral class II fusion proteins to catalyze membrane fusion. The three domains are colored red, yellow and blue (for domains I, II and III, respectively), the fusion loop orange, the stem magenta and the TM segment grey. Viral and cellular membranes are in light and dark grey, respectively. i) Prefusion arrangement, with the fusion loop buried at an oligomeric contact -a homo-dimer in the flaviviruses (as shown here, with the dimer partner striped for clarity, except that the flavivirus fusion protein spans the membrane twice) or a heterodimer with a companion protein (as in alphaviruses and bunyaviruses). ii) Triggering of the fusogenic conformational change. The acidic environment of an endosome leads to dimer dissociation and exposure of the fusion loops. iii) Extended intermediate bridging the two membranes. Multiple monomers at the viral surface insert into the target membrane with concomitant trimerization into an extended intermediate form, separating the two membranes at distances of about 15 nm. The curved arrows indicate the subsequent relocation of domain III to make a hairpin. iv) Hairpin formation and pulling of the two membranes against each other. Domain III relocation to the side of the trimer redirects the stem toward the fusion loops, thereby pulling the two membranes against each other (grey arrows) to a distance within 1nm, forcing outer leaflet dehydration to allow membrane contacts. Domain III relocation is accompanied by a domain I internal reorganization, indicated by a red square instead of a red circle. Several adjacent trimers are required to drive fusion[START_REF] Chao | Sequential conformational rearrangements in flavivirus membrane fusion[END_REF]. v) Fusion pore formation. Upon contact of the

  domain II loops are indicated by arrowheads. Black arrows at the bottom end mark the observed limits of the domain I C 0 D 0 and E 0 F 0 connections. Middle panel: Ribbon representation of the new 2.6Å resolution structure. The foreground subunit is shown in dark grey with regions not resolved previously highlighted in colors according to the domain they belong to (as in Figure 1A). A central light blue bar marks the molecular 3-fold axis of the trimer. Right panel: the foreground subunit rotated as indicated and colored by domains to allow labeling the secondary structure elements buried in the trimer. In all panels, disulfide bonds are drawn as green sticks and N-linked glycan chains as sticks colored according to atom type (carbon white, nitrogen blue, oxygen red). A brown arrow in the middle and right panels marks the region of extended conformation that corresponds to the α0 helix in the left panel. (C) C. reinhardtii HAP2 aa sequence on a background colored by domains (as in A and B) and secondary structure elements marked. Regions not resolved in the previous structure are boxed. The remaining unstructured regions are in white background. N-linked glycosylation are marked by full or empty blue stars for resolved sugar residues or disordered glycans, respectively. The grey arrow marks the last residue (592) in the HAP2e expression construct. A dashed box frames the MPER.

Figure 2

 2 Figure 2 MLR and CLL motifs in domain I. (A) The HAP2 trimer is shown at the center with boxes indicating the two insets, the E 0 F 0 (left) and C 0 D 0 (right) loops. Left panel: the dashed curved line represents the disordered central region of the E 0 F 0 loop (residues 245-270), with its mucin-like sequence displayed underneath with N-linked glycosylation sequons marked in blue. Right panel: The CLL motif in the C 0 D 0 loop with the disulfides drawn in green and labeled as in Figure 1C. (B) Sequence alignment of the CLL motif showing its conservation across green algae, with cysteines in green the disulfide connectivity indicated. See STAR methods for abbreviations. (C) Surface electrostatic potential of the CLL motif displayed on a scale of -1 to 1 kT/e, from red (acidic) through neutral (white) to basic (blue). (D) The C 0 D 0 loop CLL is shown at the center, next to an authentic cystine ladder motif as observed in θ-defensins (Conibear et al., 2012) (left panel) and to an EGF-like module. The cysteines are numbered sequentially to show the different connectivity of the disulfide bonds (green).

Figure 3 .

 3 Figure 3. The stem inserts into an adjacent subunit domain II groove. HAP2 displayed next to the viral class II glycoproteins for which the stem is resolved: RVFV Gc, (PDB code 6EGU) and RV E1 (PDB accession 4ADI) in their post-fusion conformation.

Figure 4 .

 4 Figure 4. The C. reinhardtii HAP2 membrane insertion surface (A) The tip of C. reinhardtii HAP2 domain II (left) compared to the corresponding region in

  (B) Surface representation of the HAP2 trimer tip colored by hydrophobicity. The grey stripe indicates roughly polar head groups layer of the membrane. Note the space left at the center by the projecting domain II tips of the three trimer subunits, seen on the right panel by depth cuing with white fog. (C) Membrane binding analysis of C. reinhardtii WT and fusion helix mutants of HAP2 by liposome co-flotation assays on iodixanol gradients (Optiprep TM ). Top (T) and bottom (B) fractions of the gradients were analyzed by WB for the presence of the HAP2 ectodomain. The right panels show the quantification of HAP2e detected in the gradient fractions. The values represent the average of three independent experiments ± SD, analyzed by Student's t test. Level of significance are: P<0.05, *; P<0.01, **; not significant, NS.

Figure 5

 5 Figure 5 Comparison of the C. reinhardtii HAP2 fusion surface with viral counterparts. The HAP2 fusion loops (left panel) form a platform projecting hydrophobic residues. As similar platform is formed by the fusion peptide of the class I influenza virus hemagglutinin (PDB code 2KXA). (second panel). The fusion loop of the RVFV (PDB code 6EGU) or the flavivirus fusion protein (PDB code 1OK8) (right panels) is significantly less extensive.

Table 1 . Data collection and refinement statistics.

 1

	Wavelength

Acknowledgements Funding for this project was provided by the ERC Advanced grant CelCelFus (number 340371) to FAR, as well as recurrent funding from Institut Pasteur and the CNRS. JF had a PhD fellowhsip from Ecole Polytechnique AMX.

We thank I. Fernandez for technical help and discussion; W.J. Snell for helpful discussions and for testing the effect antibodies against HAP2 on C. reinhardtii gamete fusion in vivo;

Ahmed Haouz and staff of the Pasteur crystallogenesis facility as well as the staff of Proxima-1 and -2 at the SOLEIL synchrotron for help during crystallization screening and diffraction data collection.

Data Deposition:

The atomic coordinates and structure factors have been deposited in the PDB under accession code 6E18

DATA AND SOFTWARE AVAILABILITY

The atomic coordinates and structure factors of the have been deposited in the protein data bank under the accession code 6E18.

Authors contributions: TK made the HAP2 constructs, JF optimized expression. TK and FJ immunized mice with the recombinant HAP2 ectodomain to obtain HAP2-specific monoclonal antibodies, and determined the aa sequence from the hybridomas. JF made constructs expressing Fab and EB the constructs for scFv from two of the antibodies. EB and FAR designed the HAP2 mutants in the membrane insertion region. EB obtained crystals using the HAP2/A20 scFv complex, determined the new X-ray structure and conducted the

STAR Methods

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact, Félix A. Rey (felix.rey@pasteur.fr).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Insect cell culture:

Drosophila S2 cells were maintained in flasks at 28°C in HyClone medium (GE Healthcare Life Sciences) supplemented with 50 units/mL of penicillin, 50 units/mL of streptomycin and 7 µg/mL of puromycin for selection of stable transfectans. Large-scale cultures for protein production were grown in spinner flasks at 100 rpm. No information on the sex of these cells is available.

Mammalian cell culture:

Expi293F cells were grown at 37°C in Expi293 expression medium (Thermo Fisher) with rotary agitation at 135 rpm. No information on the sex of this cell line is available.

METHOD DETAILS Expression construct for the single chain variable fragment (scFv) of monoclonal antibody A20

Monoclonal antibodies targeting C. reinhardtii HAP2 were generated as described previously [START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF]. For sequencing of the antibody variable regions total RNA was prepared from A20 hybridoma cells and cDNA was synthesized using separate constant region primers for heavy and light chain. Variable regions of heavy and light chain genes were subsequently amplified using gene-specific primers and subcloned into a plasmid for insect cell expression of scFvs described previously [START_REF] Gilmartin | High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells[END_REF].

Protein expression and Purification

Drosophila S2 stable cell lines expressing soluble C-terminally truncated Chlamydomonas reinhardtii HAP2 ectodomain (residues 23-592), were generated to obtain high yields of recombinant proteins for crystallization experiments [START_REF] Fedry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF]. The expression construct included an enterokinase cleavage site (sequence DDDDK) immediately downstream HAP2 G592. at the C-terminal end of the ectodomain, followed by a double strep-tag for purification. The expression of the protein is under the control of metallothionein promoter, which is induced by adding 4 µM CdCl2 to the cell culture medium when the cell

Design and expression of HAP2 fusion loops mutants

Recombinant HAP2 ectodomain and fusion loops mutants for liposome co-flotation experiments were obtained by transient transfection of mammalian cells, by using the pcDNA3.1(+) plasmid with the gene coding for the HAP2 ectodomain (residues 23-592) followed by a double strep-tag at the C-terminal end of the ectodomain. Mutants at positions 173, 177, 192, 193, 197, 200 and 201 were generated by standard PCR methods. Expi293 cells were transfected with WT or mutant constructs using polyethylenimine (PEI, 1 mg/ml) in a DNA:PEI ratio 1:4. Cell culture supernatants were harvested at 5 days post-transfection and protein was purified by affinity chromatography as mentioned above. As for the protein produced in S2 cells, the purified proteins were buffer exchanged to 10 mM bicine pH 9.3 and concentrated to 1 mg/ml, to minimize hexamer formation.

The yields of HAP2 WT expressed in mammalian cells was 200 µg of pure protein per 30 ml of cell culture. The yields of all HAP2 fusion loop mutants tested was in the range of 1 to 2 times the yield of the WT protein.

Liposome Co-flotation Experiments

Liposomes composed of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine), cholesterol and sphingomyelin in a molar ratio1/1/3/1, were prepared by the freeze-thaw and extrusion method. 1µM protein was mixed with1 mM freshly prepared liposomes and incubated for 1h at 25oC in 100 mL PBS. Samples were adjusted to a final concentration of 40% Optiprep TM (in PBS), loaded in centrifuge tubes and overlaid with 4.5 mL 20% Optiprep TM and 0.3 mL PBS. Centrifugations were performed overnight at 4oC and 192000 x g on SW55ti rotor. Top and bottom fractions of the gradient were analyzed by immunoblotting using anti-Strep tag antibody.

Illustrations: Cartoon representations were made with PyMol [START_REF] Schrodinger | The PyMOL Molecular Graphics System[END_REF]. The HAP2 amino acid sequence alignments displayed were made by MUSCLE [START_REF] Edgar | MUSCLE: a multiple sequence alignment method with reduced time and space complexity[END_REF]) and rendered with ESPript 3.0 [START_REF] Robert | Deciphering key features in protein structures with the new ENDscript server[END_REF]. The organisms represented in Figure 2 have the following abreviations: Chlamydomonas reinhardtii (Cr), Gonium pectoral (Gp),

Volvox carterii (forms

QUANTIFICATION AND STATISTICAL ANALYSIS

Densitometry analysis of western blots from liposome co-flotation assays was performed with ImageJ software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] and expressed as percentage of HAP2e in top and bottom fractions of iodixanol gradients. Results are shown as mean ± SD of three independent experiments (n=3). Differences in liposome binding between HAP2e WT and mutants were analyzed by a Student's t-test (P<0.05, *; P<0.01, **).


Didier Montet
email: didier.montet@cirad.fr

Jamal Eddine Hazm

Abdelouahab Ouadia

Abdellah Chichi

Mame Samba Mbaye

Michel Bakar Diop

Paul Mobinzo

Apollinaire Biloso

Isaac Diansambu

Joël Scher

Mame Samba Mbaye

Marie-Louise Scippo

Maria-Teresa Barreto Crespo

Contribution of the methodology of collective expertise to the mitigation of food safety hazards in low-or medium-income countries

Keywords: Food safety, Food safety authorities, Collective expertise, Microbial and chemical contamination, Food safety management systems Highlights

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Availability of food should be sufficient to nourish the global population. Nevertheless, according to international institutions (FAO, IFAD, UNICEF, WFP & WHO, 2017), people with hunger appear to be augmenting, affecting 11% of the global population. In addition, the number of undernourished people globally was 815 million in 2015, against 777 million in former year. Several factors, interconnected and variable, emerging and re-emerging, can explain and at the same time can intensify this continuous problem of food insecurity. Examples of these factors are fragility and conflict, civil insecurity, large-scale displacement, climate-change (i.e. with drought conditions), natural resources degradation, distribution and lack of awareness in the field of healthy nutrition.

Having food must also mean that people are nourished with safe food, so the links between food security and food safety are inextricable anywhere in the world [START_REF] Jones | What Are We Assessing When We Measure Food Security? A Compendium and Review of Current Metrics[END_REF][START_REF] King | Food safety for food security: Relationship between global megatrends and developments in food safety[END_REF]. Internal data on deaths and hospitalizations caused by food do not show that everybody is eating safe food. Recent data reveal that of the 56.4 million deaths worldwide in 2015, more than half (54%) were due to the following 10 causes: ischemic heart disease, stroke, lower respiratory infections, chronic obstructive pulmonary disease, trachea and bronchus cancers, diabetes mellitus, Alzheimer disease, diarrheal disease, tuberculosis, road injury. However, these causes of death differ according to the level of country income. Infectious diseases account for 43% of deaths in low-income countries compared to 1% in high-income countries. In particular, the diarrheal diseases cause 550 million people to fall ill and 230 000 to die every year (WHO, 2017a). Children under 5 years of age carry 40% of the foodborne disease burden, with 125 000 deaths every year.

Moreover, when looking at the data, it has to be taken into consideration that these numbers are only estimates. If in high-income countries statistical institutes collect information on deaths, causes of deaths and on people hospitalized, in many low-and middle-income countries no statistical institute exists, or, when they exists, institutes they are not entirely reliable, consequently the numbers of deaths from specific causes have to be estimated from incomplete data. Statistics on the causes of death and of hospitalizations can help the health authorities to determine the direction of their public health actions, not only because hospitalizations are very expensive for health care systems, but also foodborne diseases and food contamination can cause huge problems in tourism, trade and on families that do not have health care protection.

Regarding food related health hazards, it is important to have presented some contextual elements of food chains around the world such as, the globalization of the food supply; the increased complexity of those chains; the impact of contaminated food on human health and the impact on the economic well-being of the agri-food industry. It is therefore necessary to assess the hazards and risks associated with food products, throughout the food chains, from production to consumption. Indeed, at each stage, the risks of contamination are significant, when considering the diversity of production systems, the transformation systems, the transport and conservation systems and conditions and the preparation and consumption modes, all in the new context of climate change.

Foodborne illnesses are usually infectious or toxic in nature and caused by microbiological/biological agents or chemical substances through the consumption of contaminated food or water.

The notion of toxicity is a function of a complex relationship between the doses and the effects of an agent but time of exposure and the category of exposed population (i.e children, elderly people and sick people) are also important parameters to consider. In developed countries, it is usual to work on risks to protect population from the hazards.

In low-and middle-income countries, risk management is very weak and the population suffers from different food associated hazards. African situation will be the main focus of this article, although some of the problems mentioned here can be extrapolated to other continents.

Food hazards

Microbiological contamination

Among the more than 250 foodborne diseases, the most frequent ones are infectious diseases that are caused by the contamination of food with bacteria, fungi, virus and parasites [START_REF] Who | WHO estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group 2007-2015[END_REF] [START_REF] Koopmans | Improving food safety through a one health approach[END_REF][START_REF] Hikal | Food Related Parasitic Infection: A Review[END_REF]. Filamentous fungi (molds) produce mycotoxins that can cause acute or chronic toxicity in human and animals. Mycotoxins can accumulate in maturing corn, cereals, soybeans, sorghum, peanuts, and other food and feed crops in the field and in grain during transportation. Members of three fungal genera, Aspergillus, Fusarium, and Penicillium, are the major mycotoxin producers [START_REF] Alshannaq | Occurrence, Toxicity, and Analysis of Major Mycotoxins in Food[END_REF]. The most common mycotoxins found in food are aflatoxins, trichothecenes, zearalenone, fumonisins, ochratoxins, and patulin, all of them posing ongoing food safety problems worldwide and in special in Africa (Yacine [START_REF] Ware | Occurrence of mycotoxins in commercial infant formulas locally produced in Ouagadougou (Burkina Faso)[END_REF][START_REF] Manizan | Multi-mycotoxin determination in rice, maize and peanut products most consumed in Cote d'Ivoire by UHPLC-MS/MS[END_REF]. Other toxicologically important mycotoxins, are such as ergot alkaloids, enniatins, alternaria toxins, moniliformin, citrinin, beauvericin, cyclopiazonic acid, roquefortin C, mycophenolic acid, penitrems, verruculogen, griseofulvin or citreoviridin.

Food poisoning can be due to other types of toxins such as ciguatoxins produced by benthic eukaryotic dinoflagellates of the genus Gambierdiscus, mycotoxins produced by molds, or toxins produced by bacteria such as Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum or Bacillus cereus [START_REF] Friedman | An Updated Review of Ciguatera Fish Poisoning: Clinical, Epidemiological, Environmental, and Public Health Management[END_REF][START_REF] Martinović | Foodborne pathogens and their toxins[END_REF][START_REF] Liu | Probing and Quantifying the Food-Borne Pathogens and Toxins: from in vitro to in vivo[END_REF].

Microbial resistance to antibiotics is another type of food safety hazard because foodborne infections caused by antibiotic resistant bacteria can face possible treatment failure [START_REF] Lammie | Antimicrobial Resistance, Food Safety, and One Health: The Need for Convergence[END_REF][START_REF] Founou | Antibiotic Resistance in Food Animals in Africa: A Systematic Review and Meta-Analysis[END_REF].

Chemical contamination

Because of their usefulness, the chemicals used are diverse but can be dangerous. The chemicals used are very diverse because of their usefulness but can be dangerous. These chemicals can be: 1-products added or generated by food; 2-residues of veterinary drugs, 3-industrial and agricultural pollutants; 4-food contact materials (example of plastic materials migration products) [START_REF] Rather | The Sources of Chemical Contaminants in Food and Their Health Implications[END_REF]. Some of the chemical contaminants are compounds that accumulate in the body, and in the environment, they can be named Persistent organic pollutants. Examples of this type of compounds are dioxins or polychlorinated biphenyls (PCBs). Dioxins for instance cause reproductive and developmental problems, damage the immune system, interfere with hormones and cause cancer. Heavy metals as lead, cadmium and mercury cause neurological and kidney damage.

Legislation and Codex Alimentarius

Due to several food crisis in the 90's of 20 th century, the European Union re-accessed completely the food and feed paradigm, with the creation of an integrated approach from production, to transformation and commercialization of either food or feed, the "farm to fork" approach. In 2002, the European Parliament and the Council adopted the Regulation (EC) No 178/2002 laying down the general principles and requirements of food law (General Food Law Regulation, European Commission, 2002a). It also set up an independent agency responsible for scientific advice and support, the European Food Safety Authority (EFSA). Moreover, it created the main procedures and tools for the management of emergencies and crises and also the Rapid Alert System for Food and Feed (RASFF).

The idea behind this overarching and coherent framework for the development of food and feed legislation in each EU country and the EU as a whole was to provide safe food for human and animal consumption, but letting the market act by giving responsibility to stakeholders. All these regulatory documents are regularly updated since their first publication, meaning that all amendments have to be considered as well. The main route of exposure to these environmental contaminants is food from animal origin for dioxins and PCBs [START_REF] Malisch | Dioxins and PCBs in feed and food--review from European perspective[END_REF], marine fish for mercury (EFSA, 2010a), and food from both animal and vegetal origin for lead and cadmium [START_REF] Efsa | Cadmium in food. Scientific Opinion of the Panel on Contaminants in the Food Chain[END_REF](EFSA, , 2010b)). Furthermore, the combined exposure to mixtures of chemicals, which can act synergically, is highly suspected to induce negative health effects worst that the effect expected from the exposure to a single chemical, as it is done currently in toxicological studies [START_REF] Bopp | Current EU research activities on combined exposure to multiple chemicals[END_REF].

In low-and middle-income countries if a coherent body of legislation concerning food safety is not in place, as the one from EU mentioned above, the countries use Codex Alimentarius. Codex Alimentarius seeks to provide tools for people to have "safe and good food for everyone -everywhere", especially in the actual context of globalized food distribution and consumption. The Codex has international food standards, guidelines and codes of practice contribute to the safety, quality and fairness of this international food trade. Codex standards are based on sound science provided by independent international risk assessment bodies or ad-hoc consultations organized by FAO and WHO so they can adopted with trust (Codex Alimentarius).

WHO along the years has published numerous and very different types of documents that deal with food safety. Those guides, posters, or fact sheets on food safety are all publically available, as well as strategic plans to attack the problem of having unsafe food been eaten by the populations (WHO, 2012; WHO, 2107b; WHO, 2018a-c).

Moreover, training on basic principles of food hygiene on production, distribution, and retailing and even at home have been extensity promoted all over the world by NGOs, governments, universities, and other stakeholders.

The challenge

Reasons for continuing situation of existence of foodborne diseases and consequent deaths?

Still in many African countries, the people eat unsafe food and die daily, especially children, due to foodborne diseases. Foods contaminated with microbiological, chemical, physical or other hazards are more prone to be eaten in situations of food insecurity. Numerous reasons can be given for foodborne diseases continue to kill and sent people to the hospitals. Some of them, not pretending to be exhaustive are presented below.

The first one is the lack of data on the identification of the microbial agent or chemical agent that provoked the disease; limited data on severe or minor outbreaks of foodborne diseases. The data would allow a clearer public health policy for treatment and prevention of diseases. A solution to this nonexistence could be the creation of a health statistic agency/department at the government level and preferentially in the Ministries of health.

The second one is the importance of water. Safety of water is critical for drinking and to be used in the production, transformation and preparation process of food. Foodborne and waterborne diseases can be regarded as associated or as separated issues; nevertheless the hazards associated are in many cases the same.

Another very important problem is the public vs. private medical assistance in some of low-and middle-income countries. Public hospitals are poorly equipped and are not many; on the contrary, private hospitals have all conditions to provide good treatment.

Data from Eurostat (European Union, 2016) on the public expenditure on health (% of budget), an indicator that is expressed as a proportion of total general government expenditure (for European countries, it has been calculated as the proportion of general government health expenditure in total general government expenditure), indicate that in 2013 and 2014, the values in EU countries can be the double or more of values in some African countries (i.e. Morocco 6.0 (2013), Senegal 8.0 (2013), France 14.3 (2014)). Moreover, populations that do have social security, as in EU, access at fair price to health care. So, treating the disease rests in the responsibility of the families, if they have or not resources to go to private hospitals. The problem is more acute in rural areas where people can be very far away from hospitals, have no resources and so are left untreated [START_REF] Pariyo | Changes in utilization of health services among poor and rural residents in Uganda: are reforms benefitting the poor[END_REF].

The sensibility of the authorities is also a big problem because of the cost of the social insurances in low-income countries. Many consider that working on food safety has a cost and the return money is negligent simply because the human cost is linked directly to its level of insurance. In high-income countries, the human cost follows a complex calculation linked to the cost of the social security for sick people and the cost of insurances for death people. It is also possible to think to the culture of deaths in Africa that permit to link family (ITUC-Africa, 2012;[START_REF] Ekore | African Cultural Concept of Death and the Idea of Advance Care Directives[END_REF].

Also, the fact that different approaches to food safety are done by academics and by politicians can slow down the improvement of the situation. Academics rely, to study food safety issues, on microbiological and chemical analysis and on the interpretation of those analyses. They are the ones that master the interpretation and such can have knowledge on the hazards and risks faced by the population. Governments and government officials have to decide on food safety issues but do not know how to interpret data in real time.

There is need for the creation of national entities that manage food safety at national level. Manage means, in this context, risks analysis and expertise, proposition to deal the risks and risks communication.

Last, but no least, to face these constraints and ensure prevention and control of food contaminants, it is essential to implement Food Quality Management Systems (FQMS) and Food Safety Management Systems (FSMS), based on the application of the Hazard Analysis and Critical Control Points (HACCP) method and its prerequisites (Good Practices) throughout the food value chains. This has been the strategy of EU and several other countries (USA, Japan, Australia…) that already have these systems in place.

In some countries, namely African countries, where there is a lack of a structured organization and/or legislation for food quality management's systems, as mentioned, the implementation of Codex Alimentarius is the first resource for food safety assurance. But the implementation of some of the proposals, is not always straightforward for SME of the food sector, for local producers and local retailers. The problem is that in some countries most of the enterprises are in fact micro enterprises and people do not have information and formation or skills to establish HACCP systems, do not have financial capacity to perform analysis and national authorities do not have enough resources to accompany so many enterprises. Therefore, there is an urgent need for a systematic/proactive, cost-effective approach towards the control of biological and chemical hazards along the agri-food chains in low-and middle-income countries. Efficient FSMS should take into account the socio-economic context along with the organizational and technological capabilities of the chain stakeholders, including farmers, seed producers, cooperatives, storage and transportation infrastructures, SMEs, as well as market intermediaries and actors.

The implementation of a food safety management system turns out to be a difficult process for many food businesses managers because of the complexity of the available documents and methods. The eastern Mediterranean countries have already pinpointed the deficiencies in their systems and the need to update food safety laws and regulations, food standards and control management, effective food inspection and enforcement services, food monitoring and surveillance systems with adequate laboratory resources, foodborne disease surveillance systems, food safety education and training, and timely information and communications [START_REF] Alwan | Food safety in the Eastern Mediterranean region: time to act[END_REF]. These deficiencies can be extended to many other African and non-African countries.

Using Collective Expertise as a methodology to promote food safety

In low-and middle-income countries, knowledge on microbial and chemical contaminants in food chains exists in universities and public or private research institutes. Researchers have, for most of them, done graduate studies, post-docs or advanced training studies outside their countries around the world. Analysis that were done, even if they are not numerous, can be interpreted by those researchers/teachers and then be turned into data that can feed political decisions. Moreover, due to international scientific contacts, internet connection, agricultural and food big data and scientific literature the data from other countries and similar situations can be the source of data that can be related to local situations and analysis of local situations.

So, the proposal is to use as tool the knowledge translation. Transferring the knowledge already existing in local universities, private companies, citizen's organizations, to authorities involved in managing food safety, can be a powerful way to develop local strategies to solve problems and face the challenges of food safety and food security.

Transfer could be made through the use of Collective Expertise.

Collective expertise in the scope of this work consists in collecting, sharing and highlighting information available in the universities, in experts working in the field of food safety in private companies and other stakeholders.

Teachers by their function are confronted with all the problems related to these fields.

They supervise the internships of the students in relation with the socio-economic world, they are asked to give opinions, or to take part in the resolution of the problems of food safety, as well as various specialized (in charge specifically of the concerned domains) or not specialized institutions (local authorities, companies, NGOs). As a result, they have an informed opinion on all major challenges in the areas concerned.

Even if this approach is not based on actual studies, it has the merit of valuing a wealth of information and, precisely, makes it possible to circumvent the requirements of country-wide studies, which are inherently cumbersome, expensive and long.

If gathered and being consulted in groups, teachers from several universities in a country as well as the other stakeholders, become experts belonging to a Collective Expertise panel. These groups of experts due to their different geographical origins and scientific and technical background, will have an accurate idea of the state of food safety in the country. Such a panels will represent, for authorities, a mean of having proposals and scientific / technical advices of interest for the mitigation of the national problems, with minimal costs.

Collective Expertise can be implemented by organizing meeting of experts in different areas related to food safety like, chemistry, microbiology, social sciences, law, and so on. The experts should be around 20-25, being divided in two groups and for two days come up with a list of the major challenges of a country in terms of food safety that can be reported as:

• Identification of the challenge;

• Brief description of challenge;

• The impact -socio-economic, health, science, legal… levels, legislation… The use of Collective Expertise will allow the country authorities to respond to different challenges such as:

• Increase food safety for local and outside (exports) consumers;

• Develop or improve a local food safety policy and plan of action;

• Share responsibilities between industry, consumers and government;

• Inform consumers to help them make the best choice of food and prepare their food properly;

• Sensitize consumers to be more demanding about the quality of food;

• Strengthen the food control system from farm to table with the participation of stakeholders;

• Increase the level of credibility and competence of regulatory authorities;

• Increase industry and consumer awareness and participation in food safety.

  The main set of EU legislation produced for microbiological hazards Regulations are Commission Regulation (EC) No 2073/2005 (European Commission, 2005a) including all its amendments. The main EU regulatory texts relating to chemical hazards are Regulation (EC) No 1881/2006 on the setting of maximum levels for certain contaminants in foodstuffs (European Commission, 2006), Regulation (EC) No 470/2009 laying down Community procedures for the establishment of residue limits for pharmacologically active substances in food of animal origin (European Commission, 2009a), Regulation (EU) No 37/2010 on pharmacologically active substances and their classification with regard to the limits maximum residue levels in food of animal origin (European Commission, 2010), Regulation (EC) No 396/2005 concerning the maximum limits for pesticide residues in products of plant and animal origin (European Commission, 2005b), Regulation (EC) No 1107/2009 concerning the placing of products on the phytopharmaceutical market (European Commission, 2009b), Regulation (EC) No 1935/2004 on materials and articles intended to come into contact with foodstuffs (European Commission, 2004), Regulation (EU) No 10/2011 concerning plastic materials and articles intended to enter in contact with foodstuffs (European Commission, 2011), Regulation (EC) No. 1333/2008 on food additives (European Commission, 2008). To these texts which concern foodstuffs is added Directive 32/2002 / EC concerning undesirable substances in animal feed (European Commission, 2002b).

  The utilization of Collective Expertise was tested in the scope of the EU project Erasmus+ project (EU) entitled "Societal Challenges and Governance of African Universities: the case of ALIments in Morocco, the Democratic Republic of the Congo and Senegal (DAfrAli)", seeks to strengthen the governance capacity of African Higher Education Institutions to mobilize their resources in order to respond to major societal challenges in relation to external stakeholders. The results of using the methodology Collective Expertise pilot exercises are detailed in a second article untitled: Analysis of societal challenges in food safety by collective expertise in Morocco, the Democratic Republic of Congo and Senegal.

  . The most relevant biological contaminants are bacteria (Campylobacter jejuni, Clostridium botulinum and Clostridium perfringens, six Listeria monocytogenes, Salmonella spp., Shigella spp.,

	(EHEC); Enterotoxigenic E. coli (ETEC); Enteropathogenic E. coli (EPEC);
	Enteroaggregative E. coli (EAEC); Enteroinvasive E. coli (EIEC) and diffusely adherent
	E. coli (DAEC)), Staphylococcus aureus, Vibrio spp.; viruses (such as Norovirus, Astrovirus, Rotavirus,
	Enterovirus, Hepatitis A and E), parasites (protozoa such as Giardia duodenalis,
	Cryptosporidium parvum, Cyclospora cayetanensis, Toxoplasma gondii, Trichinella
	spiralis and amoeboid parasites such as Entamoeba histolytica) (CDC, 2018;
	Escherichia coli pathotypes (Shiga toxin-producing E. coli (STEC)-STEC also
	referred as Verocytotoxin-producing E. coli (VTEC) or enterohemorrhagic E. coli

Acknowledgements

This work was supported by the European Union, through the funding of the Eramus+, Key Action No2, project DAFRALI -"Défis sociétaux et gouvernance des universités AFRicaines: le cas des ALIments au Maroc, en République démocratique du Congo et au Sénégal, No 2015/C 347/06. This project is coordinated by AUF Belgium. MTBCrespo acknowledges iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundaça õ para a Ciência e Tecnologia / Ministe ŕio da

Authority and laying down procedures in matters of food safety. Official Journal L 31, 1.2.2002, 1-24. European Commission. (2002b) L 140, 30.5.2002, 10-22. European Commission. (2004) L 364, 20.12.2006, 5-24. European Commission. (2008) L 152, 16.6.2009, 11-22.


Elise Cleva

Maria Lalova

Thibault Astier

Anna Germe

Alexia Lamotte

Béatrice Blockelet

Christophe Debien
email: christophe.debien@chru-lille.frlesauteursdéclarentnepasavoirde

Keywords: Suicide, prévention, innovation, profession Suicide, prevention, innovation, profession

En janvier 2015, dans la lignée de plusieurs décennies de travaux scientifiques basés sur le maintien du contact, naissait un dispositif novateur en prévention du suicide : VigilanS. Pour assurer ce lien, le choix a été fait de constituer une équipe composée, à parts égales, d'infirmiers et de psychologues, installés au sein même de la régulation médicale. À présent, ces derniers sont désignés sous l'appellation de « VigilanSeur » : une entité originale qui souligne l'émergence de ce nouveau métier situé au carrefour de plusieurs disciplines.

Parler du suicide sans tabou

Que ce soit lors des appels à J10-J21, à 6 mois ou lors des appels entrants, les VigilanSeurs doivent avant tout faire preuve d'une grande sollicitude envers l'état de santé actuel du patient et vérifier l'adéquation avec le projet de soin négocié à la sortie de l'hôpital.

Lors d'un entretien semi-structuré, ils évaluent la présence d'éventuels troubles psychopathologiques puis explorent la présence et l'intensité d'idées suicidaires avec tact et bienveillance.

Le recueil réalisé (basé sur des outils validés) permet d'identifier les facteurs de risques et les facteurs protecteurs d'un éventuel nouveau passage à l'acte.

L'entretien permet d'apprécier la présence et la qualité de l'étayage amical et familial, l'accès et l'observance des soins, la notion de maladie ou de douleurs chroniques… Cette évaluation s'appuie aussi sur l'inquiétude de l'entourage ou des professionnels de santé de proximité et permet d'aborder les idées reçues sur le suicide mais aussi ses conséquences sur les proches, entre incompréhension, colère, culpabilité et peur de la récidive.

Enfin les VigilanSeurs s'assurent que le patient est en possession des coordonnées d'urgence et évaluent sa capacité à les utiliser en cas de nécessité. L'entretien clinique permet de repérer des anomalies au niveau des capacités attentionnelles, de mémorisation, d'orientation spatio-temporelle, de prise de décision et d'autonomie du patient.

Le numéro vert, fourni sur la Carte-Ressource, donne la possibilité au patient, à son entourage parfois, d'évoquer les problématiques suicidaires sans tabou, auprès d'un professionnel, au moment où il en a besoin.

On observe, de plus, un phénomène de « contamination » positive : la Carte-Ressource est confiée à un patient en crise, non par un soignant, mais par une personne qui a, auparavant, bénéficié du dispositif... et, ce, bien souvent, avant le passage à l'acte !

Le téléphone : un nouvel espace thérapeutique ?

Les appels passés et reçus représentent de véritables actes de soins fondés sur l'écoute active, l'empathie et la bienveillance [START_REF] Kraus | Voice-only communication enhances empathic accuracy[END_REF]. Ils permettent de maintenir un lien avec le sujet vulnérable et/ou son entourage inquiet et de favoriser les échanges.

Le patient est parfois agréablement surpris de cette prise de contact. D'ailleurs, l'entretien téléphonique permet au discours d'être plus libre comparativement à l'entretien présentiel, comme si la peur du jugement, l'angoisse du regard de l'autre était partiellement gommée. L'utilisation du téléphone a permis aux VigilanSeurs de développer des compétences particulières en matière d'écoute : ils portent une attention soutenue à l'intonation de la voix, la prosodie, les silences, mais aussi à la cohérence des propos, au choix des mots et aux émotions exprimées. Enfin, ils restent attentifs aux bruits de fond, à l'éventuelle présence d'une personne de l'entourage... Chaque détail compte et le lien s'en trouve d'autant plus étroit.

La relation est également différente, le patient pouvant refuser ou interrompre l'entretien à tout moment, ce qui lui donne une certaine liberté. L'entretien se fait dans l'environnement quotidien et familier du patient et les VigilanSeurs ont ainsi accès à d'autres éléments qu'en suivi classique.

Au final, le téléphone est un bon tremplin vers l'initiation d'un suivi, il balaye souvent les a priori que les patients peuvent avoir concernant les soins psychiatriques.

Mais il faut parfois cadrer les débordements de patients trop familiers, « désinhibés » par cette relation à visage nécessairement couvert, qui peut aboutir à des propositions inappropriées comme des demandes en mariage !

Vigilanseur, un nouveau métier ?

Issus d'horizons et de formations différentes, les VigilanSeurs sont des infirmiers ou des psychologues. Certains ont travaillé en milieu carcéral, d'autres en pédopsychiatrie, en libéral, en psychiatrie adulte ou encore dans le domaine de la recherche. Ce mélange de cultures permet un enrichissement mutuel.

Au sein de VigilanS, psychologue et infirmier exercent une collaboration en coresponsabilité. Il en résulte une grande richesse créative : par exemple, les psychologues apportent leurs outils spécifiques pour travailler la motivation du patient et les infirmiers, leurs connaissances des réseaux de soins. Tisser du lien est la fonction clé des VigilanSeurs afin de créer un « filet de sécurité » en cas de crise suicidaire. Qu'il s'agisse du lien entre le patient et le dispositif, du lien entre le patient, sa problématique et son environnement ou du lien entre le patient et son réseau de soin.

VigilanS ? De la prévention, de la post-vention mais surtout de l'urgence !

Si le dispositif VigilanS a été initialement pensé dans un objectif de prévention de la récidive, l'étude pré figurative ALGOS [START_REF] Vaiva | ALGOS: the development of a randomized controlled trial testing a case management algorithm designed to reduce suicide risk among suicide attempters[END_REF] avait montré l'importance autant clinique qu'éthique ou déontologique de prévoir une réponse aux situations d'urgence.

Logiquement confrontés à des urgences vitales au quotidien, que ce soient une menace de passage à l'acte imminente, un passage à l'acte effectif ou même un geste pendant l'appel, les VigilanSeurs ont développé des compétences en matière de gestion de la crise : évaluation du risque vital, conseils de premiers secours et de mise en sécurité, organisation et coordination des actions de secours. Seuls face aux patients, seuls à pouvoir agir et à prendre les justes décisions dans l'immédiat, ils doivent faire preuve de sang-froid et d'inventivité.

Car l'envoi de secours en collaboration avec la régulation médicale est probablement la chose la plus « simple » à faire : le SMUR se déplace et transporte le patient vers les urgences de proximité. Toutefois, dans la plupart des cas, les VigilanSeurs doivent « tenir la ligne » en attendant que les secours arrivent sur place : des moments difficiles qui requièrent une grande maîtrise émotionnelle. Mais c'est lorsque la situation est urgente, sans engagement du pronostic vital, que les VigilanSeurs doivent faire preuve de créativité pour trouver et négocier des solutions avec le patient. Ils déploient des trésors d'imagination pour identifier et mobiliser les ressources de proximité : famille, amis, voisins, pharmacien, infirmière libérale, médecin traitant, éducateur... Dans un premier temps, l'objectif est de sécuriser les lieux en limitant l'accès au moyen létal envisagé, puis d'organiser une rencontre avec un soignant.

Les patients sont ensuite orientés vers des professionnels en fonction de leurs besoins (assistantes sociales, aides juridiques, secteur, consultations spécifiques, associations…) : pour chaque facteur de risque identifié, les VigilanSeurs s'efforcent de co-construire une solution accessible. Par toutes ces actions, ils tentent de réouvrir le champ des possibles là où, bien souvent, le patient n'en voit plus.

Mais leur rôle ne s'arrête pas là : les VigilanSeurs peuvent aussi accompagner l'entourage et les professionnels d'une personne, hélas, décédée. Ils offrent un espace d'écoute, d'orientation parfois de débriefing à ceux qui en expriment le besoin. Au total, la mission des VigilanSeurs s'est progressivement déplacée d'une « simple » veille à un véritable travail de case management : évaluation des besoins de la personne, élaboration d'un plan d'intervention, mise en lien avec les services et les ressources disponibles, suivi des interventions proposées et monitoring.

Autonomie, prise de décision et coordination médicale

Au téléphone, le VigilanSeur est seul face au patient : il apaise, écoute, analyse, négocie avec le patient les solutions envisagées puis il prend les décisions adaptées.

Le VigilanSeur reste très autonome, apte à identifier ses capacités et ses limites. Il fait appel, si besoin, à l'expertise et au recul de ses collègues face à une situation difficile.

Par ailleurs, les VigilanSeurs disposent de plusieurs opportunités de régulation et de concertation médicale :

-En « direct », au quotidien, le coordinateur médical reste à leur disposition par téléphone pour analyser les cas difficiles.

-Lors du débriefing du vendredi midi qui constitue un temps institutionnel convivial et primordial pour la cohésion de l'équipe, la ventilation émotionnelle, l'analyse des rapports avec les réseaux de soin et la supervision des situations cliniques.

Une nécessaire mutation

Le métier de VigilanSeur n'est pas en soi facile à circonscrire : au carrefour de plusieurs disciplines, de plusieurs métiers, il nécessite de grandes capacités d'adaptation et d'organisation pour répondre aux différentes facettes de son exercice, que ce soit la veille ou le case management.

Mais dans la perspective d'une réorientation des soins centrés sur le « patient-partenaire », le métier de VigilanSeur pourrait constituer un exemple dans cette mutation qui s'amorce.

Ainsi, après trois ans d'existence, ce nouveau métier continue d'évoluer au contact des patients et des professionnels qui ont su leur accorder une place et leur confiance.

Les auteurs déclarent ne pas avoir de liens d'intérêts.

Références