R Venkat

Kalyana Sundaram

Huaizhou Jin

Feng Li

Tong Shu

Jeff Coleman

Jie Yang

Frederic Pincet

Yongli Zhang

James E Rothman
email: james.rothman@yale.edu

Shyam S Krishnakumar
email: shyam.krishnakumar@yale.edu

Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly

Synaptic vesicle fusion is mediated by SNARE proteins -VAMP2 on the vesicle and Syntaxin-1/ SNAP25 on the pre-synaptic membrane. Chaperones Munc18-1 and Munc13-1 co-operatively catalyze SNARE assembly via an intermediate 'template' complex containing Syntaxin-1 and VAMP2. How SNAP25 enters this reaction remains a mystery. Here we report that Munc13-1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single-molecule optical tweezer studies. Detailed structure-function analyses show that the binding is mediated by the Munc13-1 MUN domain and is specific for the SNAP25 'linker' region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and bound in ~1:1 stoichiometry by the self-assembled Munc13-1 nano-clusters.

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How precisely these chaperones and their homologues achieve this, be it in the highly specialized context of neurotransmission or more broadly for vesicle fusion driving intracellular protein transport, is a major open question despite important insights that have been gleaned.

Structural studies indicate that Munc18-1 binds and stabilizes monomeric Syntaxin-1 in a 'closed' (auto-inhibited) conformation [START_REF] Dulubova | Munc18-1 binds directly to the neuronal SNARE complex[END_REF][START_REF] Misura | Three-dimensional structure of the neuronal-Sec1syntaxin 1a complex[END_REF]. In doing so, it prevents its assembly into SNARE complexes or engagement into off-pathway reactions [START_REF] Shen | Selective activation of cognate SNAREpins by Sec1/Munc18 proteins[END_REF][START_REF] Dulubova | Munc18-1 binds directly to the neuronal SNARE complex[END_REF][START_REF] Misura | Three-dimensional structure of the neuronal-Sec1syntaxin 1a complex[END_REF]. This interaction is associated with Munc18-1 chaperone function and is critical for trafficking of Syntaxin-1 from endoplasmic reticulum (ER) to the PM [START_REF] Shi | Dual roles of Munc18-1 rely on distinct binding modes of the central cavity with Stx1A and SNARE complex[END_REF][START_REF] Rowe | Syntaxin 1A is delivered to the apical and basolateral domains of epithelial cells: the role of munc-18 proteins[END_REF][START_REF] Arunachalam | Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells[END_REF]. Munc18-1 has also been shown to promote SNARE complex assembly and to accelerate the resultant vesicular fusion [START_REF] Shen | Selective activation of cognate SNAREpins by Sec1/Munc18 proteins[END_REF][START_REF] Shen | SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion[END_REF]. This activator function is attributed to Munc18-1's ability to bind and sequester partiallyassembled SNARE complexes, termed SNAREpins [START_REF] Shen | Selective activation of cognate SNAREpins by Sec1/Munc18 proteins[END_REF]. Indeed, recent structural and biophysical studies suggest that Munc18-1 likely serves as a template for the formation of an early SNARE assembly intermediate (termed a 'template complex') wherein Syntaxin-1 and VAMP2 are properly oriented and aligned for nucleation into a four helix bundle [START_REF] Baker | A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly[END_REF][START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF][START_REF] Jiao | Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association[END_REF][START_REF] Sitarska | Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion[END_REF]. Thus, the dual-binding modes of Munc18-1 to monomeric Syntaxin-1 and SNARE complexes allow it to both negatively-regulate and positively-assist SNARE complex assembly.

A key function of the other dedicated chaperone, Munc13-1, is to catalyze the overall process of nucleation by "opening" the closed Syntaxin-1/Munc18 complex to enable it to rapidly assemble with VAMP2 to form the template complex [START_REF] Ma | Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release[END_REF][START_REF] Betz | Direct interaction of the ratunc-13 homologue Munc13-1 with the N terminus of syntaxin[END_REF][START_REF] Yang | Syntaxin opening by the MUN domain underlies the function of Munc13 in synaptic-vesicle priming[END_REF][START_REF] Basu | A minimal domain responsible for Munc13 activity[END_REF][START_REF] Ma | Munc13 mediates the transition from the closed syntaxin-Munc18 complex to the SNARE complex[END_REF]. This catalytic function has been mapped to the large central module within Munc13 called the MUN domain [START_REF] Yang | Syntaxin opening by the MUN domain underlies the function of Munc13 in synaptic-vesicle priming[END_REF][START_REF] Basu | A minimal domain responsible for Munc13 activity[END_REF], but the precise mechanism is not known. Independent of Munc18-1, Munc13-1 has been shown to promote the proper alignment of Syntaxin-1 and VAMP2 [START_REF] Lai | Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18[END_REF] thereby accelerating and stabilizing the Munc18/Syntaxin-1/VAMP2 template complex [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF]. The mechanism involved was recently clarified with the findings that Munc13-1 independently binds both VAMP2 [START_REF] Wang | Munc18 and Munc13 serve as a functional template to orchestrate neuronal SNARE complex assembly[END_REF] and Syntaxin-1 [START_REF] Ma | Munc13 mediates the transition from the closed syntaxin-Munc18 complex to the SNARE complex[END_REF][START_REF] Wang | Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis[END_REF] at distinct sites of its MUN domain potentially enabling it to simultaneously interact with and orient these two SNAREs in molecular proximity to Munc18-1.

Munc13-1 is a large (~20 nm) multi-domain, multi-functional protein [START_REF] Sitarska | Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion[END_REF]. Independent of chaperoning SNAREpin assembly, Munc13-1 plays a key role in local tethering of SV at dedicated active zones of the pre-synaptic PM [START_REF] Sudhof | Neurotransmitter release: the last millisecond in the life of a synaptic vesicle[END_REF][START_REF] Rizo | The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?[END_REF][START_REF] Camacho | Heterodimerization of Munc13 C2A domain with RIM regulates synaptic vesicle docking and priming[END_REF]. Local tethering of SV to the PM, occurs prior to nucleation of SNAREpins, and is required to form the pool of release ready vesicles [START_REF] Quade | Membrane bridging by Munc13-1 is crucial for neurotransmitter release[END_REF]. It has been proposed that local tethering occurs when Munc13-1 itself forms molecular bridges linking the SV and PM through interactions involving the C 1 -C 2 B region and the C 2 C domain on opposite ends of the MUN domain [START_REF] Quade | Membrane bridging by Munc13-1 is crucial for neurotransmitter release[END_REF][START_REF] Liu | Functional synergy between the Munc13 C-terminal C1 and C2 domains[END_REF][START_REF] Shin | Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis[END_REF][START_REF] Basu | Munc13-1 C1 domaina ctivation lowers the energy barrier for synaptic vesicle fusion[END_REF]. Specifically, the C 1 and C 2 B domains bind the PM through interactions with their physiological ligands diacylglycerol (DAG) and phosphatidylinositol 4,5-bisphosphate (PIP2) respectively, while the C-terminal C 2 C domain interacts with the SV membrane, at least in part via the acidic phospholipid, phosphatidylserine (PS) [33-35, 37, 38].

Overall, Munc18-1 and Munc13-1 co-operatively catalyze efficient SNAREpin nucleation via a template complex involving choreographed binding of Syntaxin-1 and VAMP-2. Of course, SNAREpins cannot nucleate without two of their four helices. Remarkably, especially for such a heavily investigated subject, how SNAP25 is chaperoned into nascent SNAREpins remains a mystery. It is known that VAMP2-containing SVs dock to PIP2enriched regions on the PM, guided by Munc13-1 and SV-associated protein, Synaptotagmin [START_REF] Park | Synaptotagmin-1 binds to PIP(2)-containing membrane but not to SNAREs at physiological ionic strength[END_REF][START_REF] Vandenbogaart | Phosphatidylinositol 4,5-bisphosphate increases Ca2+ affinity of synaptotagmin-1 by 40-fold[END_REF]. Syntaxin-1 is sequestered into these PIP2 microdomains, mediated by electrostatic interactions involving the Syntaxin-1 juxta-membrane region [START_REF] Honigmann | Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment[END_REF]. However, SNAP25, is highly dispersed on the PM surface and in fact, largely excluded from the PIP2 clusters under resting conditions [START_REF] Chamberlain | SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis[END_REF][START_REF] Salaun | The SNARE proteins SNAP-25 and SNAP-23 display different affinities for lipid rafts in PC12 cells. Regulation by distinct cysteine-rich domains[END_REF]. Exactly how SNAP25 is recruited to the site of vesicle docking to initiate SNARE complex assembly remains enigmatic.

Recently, a single-molecule force spectroscopy study revealed that the Munc13-1 MUN domain enhances the probability of SNAP25 binding to the Syntaxin-1/VAMP2/Munc18 template complex and subsequently complete assembly of the ternary SNARE complex [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF]. This suggested that Munc13-1 may play a role in recruiting SNAP25 to initiate SNARE complex formation. Here we test and confirm this hypothesis. We find that the Munc13-1 MUN domain directly and specifically binds the SNAP25 linker region and this interaction forms the molecular basis of Munc13-1 function in stimulating SNARE complex assembly both on its own and in conjunction with Munc18-1.

MATERIALS AND METHODS

Materials

The cDNA constructs used in this study include full-length mouse His 6 -SNAP25b, residues 1-206 in a pET28 vector with a N-terminal Thrombin cut site; His 12 -Munc13 L (Munc13-1 residues 529-1735 with residues 1408-1452 replaced by the sequence EF) in a modified pCMV-AN6 vector which includes a PreScission cut site following the His 12 tag, GST-MUN (residues 859-1407, △1408-1452, EF) cloned into a pGEX vector with a N-terminal Thrombin cut site (a kind gift from Dr. Josep Rizo, UT Southwestern). We used a QuikChange mutagenesis kit (Agilent Technologies, Santa Clara, CA) to generate the N1128A/F1131A in the Munc13 L background (Munc13 L NF ). SNAP25 linker domain mutants were synthesized (Genewiz Inc., Plainfield, NJ) with the SNAP25 residues 83-142 replaced with (GGGGS) 10 repeats (SNAP25 G4S ) or SNAP23 residues 83-142 (referred to as SNAP25 SNAP23 ) and cloned into the pET28 vector similar to the SNAP25 WT construct. For fluorescent labeling experiments, we introduced a Halo tag at the c-terminus of the Munc13 L and MUN constructs in the aforementioned vector background. We used a single-cysteine (S28C) version of SNAP25 WT [START_REF] Krishnakumar | Conformational dynamics of calcium-triggered activation of fusion by synaptotagmin[END_REF] and SNAP25 G4S (synthesized from Genewiz Inc., Plainfield, NJ). Constructs used in the optical tweezer experiments (cytoplasmic domains of rat syntaxin 1A residues 1-265, R198C and VAMP2 residues 1-96, N29C) and the bulk FRET experiments (VAMP2 residues 1-96, S28C, SNAP25 residues 1-206, Q20C, Syntaxin 1 residues 1-258) have been described previously [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF][START_REF] Jiao | Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers[END_REF]. Lipids, 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC), 1,2-dioleoylsn-glycero-3-phospho-L-serine) (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DOPE-PEG2000), phosphatidylinositol 4, 5-bisphosphate (PIP2), diacylglycerol (DAG) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3 benzoxadiazol-4yl) (NBD-PE) were purchased from Avanti Polar Lipids (Alabaster, AL). The polystyrene beads (2.1 μm in diameter) with anti-digoxigenin coating used in optical tweezer experiments were purchased from Spherotech (Green Oaks, IL).

Protein production and purification

The MUN domain (with and without Halo tag) and all SNARE proteins were expressed and purified as described previously [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF][START_REF] Yang | Syntaxin opening by the MUN domain underlies the function of Munc13 in synaptic-vesicle priming[END_REF][START_REF] Wang | Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion[END_REF]. Briefly, the proteins were expressed in Escherichia coli strain BL21(DE3) (Novagen, Darmstadt, Germany) and cells were lysed with a cell disruptor (Avestin, Ottawa, Canada) in lysis buffer containing 50 mM HEPES, pH 7.4, 400 mM KCl, 4% TritonX-100, 10% glycerol, 0.5 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 1 mM phenylmethylsulfonyl fluoride. Samples were clarified using a 45 Ti rotor (Beckman Coulter, Brea, CA) at 140,000 × g for 30 minutes and incubated with Ni-NTA agarose (Thermofisher, Waltham, MA) or Glutathione agarose (Thermofisher, Waltham, MA) for 4-16 h at 4°C. The resin was subsequently washed in the HEPES buffer, without TritonX-100 and supplemented with 50 mM Imidazole for the Ni-NTA purification. After the washes (three column volumes) the protein was cleaved of the resin with Thrombin for 4-16 h at 4°C. The protein was purified using gel filtration (Superdex75 or 200 Hi-load column, GE Healthcare, Chicago, IL). The peak fractions were pooled and concentrated using filters of appropriate cut-offs (EMD Millipore, Burlington, MA). NOTE: Syntaxin-1 construct used in optical tweezer studies was biotinylated in situ using previously described biotin ligase protocol [START_REF] Jiao | Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers[END_REF].

Munc13 L (with or without Halo-tag) was expressed in ExpiHEK-293 cell cultures using Expifectamine as a transfection reagent (Thermofisher, Waltham, MA). Briefly, thawed cells were passaged three times prior to transfection and were grown for 72 hours before being spun down and rinsed in ice cold PBS. The pellet was re-suspended in the HEPES lysis buffer and lysed using a Dounce homogenizer. The sample was clarified using at 140,000 × g for 30 minutes at 4°C and the supernatant incubated overnight with Ni-NTA beads, in the presence of DNAse 1, RNAse A and Benzonase to remove nucleotide contamination. The protein was further washed in the lysis buffer (without TritonX-100) before being cleaved with PreScission Protease for 2 hours at room temperature. The eluted proteins were further purified via gel filtration (Superdex200, GE Healthcare Chicago, IL).

In all cases, the protein concentration was determined using a Bradford Assay (BioRad, Hercules, CA,) with BSA as a standard and protein purity was verified using SDS/PAGE analysis with Coomassie stain. All proteins were flash-frozen and stored at -80°C for longterm storage.

The SNAP25 and Munc13-Halo proteins were fluorescently labeled as described previously [START_REF] Krishnakumar | Conformational dynamics of calcium-triggered activation of fusion by synaptotagmin[END_REF]. Typically, the proteins were incubated with 5X excess dye (AlexaFluor 488, 555 or 660 as noted) for 1h at room temperature (or overnight at 4°C) and the unbound dye was removed using spin columns (Thermofisher, Waltham, MA). The labeling efficiency was estimated using absorbance values (488 or 555 or 660 nm/280 nm) and in all cases, was verified to be >90%.

Microscale Thermophoresis Interaction Analysis

Halo-tagged Munc13-1 constructs (Munc13L, Munc13 L NF and MUN) were labeled with AlexaFluor 660 and MST analysis was carried out with 50 nM of fluorescently labeled Munc13-1 constructs in a premium-coated glass capillary with 0.5 mg/ml BSA and 0.05% Tween-20 included in the HEPES buffer (50 mM HEPES, 140 mM KCl, 1 mM TCEP, 1 mM MgCl 2 ) to prevent non-specific adsorption to the glass surface. The samples were measured in a Monolith NT.115 (Nanotemper Technologies, San Francisco, CA) and MST curves obtained with titration of SNAP25 (1 nM -125 μM range) were fitted with a non-linear curve (sigmoidal) analysis to estimate the apparent dissociation constant (K d ).

Lipid Bilayer experiments

Liposomes were made using brain physiological composition (73% DOPC, 15% DOPS, 3% PIP2, 2% DAG, 5% DOPE-PEG 2000, 2% NBD-PE) using extrusion method with HEPES buffer (50 mM HEPES, 140 mM KCl, 1 mM TCEP, pH 7.4). Lipid bilayers were created by Mg 2+ (5mM) induced bursting liposomes in ibidi glass-bottom chambers (ibidi GmbH, Germany). The bilayer was extensively washed with HEPES buffer and the fluidity of the lipid bilayer was verified using fluorescence recovery after photo-bleaching (FRAP) using the NBD fluorescence. FRAP measurements were analyzed on Wolfram Mathematica using a previously published algorithm [START_REF] Pincet | FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments[END_REF]. Consistent with a fluid bilayer, the average diffusion coefficient of the lipid was calculated to be 1.6 ± 0.3 μm 2 /sec (Supplementary Figure 1). SNAP25 and Munc13 L were added to the pre-washed supported bilayer and incubated for 30 minutes. For the clustering experiments (Figure 2), we used 160 nM SNAP25 WT or SNAP25 G4S (residue S28C) labeled with AlexaFluor555 with increasing concentrations (50 nm -2μM) of unlabeled Munc13 L . FRAP analysis showed that the membrane-bound AlexaFluor555-labeled SNAP25 was fairly mobile, with an average diffusion coefficient of 1.0 ± 0.2 μm 2 /sec (Supplementary Figure 1). For the co-localization experiments (Figure 3), we used 160 nM SNAP25 WT (residue S28C) labeled with AlexaFluor555 with either 1 μM or 50 nM of Munc13 L -Halo labeled with AlexaFluor488. Mander's coefficients were calculated using ImageJ (Fiji) with the JACOP plugin [START_REF] Manders | Measurement of Colocalization of Objects in Dual-Color Confocal Images[END_REF]. The stoichiometry analysis were carried out under 50 nM Munc13L condition. Fluorescence profiles of individual ROIs corresponding to SNAP25 (550 nm) and Munc13-1 (647) from the same cluster (~70 in total) were plotted and number of bleaching steps were manually counted. For all experiments, the chambers were washed thoroughly (after 30 min incubation) to remove any non-specifically or weakly bound proteins and the samples were imaged on a TIRF microscope (Nikon) with a 63× oil objective.

FRET Experiment

We used a FRET-based assay to track N-terminal SNARE complex formation. For sitespecific labeling with fluorophores, cysteines were introduced into cytoplasmic domains of SNAP25 (residues 20) and VAMP2 (residue 28) and was labeled with thiol-reactive fluorescent probes AlexaFluor488 maleimide and AlexaFluor555 maleimide respectively. 1.5 μM of labeled SNAP25 and unlabeled Syantaxin-1 were pre-mixed with or without 3 μM MUN domain and incubated at 37°C for 10 min. 500 nM of pre-warmed labeled VAMP2 was added to start the reaction and fluorescence at 580 nm was measured in plate reader (Molecular Devices) for 60 min with 15 sec intervals. In control experiments, we left out Syntaxin-1 from the reaction mixture.

Optical Tweezer experiments

Single molecule force spectroscopy experiments were carried out as described previously [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF][START_REF] Jiao | Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association[END_REF][START_REF] Jiao | Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers[END_REF][START_REF] Rebane | Structure-Based Derivation of Protein Folding Intermediates and Energies from Optical Tweezers[END_REF]. Briefly, ternary SNARE complexes were assembled by mixing the purified SNARE proteins and incubating at 4°C overnight. Assembled SNARE complexes were purified by binding to Ni-NTA-agarose and cross-linked with DTDP (2,2'-dithiodipyridine disulfide) treated DNA handles (50:1 molar ratio) in PBS buffer (100 mM phosphate buffer, 500 mM NaCl, pH 8.5). Dual-trap optical tweezers and basic protocols for single-molecule experiments have been described in detail elsewhere [START_REF] Jiao | Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers[END_REF][START_REF] Rebane | Structure-Based Derivation of Protein Folding Intermediates and Energies from Optical Tweezers[END_REF]. To follow SNARE assembly/ disassembly pathways, samples of the 2 DNA handles, one cross-linked with the SNARE complex and the other bound by a streptavidin molecule, were separately bound to antidigoxigenin antibody coated polystyrene beads. The beads were injected into a microfluidic channel and trapped. The 2 beads are then brought close to allow a single SNARE complex to be tethered between them. All manipulation experiments were carried out in PBS buffer supplemented with the oxygen scavenging system. All single molecules were pulled and relaxed by increasing and decreasing, respectively, the trap separation at a speed of 10 nm/s or held at constant mean forces by keeping the trap separation constant.

RESULTS

Munc13-1 MUN domain directly binds SNAP25 in solution

To test if there is direct molecular interaction between Munc13-1 and SNAP25, we employed Microscale Thermophoresis (MST), a highly sensitive technique that allows one to quantify molecular interactions with limited amounts of protein under varying buffer conditions [START_REF] Duhr | Why molecules move along a temperature gradient[END_REF][START_REF] Wienken | Protein-binding assays in biological liquids using microscale thermophoresis[END_REF]. Informed by earlier reports [START_REF] Yang | Syntaxin opening by the MUN domain underlies the function of Munc13 in synaptic-vesicle priming[END_REF][START_REF] Wang | Munc18 and Munc13 serve as a functional template to orchestrate neuronal SNARE complex assembly[END_REF][START_REF] Wang | Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis[END_REF][START_REF] Quade | Membrane bridging by Munc13-1 is crucial for neurotransmitter release[END_REF][START_REF] Liu | Functional synergy between the Munc13 C-terminal C1 and C2 domains[END_REF], we focused on the conserved C-terminal portion of Munc13-1 consisting of the contiguous C 1 -C 2 B-MUN-C 2 C domains (Munc13-1 residues 529-1735; referred to as Munc13 L ), except for residues 1408-1452 within a non-conserved loop in the MUN domain, which was deleted and replaced with EF residues to minimize dimerization/oligomerization [START_REF] Basu | A minimal domain responsible for Munc13 activity[END_REF][START_REF] Ma | Munc13 mediates the transition from the closed syntaxin-Munc18 complex to the SNARE complex[END_REF]. To facilitate the MST analysis, we introduced a C-terminal Halo tag to the Munc13 L fragment to enable covalent attachment of a fluorescent tag. Titration of full-length wild-type SNAP25 (residues 1-206, SNAP25 WT ) into AlexaFluor 660 labeled Munc13 L yielded a classical single-site doseresponse curve with a dissociation constant (K d ) of 26 ± 2 μM (Figure 1A). This indicated that that Munc13-1 directly binds SNAP25, albeit with a modest affinity under soluble conditions. This binding affinity is comparable to that reported (25-100 μM) for specific binding of the MUN domain to Syntaxin-1, VAMP2, and the fully assembled SNARE complex when these proteins are also free in solution [START_REF] Lai | Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18[END_REF][START_REF] Ma | Munc13 mediates the transition from the closed syntaxin-Munc18 complex to the SNARE complex[END_REF][START_REF] Wang | Munc18 and Munc13 serve as a functional template to orchestrate neuronal SNARE complex assembly[END_REF][START_REF] Wang | Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis[END_REF]. Therefore, the novel binding we observe between Munc13-1 and SNAP25 is similarly likely to be physiologically relevant at the high local concentrations that exist in active zones.

We then sought to identify the regions within Munc13-1 and SNAP25 responsible for their binding. The central MUN domain of Munc13-1 contains the distinct binding sites for both VAMP2 and Syntaxin-1, each of which is required for its chaperone function [START_REF] Wang | Munc18 and Munc13 serve as a functional template to orchestrate neuronal SNARE complex assembly[END_REF][START_REF] Wang | Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis[END_REF]. So, we expressed and purified the isolated MUN domain (residues 859-1531, △1408-1452, EF), and labeled it with AlexaFluor 660 at a C-terminal Halo tag. MST analysis showed that the MUN domain alone can bind SNAP25 WT with an apparent K d of 48 ± 3 μM (Figure 1B). This indicated that the MUN domain serves as the primary binding site for SNAP25 and that the flanking C 1 -C 2 B and C 2 C portions might weakly augment the primary MUN-SNAP25 binding site, be it directly or indirectly.

Binding SNAP25 involves the linker region between its two SNARE motifs

Since the isolated MUN domain promotes the nucleation of SNARE complexes [START_REF] Basu | A minimal domain responsible for Munc13 activity[END_REF], we reasoned that the Munc13-1 binding site on SNAP25 is likely outside of the SNARE helices, as these would need to be free to assemble with the SNARE motifs of VAMP2 and Syntaxin-1. This logic would make the 60 residue 'linker' region that concatenates the SNARE helical motifs the best candidate for productive binding by Munc13-1. Therefore, we designed and tested a SNAP25 mutant (SNAP25 G4S ) in which the linker region was replaced with an inert, flexible, non-specific sequence (GGGGS repeats) yet maintaining the overall length. Even though SNAP25 G4S successfully replaced SNAP25 WT in a bulk vesicle fusion assay (Supplementary Figure 2) and has been shown to assemble in SNARE complex [START_REF] Shaaban | The SNAP-25 linker supports fusion intermediates by local lipid interactions[END_REF], it failed to bind to Munc13 L even at the highest SNAP25 G4S concentration (100 μM) tested (Figure 1A). This suggests that the MUN domain likely binds to the SNAP25 linker region.

SNAP25 binding to Munc13-1 is specific and unique

To assess the specificity of binding, we replaced the SNAP25 linker region with that of its close homologue, SNAP23, producing a chimeric protein termed SNAP25 SNAP23 . SNAP23 is structurally and functionally similar to SNAP25 and is expressed in all tissues [START_REF] Kunii | Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells[END_REF][START_REF] Ravichandran | Identification of a novel syntaxin-and synaptobrevin/ VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues[END_REF][START_REF] Wang | Syndet is a novel SNAP-25 related protein expressed in many tissues[END_REF],

where it associates with Syntaxin-1 and VAMP2 and functions as part of the ubiquitous cellular fusion machinery [START_REF] Kunii | Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells[END_REF]. Notably, the linker region of SNAP23 (compared with SNAP25) is markedly less conserved (~45% identical) than either SNARE motif (~70%). Indeed, SNAP25 SNAP23 failed to bind to Munc13 L (Figure 1A), confirming that Munc13-1 binding to SNAP-25 is isoform specific and that specificity resides in the linker region.

Munc13-1 has been shown to bind and activate Syntaxin-1 via a hydrophobic pocket near the middle of the MUN domain [START_REF] Wang | Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis[END_REF]. To assess if SNAP25 binding overlaps with the Syntaxin site, we tested a previously described mutation in Munc13-1 (N1128A/F1131A; Munc13 L NF ) that disrupts binding to Syntaxin-1 [START_REF] Wang | Conformational change of syntaxin linker region induced by Munc13s initiates SNARE complex formation in synaptic exocytosis[END_REF]. These mutations had no effect and Munc13 L NF bound SNAP25 with a K d of 29 ± 10 μM (Figure 1B, purple). This indicates that the SNAP25 binding site within the MUN domain is distinct from the Syntaxin-1 binding pocket.

Munc13-1 binds SNAP25 on lipid membrane surface at 1:1 stoichiometry SNAP25 and Munc13 are both naturally concentrated within active zones of the pre-synaptic plasma membrane, where they are restricted to a common surface by distinct mechanisms. SNAP25 is anchored by the direct insertion of its multiple palmitoylated cysteines in the linker region [START_REF] Shaaban | The SNAP-25 linker supports fusion intermediates by local lipid interactions[END_REF][START_REF] Greaves | The hydrophobic cysteine-rich domain of SNAP25 couples with downstream residues to mediate membrane interactions and recognition by DHHC palmitoyl transferases[END_REF][START_REF] Nagy | The SNAP-25 linker as an adaptation toward fast exocytosis[END_REF]. As mentioned, Munc13-1 is anchored by binding to lipid headgroups by C1-C2B domains [START_REF] Liu | Functional synergy between the Munc13 C-terminal C1 and C2 domains[END_REF][START_REF] Shin | Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis[END_REF][START_REF] Basu | Munc13-1 C1 domaina ctivation lowers the energy barrier for synaptic vesicle fusion[END_REF]. We thus investigated the SNAP25-Munc13 interaction on a lipid bilayer surface. We anticipated that restriction of these two proteins to a two-dimensional surface would increase the association rate, resulting overall in markedly greater stability of the Munc13-SNAP25 complexes thereby enabling them to be imaged and quantified on the supported bilayers using total internal reflection fluorescence (TIRF) microscopy.

We created a supported lipid bilayer with functionally-relevant lipid composition (73% PC, 15% PS, 3% PIP2, 2% DAG) by bursting pre-formed vesicles onto a glass coverslip. We had anticipated that it would be necessary to use palmitoylated SNAP25 in this experiment, but instead observed that even non-palmitoylated SNAP25, added at low (150 nM) concentrations in solution, bound to the bilayer. This is likely due to the amphipathic nature of two SNARE motifs of SNAP25 [START_REF] Sutton | Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution[END_REF] as it is well-established that amphipathic peptides associate strongly to phospholipid surfaces as their hydrophobic side chains could insert into the lipid bilayers [START_REF] Wagle | Interaction of SNARE Mimetic Peptides with Lipid bilayers: Effects of Secondary Structure, Bilayer Composition and Lipid Anchoring[END_REF][START_REF] Gimenez-Andres | The Many Faces of Amphipathic Helices[END_REF]. On its own, freely-diffusing AlexaFluor 555-labeled SNAP25 molecules thus bound were monomeric and uniformly distributed on the lipid bilayer surface (Figure 2A, Supplementary Figure 1). When Munc13 L was present, SNAP25 was no longer monodisperse, but rather located in numerous distinct clusters of variable intensity (Figure 2A) and the clustering efficiency directly correlated to the concentration of Munc13 L (Figure 2A, Supplementary Figure 3).

Considering that Munc13-1 has been shown to self-associate into clusters on lipid bilayers under both in vitro and physiological conditions [START_REF] Sakamoto | Synaptic weight set by Munc13-1 supramolecular assemblies[END_REF][START_REF] Li | Vesicle capture by discrete self-assembled clusters of membrane-bound Munc13[END_REF], we reasoned that SNAP25 puncta are a consequence of its association with Munc13-1 clusters. To confirm, we co-localized AlexaFluor555-labeled SNAP25 with AlexaFluor488-labeled Munc13L using two color TIRF microscopy (Figure 3A). As expected, Munc13 L on its own formed clusters on lipid bilayer surface (Figure 3A) and SNAP25 increasingly co-localized with these clusters as the concentration of Munc13 L was increased. When Munc13 L was added at 50 nM bulk concentration the Manders' coefficient (SNAP25:Munc13 L ) was 0.2 ± 0.07, which increased to 0.6 ± 0.05 at 1 μM Munc13 L , consistent with a concentration-dependent binding process.

As a control, we employed membrane-anchored (palmitoylated) SNAP25 and obtained similar results (Supplementary Figure 4).

To establish the specificity of this binding, we employed the linker mutant, SNAP25 G4S . As expected, no clustering of SNAP25 G4S was observed, even at the highest concentration (2 μM) of Munc13 L (Figure 2B). This suggests that similar to the soluble conditions, SNAP25 binding to Munc13 L nanoclusters on membranes involves the SNAP25 linker region. Finally, to establish the stoichiometry of binding of SNAP25 to the Munc13 L nanoclusters, we performed two color photo-bleaching experiments (Fig. 3B). Reliable identification of single steps by this method can only be made when relatively small (<10) numbers of molecules are present. Therefore, we performed these experiments at a low Munc13 L concentration (50 nM) designed to limits the copy number of Munc13 L in the nanoclusters. Among these co-clusters, independent of their size, we observed a broad distribution of the apparent mole ratio of SNAP25 to Munc13 L centering on ~0.8 mole/mole (Figure 3B). Considering the high and similar efficiency of labeling of both SNAP25 and Munc13 L , the ratio of fluorescence likely reflects the actual mole ratio. This suggests that Munc13-1 binds SNAP25 at approximately 1:1 stoichiometry.

Linker-specific binding by MUN is necessary for SNAP25 to enter the SNARE complex

We then examined the potential functional relevance of the MUN-SNAP25 linker domain interaction to Munc13-1's established and independent role [START_REF] Lai | Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18[END_REF][START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF][START_REF] Liu | Functional synergy between the Munc13 C-terminal C1 and C2 domains[END_REF] in accelerating nucleation of SNAREpins. We employed a fluorescence resonance energy transfer (FRET) assay with fluorescent probes (AlexaFluor 488 & AlexaFluor 555) introduced at the Ntermini of SNAP25 (residue Q20) and VAMP2 (residue S28) respectively to directly track the effect of the MUN domain on the initiation of SNARE complex assembly.

In the absence of the MUN domain, SNAP25 WT spontaneously assembled into a complex with VAMP2 and unlabeled Syntaxin-1 over the course of an hour as evident from the gradual increase of the acceptor fluorescence intensity (Figure 4A). We did not detect FRET in control experiments when Syntaxin-1 was omitted confirming that the observed FRET signal corresponds to the formation of a ternary SNARE complex (Figure 4A). In line with the bulk-fusion assays (Supplementary Figure 2) and previous reports [START_REF] Shaaban | The SNAP-25 linker supports fusion intermediates by local lipid interactions[END_REF][START_REF] Nagy | The SNAP-25 linker as an adaptation toward fast exocytosis[END_REF], mutation of the linker region had very little effect on the spontaneous rate of SNAP25 assembly into SNARE complexes (Figure 4A), with comparable increase in acceptor (FRET) signal with SNAP25 G4S mutant (Figure 4A). The MUN domain strongly stimulated the rate of association of SNAP25 WT with the cognate SNAREs (~3-fold enhancement of FRET signal after 60 min) but had no effect on the rate of assembly of SNAP25 G4S mutant (Figures 4A and4B). This data forcefully suggests that binding of SNAP25 (via its linker) is required for Munc13-1 to chaperone SNARE complex assembly.

Linker-dependent binding by MUN recruits SNAP25 to the template complex

To independently test this conclusion in the fuller context of Munc13-1's co-operation with Munc18-1 in chaperoning SNARE complex assembly, we employed an established single molecule assay involving optical tweezers (Figure 5A) [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF][START_REF] Jiao | Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association[END_REF][START_REF] Jiao | Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers[END_REF][START_REF] Rebane | Structure-Based Derivation of Protein Folding Intermediates and Energies from Optical Tweezers[END_REF]. Here, Syntaxin-1 and VAMP2 were cross-linked at their N-termini (between Syntaxin R198C and VAMP2 N29C) via a disulfide bond and pulled from their C-termini via two DNA handles attached to two optically trapped polystyrene beads [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF][START_REF] Jiao | Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association[END_REF]. We first pulled a single SNARE complex in the presence of Munc18-1 and SNAP25 WT (0.5 μM & 0.6 μM respectively added into the solution). The resultant force-extension curve (FEC) indicated that a single SNARE complex unfolds in a characteristic stepwise manner among different states when pulled (Figure 5B, FEC #1 & #2 grey curves with different regions indicated by their associated state numbers). We observed reversible C-terminal domain (CTD) transitions at about 15 pN (between state 1 and state 2, regions marked by gray ovals), followed by an irreversible unfolding of the N-terminal domain (NTD) at ~16-17 pN (from state 2 to state 3, extension jumps marked by gray arrows), and finally irreversible unfolding of the Syntaxin-SNAP25 (t-SNARE) complex (marked by green arrows) with the attendant dissociation of SNAP-25 (state 5).

Relaxing the force applied to the SNARE proteins then revealed two reversible transitions along the relaxation curves (Figure 5B FEC #1 black curve). The first transition from 14 to 6 pN (marked by blue rectangle) was caused by the folding of partially closed Syntaxin-1 (state 6) and the lower force transition at ~3-6 pN resulted from the formation of Munc18-1/Syntaxin-1/VAMP2 template complex (state 7).

To detect the ternary SNARE complex assembly, we held the template complex at a constant mean force around 5 pN for 100 seconds to await SNAP25 WT binding (Figure 5B FEC#2 red curve). Indeed, in some cases, SNAP25 WT from the solution was able to bind to the template complex resulting in reassembly of the full SNARE complex (state 1). This reflects the intrinsic rate of productive association of free SNAP25 with the template complex.

To test whether the rate of productive association of SNAP25 to the template complex is accelerated by Munc13-1, we included the MUN domain (1 μM) in addition to Munc18-1 and SNAP25 WT in solution (Figure 5B FEC #3 & #4). Indeed, the MUN domain stabilized the template complex and promoted the rate of re-assembly of the SNARE complex from the template complex. Statistically, the MUN domain nearly doubled the probability of SNARE assembly from the template complex (Figure 5C). All of these observations are consistent with our previous reports [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF][START_REF] Jiao | Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association[END_REF].

To test whether binding of SNAP25 to Munc13-1 via the SNAP25 linker is required for Munc13-1 to chaperone SNAP25 in this context, we repeated the above experiment with linker domain mutant, SNAP25 G4S . With Munc18-1 only, the probability of SNARE complex formation with the SNAP25 G4S was similar to that observed with SNAP25 WT (Figure 5C). This further confirmed the alteration of the liker domain does not affect the inherent ability to SNAP25 helices to bind its cognate SNARE partners. Addition of the MUN domain along with Munc18-1 (Figure 5B, FEC #5, #6, #7) stabilized the template complex [START_REF] Shu | Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex[END_REF] but did not appreciably increase the probability of the SNAP25 G4S to complete SNARE assembly as compared to SNAP25 WT (p = 0.065) (Figure 5C). This strongly indicates that MUN-SNAP25 binding (via the linker domain) contributes to the enhanced assembly of the four-helix bundle from the Munc18-1/Syntaxin-1/VAMP2 template complex.

DISCUSSION

Altogether, we have identified a novel, functionally relevant interaction between the Munc13-1 MUN domain and the SNAP25 linker region. Our findings are supported by several previous studies [START_REF] Shaaban | The SNAP-25 linker supports fusion intermediates by local lipid interactions[END_REF][START_REF] Nagy | The SNAP-25 linker as an adaptation toward fast exocytosis[END_REF][START_REF] Dubuke | The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes[END_REF][START_REF] Sivaram | Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p[END_REF]. For example, it has been demonstrated that yeast exocyst protein Sec6p, which is strikingly similar to the c-terminal half of Munc13-1 MUN domain, binds Sec9 a SNAP25 homologue at the linker region. Indeed, disrupting the Sec6p-Sec9 interaction results in significant defects in exocytosis and overall cell growth [START_REF] Dubuke | The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes[END_REF][START_REF] Sivaram | Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p[END_REF]. Furthermore, the SNAP25 linker domain has been shown to play a crucial role in regulating vesicle priming and the Ca 2+ -regulated exocytosis [START_REF] Shaaban | The SNAP-25 linker supports fusion intermediates by local lipid interactions[END_REF][START_REF] Nagy | The SNAP-25 linker as an adaptation toward fast exocytosis[END_REF]. Strikingly, functional analysis in chromaffin cells revealed that SNAP25 linker-domain mutant (similar to the SNAP25 G4S used in this study) fails to rescue secretion and significantly reduces the size of primed vesicle pools even though it has the innate capacity to form productive SNARE complexes in vitro [START_REF] Shaaban | The SNAP-25 linker supports fusion intermediates by local lipid interactions[END_REF]. While not conclusive, this is consistent with impairments in Munc13-1 chaperoned SNARE complex assembly under these conditions, and in line with our FRET and optical-tweezer experiments (Figures 4 &5).

This newly uncovered feature of Munc13-1 solves an enduring mystery -how is SNAP25 chaperoned into the SNARE complex -while being compatible with the several established roles of this remarkably multi-functional protein. We now see that Munc13-1 can potentially bind all three synaptic SNAREs simultaneously to sterically position them along with Munc18-1 to maximize the rate of their productive assembly into SNAREpins [START_REF] Lai | Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18[END_REF][START_REF] Ma | Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release[END_REF]. It is well-established that the inherently cytoplasmic Munc13-1 translocates to DAG & PIP 2 -rich domains on the PM in response to neuronal activity and elevated intracellular Ca 2+ concentration [START_REF] Quade | Membrane bridging by Munc13-1 is crucial for neurotransmitter release[END_REF][START_REF] Michelassi | A C1-C2 Module in Munc13 Inhibits Calcium-Dependent Neurotransmitter Release[END_REF]. On the PM, Munc13-1 initially functions as a tethering factor for local capture of SVs [START_REF] Sudhof | Neurotransmitter release: the last millisecond in the life of a synaptic vesicle[END_REF][START_REF] Rizo | The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?[END_REF][START_REF] Quade | Membrane bridging by Munc13-1 is crucial for neurotransmitter release[END_REF][START_REF] Li | Vesicle capture by discrete self-assembled clusters of membrane-bound Munc13[END_REF]. At this stage, Munc13-1 is thought to adopt an 'erect' topology with the C 1 /C 2 B region anchored on the PM and extended (~ 15 nm) MUN domain reaching out to tether the SV via the C 2 C domain at its apex [START_REF] Sitarska | Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion[END_REF][START_REF] Quade | Membrane bridging by Munc13-1 is crucial for neurotransmitter release[END_REF][START_REF] Gipson | Morphologies of synaptic protein membrane fusion interfaces[END_REF]. However, this erect topology seems incompatible with Munc13-1 role in catalyzing the nucleation of SNAREpins because in this extended configuration, Munc13-1 would prevent the membrane from coming closer and any nucleated SNAREpin would be inherently unstable when the involved bilayers are separated by more than ~ 10 nm [START_REF] Li | Energetics and dynamics of SNAREpin folding across lipid bilayers[END_REF][START_REF] Wang | Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion[END_REF].

Munc13-1 is therefore predicted to change its orientation, ultimately to a 'flat' topology, in which the inter-membrane separation is reduced to ~5 nm (as observed in release-ready vesicles in the active zone) where SNAREpins can be meta-stably clamped at the approximately half-zippered state [START_REF] Li | Symmetrical organization of proteins under docked synaptic vesicles[END_REF][START_REF] Hua | Activity-dependent changes in partial VAMP complexes during neurotransmitter release[END_REF][START_REF] Prashad | SNARE zippering and synaptic strength[END_REF]. This reorientation is likely driven by rearrangement of the C1-C2B domains on the PM [START_REF] Liu | Functional synergy between the Munc13 C-terminal C1 and C2 domains[END_REF][START_REF] Michelassi | A C1-C2 Module in Munc13 Inhibits Calcium-Dependent Neurotransmitter Release[END_REF], and may hypothetically occur via transitional states at intermediate separations that sterically favor the concerted reaction in which three SNAREs from two membranes and two molecular chaperones can favorably align. The precise molecular details of Munc13-1's switch from a local tether to a SNARE chaperone remains to be determined. In this context, we cannot adjudicate whether Munc13-1 can sterically bind SNAP25 in the erect conformation or requires a conformational switch because the location of the binding site within the MUN domain is not yet known. Previous work has shown that that Munc18 function does not require the linker region of SNAP-25 [START_REF] Shen | SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion[END_REF], which suggests that Munc18 and Munc13 play complementary roles in chaperoning SNARE assembly.

DAG/PIP 2 binding has been shown to induce recruitment and clustering of Munc13-1 on the PM surface [START_REF] Michelassi | A C1-C2 Module in Munc13 Inhibits Calcium-Dependent Neurotransmitter Release[END_REF][START_REF] Sakamoto | Synaptic weight set by Munc13-1 supramolecular assemblies[END_REF][START_REF] Li | Vesicle capture by discrete self-assembled clusters of membrane-bound Munc13[END_REF] and nanoclusters containing ~5-10 Munc13-1 molecules have been shown to be intimately associated with SV in the readily-releasable pool by super-resolution microscopy [START_REF] Sakamoto | Synaptic weight set by Munc13-1 supramolecular assemblies[END_REF][START_REF] Reddy-Alla | Stable Positioning of Unc13 Restricts Synaptic Vesicle Fusion to Defined Release Sites to Promote Synchronous Neurotransmission[END_REF]. Recently, we have shown that possibly similar nanoclusters form by self-assembly of Munc13-1 protomers [START_REF] Li | Vesicle capture by discrete self-assembled clusters of membrane-bound Munc13[END_REF]. Local multi-valency likely augments Munc13's chaperone functions as it does vesicle capture [START_REF] Quade | Membrane bridging by Munc13-1 is crucial for neurotransmitter release[END_REF][START_REF] Li | Vesicle capture by discrete self-assembled clusters of membrane-bound Munc13[END_REF]. It is easy to imagine that Munc13-1 clusters could help concentrate SNAP25 molecules at the vesicle docking site, similar to the co-cluster we observe (Figure 3) thus increasing the probability of productive SNARE complex formation. While the precise molecular details of Munc13-1 collective and overlapping interaction with the individual SNARE proteins remains to be determined, we address a long-standing question concerning the recruitment of SNAP25 to the vesicle docking site and in turn, highlight the pivotal role of Munc13-1 in orchestrating SNARE complex assembly. It is worth noting that Munc13 family of proteins are not universal for vesicular exocytosis, rather other Munc13-like factors (e.g. CAPS) have been implicated in tethering/chaperoning process. Thus, it would be interesting to also examine if the ability to bind SNAP25 (and other SNARE proteins) are conserved across different exocytosis pathways.

Figure 1 .

 1 Figure 1. Dissecting Munc13-1 interaction with SNAP25 using Microscale Thermophoresis (MST) analysis. (A) Titration of full-length SNAP25 into AlexaFluor 660 labeled Munc13 L (C1-C2B-MUN-C2C domains) produced a concentration-dependent change in the MST signal, yielding an apparent dissociation constant (K d ) = 26 ± 2 μM implying that Munc13 L directly binds to SNAP25. Munc13-1 domain likely binds to the SNAP25 linker region as replacing the SNAP25 linker domain that connects that SNARE helical motifs with a GGGGS sequence (SNAP25 G4S ) completely abrogates Munc13-SNAP25 interaction (red curve). This interaction is highly specific as swapping the SNAP25 linker region with closely associated SNAP23 linker (SNAP25 SNAP23 ) also disrupts the binding (blue curve).(B) MUN domain (orange curve) alone is sufficient to bind SNAP25 albeit with slightly reduced affinity (K d = 48 ± 3 μM) and SNAP25 binding site is unique and different from the Syntaxin-1 binding site, as the Syntaxin non-binding Munc13-1 mutant (Munc13 L NFAA ) is capable of binding SNAP25 (purple curve). Average and standard deviations from a minimum of three independent experiments are shown.

Figure 2 .

 2 Figure 2. Munc13-1 binds and clusters SNAP25 on lipid bilayer surface. (A) Distribution of AlexaFluor 488 labeled SNAP25 WT (~150 nM) on supported lipid bilayer under physiological lipid and buffer composition in the absence or presence of Munc13 L was visualized using TIRF microscopy. Munc13L induces clustering of SNAP25 WT molecules and the number/size of the SNAP25 clusters directly correlates to concentration of Munc13 L added. (B) Munc13 L fails to cluster SNAP25 linker domain mutant (SNAP25 G4S ) even at the highest concentration (2 μM) tested. This suggests that MUN-SNAP25 linker domain interaction is feasible on lipid bilayers surfaces and results in clustering of otherwise diffusely distributed SNAP25 molecules. Representative fluorescent images are shown.

Figure 3 .

 3 Figure 3. Munc13-1 co-clusters with SNAP25 at 1:1 stoichiometry. (A) Representative fluorescence images showing that AlexaFluor 555 labeled SNAP25 WT co-localizes with clusters of AlexaFluor 488 labeled Munc13 L on lipid bilayer surface and the extent of co-localization depends on the concentration of Munc13 L added. SNAP25 (160 nM) shows limited co-localization (Manders' coefficient ~ 0.2) at 50 nM of Munc13 L but has high overlap (Manders' coefficient ~ 0.7) at 1μM of Munc13 L . (B) Stoichiometry of SNAP25 and Munc13 L in the co-clusters under low Munc13L concentration was estimated using step-bleaching analysis. Representative step-bleaching traces for SNAP25 (red) and Munc13 L (green) are shown and the ratio of SNAP25:Munc13 L quantified from ~70 coclusters is shown as the histogram. Under these conditions, each cluster contains ~5-7 molecules at almost 1:1 ratio of Munc13 L :SNAP25. This indicates that molecular interaction between the MUN domain and the SNAP25 linker region is likely responsible for the observed co-clustering of Munc13-1 and SNAP25.

Figure 4 .

 4 Figure 4. Munc13 interaction with SNAP25 is essential for its chaperone function.FRET between AlexaFluor 488 labeled SNAP25 and AlexaFluor 555 labeled soluble VAMP2 introduced in the N-terminus (SNAP25 residue 20 & VAMP2 residue 28) shows that in the presence of cytoplasmic Syntaxin-1, the MUN domain activates the initial engagement of the SNARE complex. Negative control wherein Syntaxin-1 was left out confirms the observed FRET signal corresponds to formation of the ternary SNARE complex. The stimulatory effect of MUN domain was not observed with SNAP25 linker mutant (SNAP25 G4S ) indicating that the MUN-SNAP25 linker domain interaction is crucial for Munc13-1 ability to promote SNARE complex formation. Representative fluorescence traces for first 30 min of the reaction (left) and end-point quantification at 60 min (right) from two independent trials are shown. Statistical significance was determined using student t-test (** p<0.005).

Figure 5 :

 5 Figure 5: Munc13-1 recruits SNAP25 to Munc18-1/Syntaxin-1/VAMP2 template complex.(A) Experimental setup for optical tweezers. A single SNARE complex was pulled from the C termini of Syntaxin 1A (red) and VAMP2 (blue) via two DNA handles attached to two optically trapped polystyrene beads. The N-termini of Syntaxin 1A and VAMP2 were crosslinked via a disulfide bond. Munc18-1 (gray) and the MUN domain of Munc13-1 (yellow) were added in the solution. (B) Representative force extension curves (FECs) obtained for SNARE complexes assembled with SNAP25 WT or SNAP25 G4S in the presence of Munc18-1 and MUN domain as indicated. The Syntaxin-VAMPs conjugate was pulled (grey FECs) or relaxed (black FECs) by changing the separation between two optical traps at a speed of 10 nm/s or held at constant mean force around 5 pN (red FECs). The molecular states associated with different FEC regions are indicated by the corresponding state numbers (see Supplementary Figure5for their molecular diagrams) (C) Probability of full SNARE complex assembly from the Munc18-1/Syntaxin-1/VAMP2 template complex observed

FEBS Lett. Author manuscript; available in PMC 2021 April 24.

ACKNOWLEDGEMENT

This work was supported by National Institute of Health (NIH) grant DK027044 to JER/SSK and GM131714 to YZ.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Maxime Wack

Hélène Péré

Nathalie Demory-Guinet

Najiby Kassis-Chikhani

Laurence Janot

Benoit Vedie

Laure Izquierdo

Laurent Bélec

Pharm D David Veyer
email: david.veyer@aphp.fr

Nathalie Demory Guinet

Kassis Chikhani Najibi

No SARS-CoV-2 reinfection among staff health-care workers: Prospective hospital-wide screening during the first and second waves in Paris

Keywords: SARS-CoV-2, reinfection, health-care workers, vaccination, epidemiology

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

Introduction

Health-care workers (HCWs) constitute a vulnerable population at high risk of SARS-CoV-2 infection [START_REF] Contejean | Comparing Dynamics and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 Transmissions Among Healthcare Workers of Adult and Pediatric Settings in Central Paris[END_REF][START_REF] Hunter | First experience of COVID-19 screening of health-care workers in England[END_REF][START_REF] Amanat | A serological assay to detect SARS-CoV-2 seroconversion in humans[END_REF]. During the first wave of COVID-19 epidemic in France, as of May 13, 2020, more than 4,500 professionals were infected and 4 dead from COVID-19 in the Assistance Publique-Hôpitaux de Paris (AP-HP, which represents the largest group of university hospitals in Europe, accounting for about 100 000 employees). SARS-CoV-2 infection elicits both humoral and cellular, mucosal and systemic specific immunity [START_REF] Canedo-Marroquín | SARS-CoV-2: Immune Response Elicited by Infection and Development of Vaccines and Treatments[END_REF][START_REF] Galipeau | Humoral Responses and Serological Assays in SARS-CoV-2 Infections[END_REF](6). A key question yet to be addressed is whether SARS-CoV-2 infection induces long-lasting protective or sterilizing immunity. Our understanding of the immune correlates of protection for SARS-CoV-2 infection and their durability remains limited and depends mainly on previous knowledge gained from SARS-CoV-1, the most closely related virus known to affect humans [START_REF] Galipeau | Humoral Responses and Serological Assays in SARS-CoV-2 Infections[END_REF][START_REF] Bert | SARS-CoV-2specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls[END_REF]. Furthermore, a number of cases of SARS-CoV-2 reinfection in humans have now been reported a few months after initial infection [START_REF] Babiker | The Importance and Challenges of Identifying SARS-CoV-2 Reinfections[END_REF][START_REF] Cohen | Reinfection with SARS-CoV-2: Implications for Vaccines[END_REF], including HCWs [START_REF] Gupta | Association Between Early Treatment With Tocilizumab and Mortality Among Critically Ill Patients With COVID-19[END_REF], challenging the possibility of durable protective immunity. These findings may have implications for the need of continued protective measures and of further vaccination for persons previously infected with SARS-CoV-2 [START_REF] Cohen | Reinfection with SARS-CoV-2: Implications for Vaccines[END_REF].

In France, the SARS-CoV-2 epidemic officially began on January 24, 2020, with the first confirmed case of COVID-19 imported from China. Early April, the number of deaths from coronavirus increased dramatically, with more than 10,000 people dying during that period of time.

The first wave of the COVID-19 epidemic was contained due to the first national lockdown that extended from March 17, 2020 until May 10, 2020, the peak of the first wave being reached on the 1 st of April 2020. During August 2020, COVID-19 cases began to rise again. On October 28, France entered a second nationwide lockdown with progressive lifting starting from December 15. The peak of the second epidemic wave was reached on the 20 th of November 2020, but the epidemic has since remained at a high plateau level. The duration period between the peaks of the first and second waves was 7.8 months (234 days).

Our institution, the Assistance Publique-Hôpitaux de Paris, has largely promoted both molecular and serological testing for SARS-CoV-2 among healthcare staff, from the first wave of the COVID-19 epidemic [START_REF] Contejean | Comparing Dynamics and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 Transmissions Among Healthcare Workers of Adult and Pediatric Settings in Central Paris[END_REF]. Molecular testing for SARS-CoV-2 of symptomatic hospital employees with COVID-19-related symptoms started on February 24, 2020. In addition, starting early April 2020, the Assistance Publique-Hôpitaux de Paris offered HCWs serological screening for SARS-CoV-2 to assess the prevalence of the infection during the first epidemic peak.

The intensity of COVID-19 epidemic in France in 2020 with a high risk of re-exposure to SARS-CoV-2 infection for HCWs, as well as the availability of our centralized data-capture system of all molecular and serological SARS-CoV-2 tests results, prompted us to assess the risk of SARS-CoV-2 reinfections in a real-life setting in HCWs from our institution.

Materiel and method

Study design and participants recruitment.

The study was designed as an observational cohort with longitudinal analysis, focused on molecular detection of SARS-CoV-2 RNA and immune response to SARSCoV-2 in volunteers HCWs from the Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, tested during the first and second epidemic waves for suspected SARS-CoV-2 infection, contact tracing and SARS-CoV-2 infection serological surveillance. Data included HCW ages, SARS-CoV-2 PCR and SARS-CoV-2 serology sampling dates and results. HCWs gender was not available. All molecular and serological results were fully pseudonymized. July 15 2020 was considered as the limit between the first and second epidemic waves.

During the first epidemic wave, the HCW participants were categorized into two groups according to their molecular and serological results: positive if any SARS-CoV-2 PCR test and/or serology were positive, and negative if every SARS-CoV-2 PCR test and serology were negative.

During the second epidemic wave, the participants were categorized into two groups according to their molecular results, as positive if any SARS-CoV-2 PCR test was positive, and negative if every SARS-CoV-2 PCR test were negative. In addition, HCW participants included during the first wave but not tested during the second wave, and those included during the second wave but not tested during the first wave were categorized as participants with not available results (NA). Finally, reinfection was defined as a positive result during the first wave, either by serology or PCR, followed by a positive PCR during the second wave. If iterative PCR sampling occurred, reinfection was considered if a negative result was surrounded by 2 positive results. Furthermore, if 2 PCRs were positive with more than 30 days between the sample dates without any negative results in between, results were individually assessed by a virologist to decide if reinfection was confirmed or if remnant RNA from the original infection was still detected when the cycle threshold (Ct) value of the PCR was over 33 [START_REF] Yu | Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients[END_REF]. Positive serology during the first wave followed by positive PCR during the second wave was also considered a reinfection.

SARS-CoV-2 testing.

Molecular detection of SARS-CoV-2 was carried out from flocked nasopharyngeal swab samples using Allplex™ 2019-nCoV Assay (Seegene, Seoul, Korea), a multiplex real-time PCR assay that detects three coronavirus target genes (E gene, RdRP gene and N gene) in a single tube, according to the manufacturer's instructions, as previously described [START_REF] Péré | Nasal Swab Sampling for SARS-CoV-2: a Convenient Alternative in Times of Nasopharyngeal Swab Shortage[END_REF].

Abbott SARS-CoV-2 IgG assay detecting IgG against SARS-CoV-2 nucleoprotein was used on Architect analyzer (Abbott Architect™ i2000), according to manufacturer's instructions, as previously described [START_REF] Péré | Unexpected diagnosis of COVID-19-associated disorders by SARS-CoV-2-specific serology[END_REF]. Index value threshold for positivity was 1.4. Qualitative results were used for analysis.

Statistical analysis. Categorical variables are presented as number (percentage and its [95%

confidence interval]), and numeric variables as median or mean (SD or IQR). The number of test results was computed on a weekly basis. The evolution of the status for COVID-19 of HWCS between the two waves was assessed using a Sankey diagram, as described [START_REF] Huang | A richly interactive exploratory data analysis and visualization tool using electronic medical records Clinical decision-making, knowledge support systems, and theory[END_REF].

Ethics statement. Our observational study was carried out in accordance with the Declaration of Helsinki with no sampling addition to usual procedures. Swab and serum specimens were obtained only for standard diagnostic following medical prescriptions in the service of occupational medicine of our institution, and further eventual care. Under these conditions, the study was exempt from informed consent application, according to the French public health code (Code de la Santé Publique, article L 1121-1.1; https://www.legifrance.gouv.fr/). The dataset was completely anonymous and did not contain any identifiable personal health information. Overall, 4579 PCR and 3186 serology tests were performed with positivity rates of 10.7% and 12.1%, respectively. A total of 4168 HCWs (median age, 34.3 years; IQR, 20.9 years) had at least one result, either PCR or serology, during the entire period with a positivity rate of 15.9%. There were 690 HCWs with at least one PCR result during the first wave versus 2340 during the second wave with a positivity rate of 21.3% and 14.1%, respectively. Conversely, 2737 HCWs had a serology result during the first wave versus 417 during the second one, with a positivity rate of 9.8% and 27.8%, respectively (Table 1).

Results

A

Among the 302 positive HCWs (either by serology or PCR) during the first wave, 102 were tested by PCR at least once during the second wave (154 total PCRs), and all their PCR results were negative (Figure 2). Furthermore, among the 330 PCR-positive HCWs during the second wave, 190 had a previous result from the first wave. Among those 190 that were tested in the first wave, there were 9 positive PCRs and 14 positive serology (15 total HCWs), and 170 negative serology and 45 negative PCRs (175 HCWs). All those 15 positive HCWs in the first wave were only positive in serology in the second wave. Among the 2657 negative HCWs during the first wave, 175 had a positive PCR result (185 tests) and 965 (1859 tests) had a negative result during the second wave.

Overall, no case of reinfection during the second wave could be observed among the HCWs population in our hospital. The observed reinfection incidence was thus 0% [95% CI: 0 -3.55%].

Discussion

We herein assessed the incidence rate of SARS-CoV-2 reinfection in the real-life setting of a longitudinal observational cohort of HCWs from one of the major university hospitals of the Assistance Publique-Hôpitaux de Paris, France, during the first and second waves of COVID-19 epidemic in 2020. The longitudinal prevalence rates of SARS-CoV-2 infection among HCWs, mostly symptomatic infections due to the design of the study, mimicked the curves of both waves of the COVID-19 epidemics in France with an overall rate of SARS-CoV-2 infection around 16%, demonstrating possible occupational risk for SARS-CoV-2 infection in HCWs despite barrier protection measures within the community and hospital settings. Among the included SARS-CoV-2 infected HCWs tested for SARS-CoV-2 PCR swab and/or IgG serology in both the first and second waves of the COVID-19 epidemic in Paris, no case of SARS-CoV-2 reinfection could be evidenced. Thus, the risk of SARS-CoV-2 reinfection among HCWs appears negligible, despite the high level of exposure to the virus during epidemic waves. Taken together, these observations demonstrate that HCWs are at high risk of SARS-CoV-2 infection. SARS-CoV-2 reinfection was infrequent in study HCWs, highly suggestive of a protective immunity against reinfection lasting at least 8 months following the primary infection. [START_REF] Contejean | Comparing Dynamics and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 Transmissions Among Healthcare Workers of Adult and Pediatric Settings in Central Paris[END_REF][START_REF] Hunter | First experience of COVID-19 screening of health-care workers in England[END_REF][START_REF] Amanat | A serological assay to detect SARS-CoV-2 seroconversion in humans[END_REF][START_REF] Péré | Sequential SARS-CoV-2 IgG assays as confirmatory strategy to confirm equivocal results: Hospitalwide antibody screening in 3,569 staff health care workers in Paris[END_REF] More than a seventh (15.9%) of HCWs showed at least one positive biological marker for SARS-CoV-2 during the nearly 8 months of study inclusion. The risk of SARS-CoV-2 infection in included HCWs strictly followed the waves of the COVID-19 epidemic in France, during which between 3% and 7% of French people will have been infected by SARS-CoV-2 ( 16). Thus, our findings suggest that HCWs are likely at supplementary risk for occupational SARS-CoV-2 infection, as previously reported in Paris and other settings [START_REF] Contejean | Comparing Dynamics and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 Transmissions Among Healthcare Workers of Adult and Pediatric Settings in Central Paris[END_REF][START_REF] Hunter | First experience of COVID-19 screening of health-care workers in England[END_REF][START_REF] Amanat | A serological assay to detect SARS-CoV-2 seroconversion in humans[END_REF][START_REF] Péré | Sequential SARS-CoV-2 IgG assays as confirmatory strategy to confirm equivocal results: Hospitalwide antibody screening in 3,569 staff health care workers in Paris[END_REF].

The possibility of SARS-CoV-2 reinfection has raised important issues about the strength and durability of the immune response to primary infection, which are key factors in predicting the course of the pandemic [START_REF] Babiker | The Importance and Challenges of Identifying SARS-CoV-2 Reinfections[END_REF]. We herein show SARS-CoV-2 reinfection was infrequent or absent among HCWs infected during the first wave of COVID-19 epidemic, despite their community and occupational re-exposure during the second wave. Our observations are in agreement with the low risk of SARS-CoV-2 reinfection generally reported [START_REF] Babiker | The Importance and Challenges of Identifying SARS-CoV-2 Reinfections[END_REF][START_REF] Abu-Raddad | Assessment of the risk of SARS-CoV-2 reinfection in an intense re-exposure setting[END_REF], although the rates of reinfection with SARS-CoV-2 can vary widely, depending on the criteria used, and also because it is difficult to make the diagnosis with certainty, as previously pointed [START_REF] Tomassini | Setting the criteria for SARS-CoV-2 reinfectionsix possible cases[END_REF]. By including the stringent criteria of viral genomic data to distinguish reinfection from persistent viral carriage, only 16 documented individual cases of reinfection confirmed by sequencing have been reported in the literature at the end of 2020 [START_REF] Babiker | The Importance and Challenges of Identifying SARS-CoV-2 Reinfections[END_REF][START_REF]Investigative Criteria for Suspected Cases of SARS-CoV-2 Reinfection (ICR)[END_REF]. Recently in Qatar, the risk of SARS-CoV-2 reinfection was estimated to be as low as 1 case of reinfection per 5000 infected individuals [START_REF] Abu-Raddad | Assessment of the risk of SARS-CoV-2 reinfection in an intense re-exposure setting[END_REF]. However, a 2,800 person study found no symptomatic re-infections over a ~118 days window [START_REF] Wyllie | SARS-CoV-2 responsive T cell numbers are associated with protection from COVID-19: A prospective cohort study in keyworkers[END_REF], and a 1,246 person study observed no symptomatic re-infections over 6 months [START_REF] Lumley | Antibodies to SARS-CoV-2 are associated with protection against reinfection[END_REF]. Overall, SARS-CoV-2 re-infection is considered a possible but rare event [START_REF] Babiker | The Importance and Challenges of Identifying SARS-CoV-2 Reinfections[END_REF], in keeping with our observational study based on field results from a well-documented hospital cohort. SARS-CoV-2 infection consistently elicits neutralizing antibodies targeting the spike protein (in addition to other viral antigens), as well as CD8+ and CD4+ T cell responses (4-6). However, the duration of effective immunity to SARS-CoV-2 remains unknown, and the issue of waning immunity and reversion to a SARS-CoV-2 susceptible state over months to years is raised [START_REF] Overbaugh | Understanding protection from SARS-CoV-2 by studying reinfection[END_REF]. Our observations of infrequent SARS-CoV-2 reinfection in hospital setting during the first and second waves of COVID-19 epidemic in Paris are strongly suggestive of efficient and protective immunity against reinfection that lasts at least 8 months post primary infection. These observations are in keeping with measurable immune memory in the three major branches of adaptive immunity (CD4+ T cell, CD8+ T cell, and humoral immunity) in ~95% of subjects 5 to 8 months post symptom onset, indicating that durable immunity against secondary COVID-19 disease is a possibility in most individuals [START_REF] Dan | Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection[END_REF]. Obviously, further follow-up of SARS-CoV-2-infected HCWs over time may allow characterization of potential effects of SARS-CoV-2-specific immunity waning.

The need to vaccine previously SARS-CoV-2-infected individuals remains the matter of debate [START_REF] Cohen | Reinfection with SARS-CoV-2: Implications for Vaccines[END_REF][START_REF] Chung | COVID-19 vaccines: The status and perspectives in delivery points of view[END_REF]. It is possible that a fraction of the SARS-CoV-2-infected population with low immune memory would become susceptible to re-infection relatively soon [START_REF] Dan | Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection[END_REF]. Current recommendations for COVID-19 vaccination from the World Health Organization [START_REF] Organization | WHO SAGE values framework for the allocation and prioritization of COVID-19 vaccination[END_REF] and the Centers for Disease Control and Prevention (USA) [START_REF] Dooling | The Advisory Committee on Immunization Practices' Interim Recommendation for Allocating Initial Supplies of COVID-19 Vaccine -United States, 2020[END_REF] do not consider the fact of having been infected with SARS-CoV-2. The National Health Service (UK) recommends to temporarily postponing vaccination for 4 weeks after the onset of COVID symptoms [START_REF]COVID-19: the green book[END_REF]. In France, the Haute Autorité de Santé, Saint-Denis, has recommended that there is no need to systematically vaccinate people who have already developed a symptomatic form of COVID-19 [START_REF]Haute Autorité de Santé -Stratégie de vaccination contre le Sars-Cov-2 -Recommandations préliminaires sur la stratégie de priorisation des populations à vacciner[END_REF]. Our observations of HCWs infected by SARS-CoV-2 harboring durable protection against reinfection for at least 8 months indicate that COVID-19 vaccination does not a priori constitute a priority in case a previous infection, especially if vaccine doses are limited. As vaccine candidates advance worldwide, serostatus for COVID-19 could be relevant in HCWS before vaccination. If it is determined that a single SARS-CoV-2 exposure induces long-lasting protective immunity, a positive serology test could indicate that an individual does not require vaccination or should not receive priority for vaccination [START_REF] West | COVID-19 Antibody Tests: A Valuable Public Health Tool with Limited Relevance to Individuals[END_REF]. Finally, in case of vaccination in HCWS who have already been infected, the vaccination schedule could also possibly be simplified to a single dose.

Our study has some limitations. Except for HCWs that were recently exposed to positive patients or colleagues (contact tracing), only HCWs volunteering for testing were included and probably most of them had symptomatic infection. Furthermore, the possibility could be that reinfections (if they occur) would have been asymptomatic or well tolerated and that they went unnoticed without a biological diagnosis. Finally, HCWs in our institution were exposed to the SARS-CoV-2 variants circulating in France until December 2020, and our study does not evaluate the possibility of reinfection by other variants, such as the new U.K. variant of SARS-CoV-2, which various modeling exercises have estimated to be up to 70% more transmissible than the previously circulating form of the virus [START_REF] Kirby | 9 -17.1HCWs tested by both PCR and serology [n] 525 251 1565 [percent of positivity[END_REF].

In conclusion, SARS-CoV-2 reinfection appears to be a rare phenomenon, if it exists, in HCWs despite their high level of exposure to the virus during epidemic peaks. This feature may suggest that effective immunity against SARS-CoV-2 infection develops after primary infection and lasts for at least 8 months, and that it is able to protect against reinfection. Finally, HCWs already infected with SARS-CoV-2 do not constitute a priority population for vaccination, especially as vaccine doses remain rationed.

  total of 7765 tests, including PCR and serology, were collected from March 5, 2020 to December 4, 2020 by the occupational medicine service at the Hôpital Europèen Georges Pompidou, Paris. As depicted in the Figure 1, the weekly number of PCR tests evolved in accordance to the two waves of the epidemic of cases of SARS-CoV-2 infection in Paris. The number of serology tests per week evolved according to the curve resembling the first wave of the epidemic due to an institutional campaign for SARS-CoV-2 serology testing among HCWs between May and July.

Figure 1 .

 1 Figure 1. PCR and serology results during the first and the second wave of the COVID-19

Figure 2 .

 2 Figure 2. Health-care workers COVID-19 status evolution between the first and the second wave of

Table 1 .

 1 SARS-CoV-2 RNA PCR and SARS-CoV-2-specific IgG serology results carried out among HCWs in Hôpital Européen Georges Pompidou, Paris, France, during the first and second waves of the COVID-19 epidemic in 2020.

	First wave	Second wave	Total

Acknowledgments. The authors gratefully thank the department of clinical biochemistry, Hôpital

Européen Georges Pompidou, Paris, especially the technicians from the serological platform.