Nicolas Flaugnatti

Chiara Rapisarda

Martial Rey

Viet Solène G Beauvois

Anh Nguyen

Stéphane Canaan

Eric Durand

Julia Chamot-Rooke

Eric Cascales

Rémi Fronzes
email: r.fronzes@iecb.u-bordeaux.fr

Viet Anh Nguyen

Laure Journet
email: ljournet@imm.cnrs.fr

Structural basis for loading and inhibition of a bacterial T6 SS phospholipase effector by the VgrG spike

Keywords: type VI secretion system, bacterial competition, cryo-electron microscopy, bacterial toxin, protein secretion

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

INTRODUCTION

The Type VI secretion system (T6SS) is a multiprotein secretion nano-machine used by many Gram-negative bacteria to deliver toxins in a contact-dependent manner directly into target cells. The T6SS can target both eukaryotic and prokaryotic cells, secreting effectors that degrade the peptidoglycan (amidases, glycoside hydrolase), membranes (phospholipases A 1 , A 2 and D), or DNA (DNase) [START_REF] Russell | Type VI secretion system effectors: Poisons with a purpose[END_REF][START_REF] Durand | Tle, and beyond: the versatile arsenal of Type VI secretion effectors[END_REF][START_REF] Cianfanelli | Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon[END_REF][START_REF] Hachani | Type VI secretion and anti-host effectors[END_REF]. Some anti-host effectors interfere with eukaryotic cytoskeleton dynamics (actin, microtubules), leading to phagocytosis inhibition or promoting invasion. [START_REF] Russell | Type VI secretion system effectors: Poisons with a purpose[END_REF][START_REF] Durand | Tle, and beyond: the versatile arsenal of Type VI secretion effectors[END_REF][START_REF] Cianfanelli | Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon[END_REF][START_REF] Hachani | Type VI secretion and anti-host effectors[END_REF]. The T6SS secretion of anti-bacterial effectors allows bacteria to eliminate competitors, to acquire nutrients, new DNA or metal, thus favoring the colonization of specific niches [START_REF] Russell | Type VI secretion system effectors: Poisons with a purpose[END_REF][START_REF] Durand | Tle, and beyond: the versatile arsenal of Type VI secretion effectors[END_REF][START_REF] Cianfanelli | Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon[END_REF][START_REF] Hachani | Type VI secretion and anti-host effectors[END_REF].

T6SS biogenesis needs at least 14 different proteins. These subunits assemble two subcomplexes: a membrane complex anchored in the envelope [START_REF] Durand | Biogenesis and structure of a type VI secretion membrane core complex[END_REF][START_REF] Rapisarda | In situ and high-resolution cryo-EM structure of a bacterial type VI secretion system membrane complex[END_REF], on which is docked a platform that controls the assembly of a contractile tail tube/sheath structure [START_REF] Brunet | The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization[END_REF][START_REF] Logger | Molecular Dissection of the Interface between the Type VI Secretion TssM Cytoplasmic Domain and the TssG Baseplate Component[END_REF][START_REF] Taylor | Structure of the T4 baseplate and its function in triggering sheath contraction[END_REF][START_REF] Nguyen | Type VI secretion TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex[END_REF][START_REF] Nazarov | Cryo-EM reconstruction of Type VI secretion system baseplate and sheath distal end[END_REF][START_REF] Basler | Type VI secretion requires a dynamic contractile phage tail-like structure[END_REF]. The tail tube/sheath complex comprises an inner tube made of Hcp hexamers covered by a sheath and topped by a needle spike constituted of a VgrG trimer and a PAAR-domain protein [START_REF] Zoued | Architecture and assembly of the Type VI secretion system[END_REF][START_REF] Ho | A View to a Kill: The Bacterial Type VI Secretion System[END_REF].

The tail sheath assembles in an extended conformation and its contraction propels the inner tube, the spike and toxins towards target cells. Self-protection is assured by immunity proteins that specifically bind and inhibit their corresponding effectors. A few T6SS effectors are fused to the Hcp, VgrG, or PAAR proteins and are therefore delivered into the recipient cell as domains of syringe components [START_REF] Pukatzki | Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system[END_REF][START_REF] Blondel | Comparative genomic analysis uncovers 3 novel loci encoding type six secretion systems differentially distributed in Salmonella serotypes[END_REF][START_REF] Suarez | A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin[END_REF][START_REF] Brooks | Lytic Activity of the Vibrio cholerae Type VI Secretion Toxin VgrG-3 Is Inhibited by the Antitoxin TsaB[END_REF][START_REF] Dong | Identification of T6SSdependent effector and immunity proteins by Tn-seq in Vibrio cholerae[END_REF][START_REF] Shneider | PAAR-repeat proteins sharpen and diversify the type VI secretion system spike[END_REF][START_REF] Ma | The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems[END_REF]. However, most T6SS effectors are independent polypeptides. Upon T6SS assembly these effectors are loaded within the lumen of the Hcp tube or bind the VgrG/PAAR spike and are then transported as cargo [START_REF] Durand | Tle, and beyond: the versatile arsenal of Type VI secretion effectors[END_REF][START_REF] Cianfanelli | Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon[END_REF][START_REF] Ho | A View to a Kill: The Bacterial Type VI Secretion System[END_REF]. Small effectors (< 20 kDa), such as the Pseudomonas aeruginosa Tse2 toxin, have been shown to fill the internal hole of Hcp hexamers [START_REF] Silverman | Haemolysin Coregulated Protein Is an Exported Receptor and Chaperone of Type VI Secretion Substrates[END_REF] whereas others have been demonstrated to indirectly interact with VgrG. Binding of these effectors is mediated by specific adaptors (Alcoforado [START_REF] Diniz | Intraspecies Competition in Serratia marcescens Is Mediated by Type VI-Secreted Rhs Effectors and a Conserved Effector-Associated Accessory Protein[END_REF][START_REF] Liang | Identification of divergent type VI secretion effectors using a conserved chaperone domain[END_REF][START_REF] Unterweger | Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae[END_REF][START_REF] Bondage | VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex[END_REF]. These adaptors are not secreted components but are rather required for the secretion of their cognate effectors, suggesting a role in loading effectors to VgrG prior to translocation. A complex constituted of the PAAR-fused Tse6 effector, the EagT6 adaptor, the immunity to Tse6 and EF-Tu was recently visualized by negative stain electron microscopy (EM). It forms a horseshoe-like structure at the tip of the VgrG trimer [START_REF] Whitney | An Interbacterial NAD(P)+ Glycohydrolase Toxin Requires Elongation Factor Tu for Delivery to Target Cells[END_REF]. The cryo-EM structure of Tse6-EagT6 in complex with VgrG was further determined [START_REF] Quentin | Mechanism of loading and translocation of type VI secretion system effector Tse6[END_REF].

We recently identified Tle1, a periplasmic-acting phospholipase A 1 (PLA1) antibacterial effector of the enteroaggregative E. coli (EAEC) T6SS-1 [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. We showed that Tle1 is transported into the periplasm of target cells using the VgrG spike protein as carrier. By contrast to other VgrG-dependent effectors, Tle1 interacts directly with VgrG, without the need for a PAAR or adaptor protein. VgrG from EAEC possesses a C-terminal extension that comprises a DUF2345 and a transthyretin-like domain (TTR) responsible for the interaction with Tle1 [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. Here, we report the purification of the VgrG-Tle1 complex and show that three Tle1 effectors bind to the VgrG trimer. Our data reveal that VgrG inhibits Tle1 PLA1 activity and hence that Tle1 needs to be dissociated from its carrier once in the target cell. Using a combined approach of cross-linking mass spectrometry, deletion studies and functional assays, we identify the motifs involved in VgrG-Tle1 interaction, confirming the importance of the VgrG TTR domain and highlighting an Nterminal interaction motif within Tle1. Finally, we present the cryo-EM structure of Tle1 bound to the VgrG needle.

RESULTS

Purification of the VgrG-Tle1 complex reveals that three Tle1 bind to the VgrG trimer

We previously showed that Tle1 from EAEC interacts directly with the C-terminal domain extension of the VgrG spike protein [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. To gain further insight into the VgrG-Tle1 complex, affinity-tagged VgrG and Tle1 proteins were co-produced in E.

coli. The position of the affinity tag on each protein was rationally chosen to maintain the VgrG-Tle1 interaction: a Strep-tagII was fused to the N-terminus of VgrG ( S VgrG) to maintain the C-terminal Tle1-interacting motif available, and a 6×His tag was fused to the Tle1 C-terminus (Tle1 H ), as previous bacterial two-hybrid (BACTH) analysis suggested that T25/T18 fusion at the N-terminus of Tle1 protein impaired the interaction with VgrG [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. A complex containing both S VgrG and Tle1 H was purified to homogeneity using a two-step affinity purification and gel filtration (Fig EV1A). When subjected to size exclusion chromatography analyses, S VgrG separated with an apparent mass of ~ 500 kDa (Fig EV1B,black line;Appendix Fig S1), which is higher than the 280-kDa theoretical mass of a VgrG trimer. This could be due to an artefact of the gel filtration technique, the elongated-shape structure of the VgrG β-helix [START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF][START_REF] Uchida | Structure and properties of the Cterminal β-helical domain of VgrG protein from Escherichia coli O157[END_REF][START_REF] Spínola-Amilibia | The structure of VgrG1 from Pseudomonas aeruginosa , the needle tip of the bacterial type VI secretion system[END_REF], or this species may correspond to a dimer of a VgrG trimer. Once bound to Tle1 H , we observed an increase in the apparent molecular mass (Fig EV1B, blue line). Native-purified Tle1 was previously shown to be monomeric in solution, with an apparent molecular mass of 66 kDa [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. Taken together, these results suggest that several Tle1 proteins are bound to the VgrG trimer. To provide further information on the stoichiometry of the S VgrG-Tle1 H complex, we performed quantitative ingel SYPRO Ruby staining. This analysis of the purified S VgrG-Tle1 H complex gave a 1:0.92 molar ratio (Fig EV1C). The approximate 1:1 stoichiometry suggests that three Tle1 effectors are bound to a VgrG trimer.

High-resolution cross-linking mass spectrometry (XL-MS) mapping identifies VgrG-Tle1 sites of contact

To provide detailed information on the VgrG/Tle1 interaction, the S VgrG-Tle1 H complex was subjected to cross-linking with the NNP9 cross-linker [START_REF] Nury | A Novel Bio-Orthogonal Cross-Linker for Improved Protein/Protein Interaction Analysis[END_REF]. NNP9 was specifically engineered to carry 2 NHS carbamate reactive groups, which are less prone to hydrolysis than widely used NHS-esters, thus improving cross-linking efficiency and performance compared to commercial ones [START_REF] Nury | A Novel Bio-Orthogonal Cross-Linker for Improved Protein/Protein Interaction Analysis[END_REF]. Mass spectrometry analysis of cross-linked peptides revealed that VgrG and Tle1 establish numerous contacts (Fig 1A,Dataset EV1). In agreement with previous findings demonstrating that Tle1 binds the VgrG C-terminal TTR extension as well as a second upstream motif [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF] Cryo-EM structure of the VgrG-Tle1 complex. Architecture of the VgrG spike-Tle1 complex. To gain further information on the structure of the VgrG-Tle1 complex, the purified S VgrG-Tle1 H complex was analysed using single particle cryo-EM. The structure of the complex at 2.7-Å resolution overall was obtained by applying no symmetry (Fig EV2A andB). The 2D classes resembled violin bodies and were found in different orientations (Fig EV2A). From the unmasked cryo-EM density we can observe the presence of a D3 symmetry with the dihedral rotational centre found between the two prism-like densities and further strengthened by opposite flexible densities interacting with each other (Fig EV2B andC). This large symmetric complex is 225-Å tall and 150-Å large, and consists of 6 VgrG and 6

Tle1 densities as inferred from the gel filtration (Fig EV1B,Fig EV2B and C). This oligomeric state is likely not physiological. The absence of the PAAR capping protein may lead to this tip-to-tip VgrG needle spike interaction. We performed an unmasked refinement to 2.7 Å (Fig EV2B -D) followed by a local refinement on what we will hereby refer to as the physiological complex, consisting of 3 Tle1 and 3 VgrG molecules, and obtained a resolution No interpretable cryo-EM density was found for VgrG amino-acids 1-490. This region which is homologous to the gp27-like base followed by the gp5-OB fold-like in the T4 phage, appears to be disordered. Such flexibility may be stabilised by binding of PAAR, and/or by the binding to baseplate components. This region is well conserved amongst the VgrG proteins from different bacterial species and phage families [START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF]. We therefore built a model using Phyre2 [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF], based on the sequence homology (42.5% identity) to the VgrG N-terminus from uropathogenic E. coli CFT073, whose structure has been solved by X-ray crystallography (Leiman et al, 2009) (Fig 2G). of 5 and3 strands respectively at aa 491-549 and726-747 (Fig EV3A).

Structure of the gp5 needle-like domain of

Unique to the EAEC VgrG (VgrG EAEC ) is also the presence of two long helices that break the β-prism at aa 576-622. The rest of the β-prism is composed of parallel β-sheets that intertwine around each other via sharp β-turns (Fig EV3A). There is no cavity at the centre of the βprism and the structure is maintained by hydrophobic interactions (Fig 2H). The interaction between the monomers of VgrG, as calculated using PISA [START_REF] Krissinel | Inference of Macromolecular Assemblies from Crystalline State[END_REF], is very strong with a mean surface of interaction of 7150 Å 2 .

Comparison with the

Tle1 binding to VgrG inhibits its phospholipase activity

The Tle1 effector exhibits PLA1 activity in vitro [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. In the cryo-EM structure of VgrG-Tle1, the catalytic module of Tle1 is well defined. It contains the phospholipase catalytic triad in an active conformation as the Ser197 and Asp245 residues are at a <3-Å distance from His310 (Fig 3B). The catalytic triad is located within a cavity that is positively charged at its end, with a hydrophobic entrance (Fig EV5A -B). However, comparison of the cryo-EM structure of Tle1 EAEC bound to VgrG to the P. aeruginosa Tle1 (Tle1 PA ) crystal structure suggests that Tle1 EAEC PLA1 activity is inhibited by VgrG. In the Tle1 PA structure, a specific pocket allows binding of the phosphatidylglycerol moiety of the substrate at close proximity to the active site (Fig 4A) [START_REF] Hu | Structure of the type VI secretion phospholipase effector Tle1 provides insight into its hydrolysis and membrane targeting[END_REF]. By contrast, in the Tle1 EAEC structure, this pocket is partially occluded by the lid domain. Remarkably, the position of the lid domain is constrained by the binding of its tip (helix 389-393) to VgrG (Fig 4B). In agreement with this observation, PLA1 activity of the purified S VgrG-Tle1 H complex was undetectable compared to the purified isolated Tle1 H protein (Fig 5A ), suggesting that Tle1 binding to VgrG inhibits its activity. To confirm this VgrG-mediated inhibition, isolated Tle1 H purified protein was incubated with increasing amounts of purified S VgrG. Taken together, these results show that the Tle1 phospholipase activity is inhibited by VgrG in the S VgrG-Tle1 H complex, and hence that the Tle1 effectors have to be dissociated from VgrG to exhibit toxic PLA1 activity in the target cell.

DISCUSSION

In this study, we provide a comprehensive picture of a complex between the T6SS VgrG spike protein and the phospholipase effector it recruits for translocation into target cells.

We show that these two proteins form a complex with a 1:1 stoichiometry comprising three Tle1 effectors bound to one VgrG trimer, suggesting that three Tle1 effectors are translocated into the target cell at each T6SS firing event. We further show that Tle1 recruitment to VgrG causes the inhibition of its activity. Bioinformatics, high-resolution mass spectrometry mapping and deletion studies combined with protein-protein interaction and functional assays demonstrated the importance of the N-terminal portion of Tle1 as a critical determinant for the interaction with the VgrG C-terminal TTR extension. Finally, we report the highresolution cryo-EM structure of the Tle1 effector in complex with the needle/TTR portion of the VgrG spike. This structure confirms the importance of the TTR domain of VgrG for its interaction with the N-terminal finger domain of Tle1, and further highlights two previously unidentified regions of contact at the base and at the centre of the needle. Moreover, we provide a structural explanation for Tle1 inhibition by VgrG.

Structural model of the EAEC T6SS baseplate in a pre-firing state

VgrG is not only the spike of the needle but also the hub of the T6SS baseplate (BP) [START_REF] Taylor | Structure of the T4 baseplate and its function in triggering sheath contraction[END_REF][START_REF] Brunet | The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization[END_REF]. The baseplate is the assembly platform for the tail tube/sheath complex and is docked to the TssJLM membrane complex [START_REF] Brunet | The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization[END_REF][START_REF] Zoued | Priming and polymerization of a bacterial contractile tail structure[END_REF]. The structure of the TssKFGE wedge complex of the baseplate was recently determined by cryo-EM [START_REF] Cherrak | Biogenesis and structure of a type VI secretion baseplate[END_REF]. The structure of the fully assembled baseplate, including the VgrG hub surrounded by six wedges, was presented, based on the high-resolution wedge structure [START_REF] Cherrak | Biogenesis and structure of a type VI secretion baseplate[END_REF] and the baseplate lower resolution structure [START_REF] Nazarov | Cryo-EM reconstruction of Type VI secretion system baseplate and sheath distal end[END_REF]. We propose here a structural model of the EAEC T6SS baseplate including . Interestingly, the baseplate can readily accommodate the three Tle1 effectors (Appendix Fig S6). We propose that the VgrG-Tle1 structure represents the "pre-firing complex".

A new mode of T6SS substrate loading.

The VgrG-Tle1 complex represents the first example of direct binding of an effector on VgrG, which in general requires the PAAR subunit or an adaptor protein [START_REF] Shneider | PAAR-repeat proteins sharpen and diversify the type VI secretion system spike[END_REF]Alcoforado Diniz & Coulthurst, 2015;[START_REF] Liang | Identification of divergent type VI secretion effectors using a conserved chaperone domain[END_REF][START_REF] Unterweger | Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae[END_REF][START_REF] Bondage | VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex[END_REF]. Tle1 binds to three different regions of VgrG (bottom, centre and top), and the three Tle1 molecules are located on the side of the needle rather than on its tip. This contrasts to the position of PAAR-fused Tse6 effector/adaptor in the P. aeruginosa VgrG tip complex EM structure [START_REF] Whitney | An Interbacterial NAD(P)+ Glycohydrolase Toxin Requires Elongation Factor Tu for Delivery to Target Cells[END_REF][START_REF] Quentin | Mechanism of loading and translocation of type VI secretion system effector Tse6[END_REF]) (Fig EV3B andE). Tle1 positioning on the VgrG sides and the multiple attachment zones tightly tether Tle1 to the spike, which will be released by some unknown mechanism in the periplasm of the target cell.

The non-covalent nature of the bonds may also allow Tle1 to be separated from the VgrG spike to elicit its function. The oxidative nature of the periplasm or the presence of specific chaperones might provide the source for Tle1 dissociation. Further work will need to be carried out to understand the implication of such flexible interactions in VgrG-Tle1 dissociation.

The EAEC T6SS-1 cluster also encodes a PAAR protein downstream the Tli1 immunity encoding gene. However, in the VgrG-Tle1 complex characterized here, no PAAR is needed for the three Tle1 proteins to bind to the VgrG trimeric spike. The tip of the needle is free to accommodate a PAAR protein necessary for target cell penetration. In the future, it would be of particular interest to co-purify and image PAAR associated to the VgrG-Tle1 complex.

In contrast to canonical VgrGs, such as VgrG PA , VgrG EAEC bears a C-terminal extension (CTD) comprising DUF2345 and TTR domains, linked to the gp27/gp5 base by two long helices (Fig 2G). As shown in this study, the DUF2345 domain folds as a β-prism and hence extends the length of the VgrG EAEC gp5-like β-helix. This extension, as well as the TTR domain, corresponds to the major binding regions of Tle1. VgrG EAEC is therefore a fusion between a prototypical VgrG and DUF2345/TTR extensions that serve as internal adaptor domains [START_REF] Bondage | VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex[END_REF][START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF][START_REF] Wettstadt | Delivery of the Pseudomonas aeruginosa Phospholipase Effectors PldA and PldB in a VgrG-and H2-T6SS-Dependent Manner[END_REF]. However, there is an additional contact between Tle1 and the base of gp5-C domain of VgrG. The direct binding of Tle1 on VgrG EAEC therefore represents the first report of a new mode of effector loading on the T6SS spike. Similar modes of binding might be predicted. This organization of VgrG C-terminal extension (i.e., the presence of two long helices followed by the DUF2345 and the TTR domains) corresponds to the COG4532 found at the C-terminal domain of a subgroup of VgrG proteins in Proteobacteria [START_REF] Boyer | Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?[END_REF]. This group includes proteins such as the VgrG encoded upstream of a putative Tle3 effector in uropathogenic E. coli CFT073, or VgrG2a from P. aeruginosa encoded upstream the Tle4-like tlpE effector [START_REF] Wood | PAAR proteins act as the 'sorting hat' of the type VI secretion system[END_REF] (Appendix Fig S7). The structural organization of these VgrG proteins and their genetic link to phospholipase-encoding genes suggest that these phospholipases might also be directly recruited to the spike extension for delivery.

Tle1 inhibition by VgrG

It is noteworthy that the 3' of the vgrG gene overlaps with the tle1 5' in EAEC (Flaugnatti et al, 2016), suggesting translational coupling of the VgrG and Tle1 proteins, a mechanism evolved to promote protein stabilization or to prevent protein toxicity. Indeed, our results

show that the VgrG-bound Tle1 phospholipase is inactive as the catalytic site of Tle1 is masked by the mode of binding to VgrG. Production of the Tle1 phospholipase may have deleterious effect on the cytoplasmic membrane phospholipids, and VgrG-mediated inhibition could be regarded as a surveillance mechanism to prevent phospholipid degradation in the attacker cell. However, previous results have shown that Tle1 cytoplasmic overproduction has no impact on the growth of E. coli K-12 cells [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. Another hypothesis is that there is no need to inhibit Tle1 but rather a need to protect the catalytic site and to prevent exposition of the hydrophobic entrance. Nevertheless, Tle1 needs to dissociate from VgrG once in the periplasm of the target cell.

In conclusion we have identified a novel mechanism of loading and inhibition in the T6SS

Tle1 toxin that establishes a new relationship between the spike and its carrier effector.

MATERIALS AND METHODS

Bacterial strains, growth conditions and chemicals.

Strains used in this study are listed in Appendix Table S1. E. coli DH5α, BL21(DE3), and W3110 were used for cloning procedures, protein production, and co-immunoprecipitation, respectively. EAEC strain 17-2 and its isogenic derivatives were used for in vivo studies.

Cells were grown at 37°C with aeration in LB or in T6SS-1-inducing medium (SIM; M9 minimal medium, 0.2% glycerol, 1 μg/mL vitamin B1, 100 μg/mL casaminoacids, 10% LB, supplemented or not with 1.5% bactoagar, ampicillin (100 μg/mL), kanamycin (50 μg/mL), or chloramphenicol (30 μg/mL). Gene expression from pBAD [START_REF] Guzman | Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter[END_REF] and pOK12 [START_REF] Vieira | New pUC-derived cloning vectors with different selectable markers and DNA replication origins[END_REF] derivatives was induced by the addition of 0.02-0.2% of Larabinose (Sigma-Aldrich) and 100 µM isopropyl-thio-β-D-galactopyrannoside (IPTG, Eurobio), respectively. Gene expression from pETDuet-1 and pRSFDuet-1 (Novagen) derivative vectors was induced by the addition of 1 mM IPTG.

Cloning procedures.

Plasmids and primers used in this study are listed in Appendix Table S1. PCRs were performed using a Biometra thermocycler using the Q5 high-fidelity DNA polymerase (New England Biolabs) and EAEC 17-2 chromosomal DNA as a template. Custom oligonucleotides (Sigma-Aldrich) are listed in Appendix Table S1. pRSF-Tle1 H and pET-S VgrG were constructed by a restriction-ligation procedure. The sequence encoding Tle1 was amplified by PCR using 5-NdeI-Tle1 and 3-FseI-Tle1 H primers introducing NdeI and FseI sites, respectively and cloned into the pRSFDuet-1 (Novagen) multiple cloning site 2 (MCS2) to yield pRSF-Tle1 H . The 3-FseI-Tle1 H primer introduces a sequence encoding a 6×His-tag to allow in-frame fusion of Tle1 with a C-terminal hexa-histidine extension. The sequence encoding VgrG was amplified by PCR using primers 5-BamHI-S VgrG and 3-HindIII-VgrG introducing BamHI and HindIII sites, respectively and cloned into the pETDuet-1 MCS1 to yield pET-S VgrG. The 5-BamHI-S VgrG primer introduces a sequence encoding a Strep-tagII to allow in frame fusion of VgrG with a N-terminal Strep-tagII. The same cloning strategy was used for pET-S VgrG ∆CTD and pET-S VgrG 1-490 , with the use of primers 5-BamHI-S VgrG and 3-HindIII-VgrG ∆CTD for pET-S VgrG ∆CTD , and primers 5-BamHI-S VgrG and 3-HindIII-VgrG 1-490 for pET-S VgrG 1-490 . For pBAD18-Tle1∆1-26 VSVG, pBAD18-Tle1∆1-34 VSVG and pRSF-Tle1 ∆1-26 H constructions, the sequence encoding amino-acids 1 to 26 (or 1 to 34) of Tle1 has been deleted from pBAD18-Tle1 VSVG [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF] or pRSF-Tle1 H by Quick-change PCR-based targeted mutagenesis using complementary pairs of oligonucleotides and the Pfu Turbo high-fidelity polymerase (Agilent Technologies). Similarly, the K28-to-E/K29to-E/K30-to-E substitutions were introduced in the pBAD18-Tle1 VSVG plasmid by QuickChange PCR-based targeted mutagenesis. All constructs were verified by restriction analysis and DNA sequencing (Eurofins, MWG).

Antibacterial competition assay.

Antibacterial competition assays were performed as described previously [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF], using wild-type E. coli W3110 cells transformed with the pUA66-rrnB plasmid [START_REF] Zaslaver | A comprehensive library of fluorescent transcriptional reporters for Escherichia coli[END_REF] as a prey. In addition to conferring resistance to kanamycin, the pUA66-rrnB plasmid allows a strong constitutive expression of the green fluorescent protein.

The experiments were done in triplicate, with identical results, and the results of a representative experiment is shown here.

Co-immunoprecipitation.

W3110 cells producing Tle1, Tle1 ∆1-26 or VgrG proteins from independent plasmids were subjected to co-immunoprecipitation using ANTI-FLAG ® M2 affinity gel (Sigma-Aldrich), as described previously [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. Anti-FLAG (clone M2, Sigma-Aldrich) or anti-VSV-G (clone P5D4, Sigma-Aldrich) monoclonal antibodies were used for Western-blot analyses.

VgrG-Tle1 complex production and co-purification.

The pRSF-Tle1 H and pET-S VgrG plasmids were co-transformed into BL21(DE3). Following growth at 37°C in LB medium to an OD 600nm of 0.7, the expression of tle1 and vgrG genes was induced with 1 mM IPTG at 16°C for 16 h. Cell were pelleted , resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 100 µg/mL DNase I, 100 µg/mL lysozyme, cOmplete EDTA-free protease inhibitor cocktail (Sigma) and 10 mM

MgCl 2 ) and broken using an Emulsiflex-C5 (Avestin). After centrifugation at 38,500 × g for 30 min, the supernatant was loaded onto a 5-mL StrepTrap HP column (GE Healthcare) at 4°C and the column was washed with affinity buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl) at 4°C. The S VgrG-Tle1 H complex was then eluted directly into a 5-mL HisTrap HP (GE Healthcare) column with affinity buffer containing 2.5 mM desthiobiotin (IBA). After a washing step in affinity buffer supplemented with 20 mM imidazole, the S VgrG-Tle1 H complex was eluted in affinity buffer supplemented with 500 mM imidazole. The fractions corresponding to the peak were pooled and loaded onto a Superose 6 10/300 column (GE Healthcare) equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl buffer. The S VgrG-Tle1 H complex eluted as a single monodisperse peak. The same production conditions and purification protocol were applied for the purification of S VgrG, S VgrG 1-490 , Tle1 H and Tle1 ∆1- 26 , except that only one affinity column was performed (StrepTrap HP for S VgrG and S VgrG 1- 490 , HisTrap for Tle1 H and Tle1 ∆1-26 ) followed by a gel filtration as described above. For Tle1 inhibition studies, S VgrG and S VgrG 1-490 eluted from the StrepTrap column were used directly (without a gel filtration step), due to the tendency of S VgrG to precipitate during or after the gel filtration step when purified alone. A representative SDS-PAGE followed by Instant blue staining of the resulting purified proteins is presented in Fig 5C .      Sypro-Ruby staining.

Aliquots of 2.5, 5, and 10 µL of the VgrG-Tle1 complex from two different peak fractions of the gel filtration column were subjected to SDS-PAGE. The gel was then stained with Sypro-Ruby as recommended by the manufacturer (Invitrogen). Protein bands were visualized using a Biorad Chemidoc MP Imaging system, using a 605/50 Filter (excitation wavelength = 488 nm, emission wavelength = 605 nm) with 0.5 sec exposition, and the fluorescence intensity was quantified using the Fuji Image software. Intensities were divided by the molecular weight of each protein and the quantification is expressed as the mean +/-standard deviation relative to VgrG bands intensity

Cryo-EM sample preparation and data collection

UltraAuFoil Holey gold Film grids (Quantifoil) were glow discharged for 20 s at 2 mA in an ELMO glow discharge cleaning system (Agar Scientific, UK). Four µl of sample at 600 µg/mL were loaded on the glow discharged grid at 100 % humidity and 4°C in a Vitrobot (Thermo Fisher Scientific, USA). Following a 5 s pre-blot the grid was blotted for 4 s and plunged into liquid ethane. Grids were screened on a Talos Arctica electron microscope equipped with a Falcon 3EC camera (Thermo Fisher Scientific, USA), and the final data were collected on a K2-Summit detector through a Gatan Quantum 967 LS energy filter using a 20 eV slit width in zero-loss mode (Gatan, Japan) on a Titan Krios transmission electron microscope at 300 kV (Thermo Fisher Scientific, USA). Table 2 summarises the acquisition parameters.

Cryo-EM data processing

The 40 frames for each movie collected were aligned for each of the 11,600 movies using MotionCor2, binned by 1.28 with dose weighting (1.24 e-/Å2/frame) and with 5×5 patches applied [START_REF] Zheng | MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy[END_REF]. gCTF was used to estimate the CTF parameters [START_REF] Zhang | Gctf: Real-time CTF determination and correction[END_REF].

Cryosparc [START_REF] Punjani | cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination[END_REF] In Relion, CTF Refine and the Bayesian polishing steps [START_REF] Zivanov | A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis[END_REF]) yielded a final resolution of 2.3 Å for the masked VgrG density and was used for model building and real space refinement (Fig EV2F andG). The quality of the maps for the Tle1/TTR densities were worse than those obtained with cryosparc and were thus not used. The resolution for the Tle1

and VgrG densities was calculated by cryosparc and Relion postprocess respectively according to the "gold standard" method using 0.143 as the FSC value cut-off [START_REF] Rosenthal | Optimal Determination of Particle Orientation, Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy[END_REF]. The local resolution was calculated by cryosparc [START_REF] Punjani | cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination[END_REF] or

Relion with a cut-off of 0.5 (Appendix Fig S5A -C).

Model building and validation

Model building proceeded differently for VgrG and Tle1, but for both no homologous protein could be docked in the density and the Phenix autosharpened (Terwilliger et al, 2018b) cryo-EM densities were used. For Tle1, a partial initial model based on sequence was built using phenix.map_to_model (Terwilliger et al, 2018a). The model was subsequently corrected and extended manually in Coot [START_REF] Emsley | Features and development of Coot[END_REF]. For VgrG, the Phenix automatic model building was not successful. The model was built starting with the only 2 α-helices and using the cryo-EM density to place manually all the amino acid sequence. For the TTR domain that interacts with Tle1, we combined the use of bulky side-chains as guides for model building, secondary structure and residue contact predictions according to the Phyre2 [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF] and the RaptorX [START_REF] Källberg | Template-based protein structure modeling using the RaptorX web server[END_REF] servers respectively. The VgrG models were both refined using phenix.real_space_refine [START_REF] Afonine | Real-space refinement in PHENIX for cryo-EM and crystallography[END_REF] and manual adjustments were done with Coot [START_REF] Emsley | Features and development of Coot[END_REF]. Each model was initially validated using the protocol implemented in Refmac5 (Murshudov et al, 2011) and visually (Appendix Fig S8).

The FSC map-to-model was calculated with the autosharpened maps (FSC sum ). 2). Pore radius calculations were carried out using the MOLE server [START_REF] Berka | MOLEonline 2.0: interactive web-based analysis of biomacromolecular channels[END_REF] and the protein interfaces were analysed with PISA [START_REF] Krissinel | Inference of Macromolecular Assemblies from Crystalline State[END_REF]. Unless stated otherwise, all of the figures were generated either using Chimera [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF], ChimeraX [START_REF] Goddard | UCSF ChimeraX: Meeting modern challenges in visualization and analysis[END_REF] and Coot [START_REF] Emsley | Features and development of Coot[END_REF].

Cross-linking mass spectrometry. Cross-linking of the purified complexes was carried out with freshly prepared NNP9 (10 mM in DMSO) using 10:1 cross-linker/protein molar ratio.

Cross-linking was performed at 4°C for 30 min and stopped by the addition of ammonium bicarbonate (AB, final concentration 50 mM) for 15 min at 4°C. The samples were transferred into a molecular filter device (Amicon ultra 0.5 mL with 30 KDa molecular cut-off) and centrifuged at 10,000×g for 5 min. The concentrate was then washed 4 times by concentration-dilution cycle with AB 50 mM to remove the excess of cross-linker. The labelled protein was digested overnight at 37°C by addition of Trypsin Gold, Mass Spectrometry Grade (Promega) at an enzyme:protein ratio of 1:50 (w:w) under 900 rpm shaking. The peptides were recovered through the molecular filtering device by centrifugation. 50 µL of 0.1% (v:v) formic acid were added onto the filter and a second centrifugation was performed to improve the peptide recovery. Cross-linked protein digests were analyzed by nanoLC-MS/MS on an Orbitrap Q Exactive Plus mass spectrometer (Thermo-Scientific). Briefly, peptides were separated on homemade C18 column using a 90 min water/acetonitrile gradient. The 10 most intense ions with at least 3 charges were selected for HCD fragmentation (NCE 24). NanoLC-MS/MS data were processed automatically using Mass Spec Studio v2.0 [START_REF] Sarpe | High Sensitivity Crosslink Detection Coupled With Integrative Structure Modeling in the Mass Spec Studio[END_REF] with methionine oxidation as a variable modification and NNP9 modification sites: lysine, serine, threonine, and tyrosine. Mass modifications were set to 314.1127 Da for cross-linked peptides, 288.1335 and 331.1393 for dead-end modifications when NNP9 reacted respectively with water or ammonium molecule.

Cross-linked peptides were searched using the parameter file provided in supplemental data (Dataset EV2). MS/MS spectra of all crosslinked candidates were further manually checked to confirm their identification. Experiments were performed in duplicate (using two different complex preparations) and only cross-linked peptides identified in both experiments were considered.

Phospholipase A 1 fluorescent assays.

PLA1 activities of Tle1, Tle1 ∆1-26 , VgrG and the VgrG-Tle1 complex were monitored using BODIPYVR dye-labeled phospholipids: PED-A1(N-((6-(2,4-DNP)Amino)Hexanoyl)-(BODIPYVRFL C5)-2-Hexyl-sn-Glycero-3-Phosphoethanolamine [START_REF] Farber | Genetic Analysis of Digestive Physiology Using Fluorescent Phospholipid Reporters[END_REF][START_REF] Darrow | A novel fluorogenic substrate for the measurement of endothelial lipase activity[END_REF], as described previously [START_REF] Flaugnatti | A phospholipase A 1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery[END_REF]. Enzyme activities were assayed at room temperature in 50 mM HEPES pH 7.5, 150 mM NaCl for 25 min in a final volume of 200 µL containing 5 µM of the substrate and 1.18 µM of Tle1 H , 1.18 µM of S VgrG or 0.393 µM of S VgrG-Tle1 H complex (corresponding to 1.18 µM of Tle1 considering a 3:3 stoichiometry) purified proteins (from 1-2 mg/mL stock solutions). Thermomyces lanuginosus lipase (TLL, Sigma-Aldrich) was used as a positive control for PLA1 activity. Inhibition assays were performed in desthiobiotin containing buffer (Tris-HCl 50 mM pH 8.0, NaCl 150 mM, desthiobiotin 2.5 mM) using S VgrG or S VgrG 1-490 purified at 1.5-2 mg/mL, after checking that desthiobiotin did not affect Tle1 PLA1 activity. Lysozyme from chicken egg white (Sigma) was used as a negative control. Differences in Tle1 activities between groups were examined by unpaired two-sample Fisher-Pitman permutation test.

Computer algorithms.

EAEC Tle1 orthologs (EC042_4534; NCBI Gene Identifier (GI): 284924255) were detected using BlastP analysis against the KEGG genes database and aligned using ClustalW and draw alignment tool in KEGG [START_REF] Kanehisa | KEGG: new perspectives on genomes, pathways, diseases and drugs[END_REF], or Multialin [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF][START_REF] Robert | Deciphering key features in protein structures with the new ENDscript server[END_REF] Protein domains were identified using Pfam [START_REF] Sonnhammer | Pfam: A comprehensive database of protein domain families based on seed alignments[END_REF] or CDart [START_REF] Geer | CDART: Protein Homology by Domain Architecture[END_REF].

  , XL-MS showed that Tle1 binds the VgrG TTR motif and a second motif located in the C-terminal part of the gp27-like domain (Fig 1A). Accordingly, Tle1 H no longer co-purified with a S VgrG variant lacking the C-terminal 616-841 residues ( S VgrG ∆CTD ) (Appendix Fig S2A and B). More importantly, several cross-links involving Lys residues in the VgrG TTR domain and at the Tle1 N-terminus (K29, K30) were detected (Fig 1A and Appendix Fig S3A), suggesting that the VgrG TTR binds the Tle1 N-terminus. Interestingly, a sequence alignment of Tle1 orthologs reveals a striking difference at their N-termini. Compared to specialized Tle1 orthologs that are fused to Hcp, PAAR and VgrG proteins, isolated Tle1 proteins bear an N-terminal 26-amino-acid extension (NT) (Fig 1B and Appendix Fig S3B). As deletion of the Tle1 NT region (Tle1 ∆1-26 ) did not impact PLA1 specific activity (Appendix Fig S4), this region is not necessary for Tle1 folding and activity. Tle1 NT extension is followed by a Lysrich linker that precedes the Tle1-domain region (Appendix Fig S3). Substitution of these Lysines by Glutamate residues (K28K29K30-to-E28E29E30) did not impair Tle1 antibacterial activity against E. coli K12 (Fig 1C), suggesting that the K29 and K30 residues identified by XL-MS are not directly involved in the interaction with VgrG. Rather, the XL-MS data highlight the proximity of the NT and the TTR region in the complex and we hypothesized that the NT region might be an important determinant of a Tle1 secretion signal allowing VgrG recognition and binding. Evidence for the crucial role of this extension was provided by co-immunoprecipitation (Fig 1D) and co-purification (Appendix Fig S2C) assays, which demonstrated that deletion of the Tle1 N-terminal 26 amino-acids strongly affects the interaction with VgrG. To further test the importance of this VgrG interaction motif for the delivery of Tle1, we tested the ability of Tle1 ∆1-26 to complement a tle1 knockout mutant in antibacterial competition (Fig 1C). While the production levels of Tle1 and Tle1 ∆1- 26 were comparable, only Tle1 restored the killing defect of the tle1 knockout mutant. Taken together these results showed that Tle1 binds two regions of VgrG including the C-terminal TTR extension, whereas the Tle1 N-terminal 26-amino-acid region is required for VgrG binding and translocation into target cells.

  of 2.6 Å (Fig 2A-C, Fig EV2E). We further locally refined the prism-like density corresponding to VgrG (to 2.3-Å resolution), and one external density belonging to Tle1 (to 2.5-Å resolution) (Fig EV2F and G). Thanks to the high resolution of the cryo-EM densities obtained (Fig EV2E-G), we could build de novo an atomic model of VgrG amino-acids (aa) 491-834 and of the full Tle1 (Fig 2D-H and Fig 3A and B), whose structures were previously unknown.

  VgrG from EAEC. The atomic model of VgrG (aa 491-834) includes the gp5-C-like, DUF2345 and TTR domains predicted by bioinformatic analyses (Flaugnatti et al, 2016) (Fig 1A and Fig 2G-H). The region from aa 491 to 760 assembles a gp5 needle-like structure corresponding to a three-stranded β-prism with a width of 30 Å at the tip and 45 Å at the bottom (Fig 2G-H). The β-prism contains 2 antiparallel βsheets consisting

  Fig EV4A).A new mechanism for T6SS effector loading on the VgrG spike. In contrast to the VgrGchaperone-effector in P. aeruginosa, where the effector is fused to PAAR and located at the tip of the needle complex (Fig EV3E), the loading of Tle1 proteins on the VgrG spike is independent of PAAR. Moreover, three molecules of Tle1 are loaded on the VgrG trimer, on the sides of the β-prism (Fig 3F-H and Supplementary Fig EV3D). Tle1 binds VgrG in three different regions (bottom, centre and top) and every VgrG chain interacts with each Tle1 monomer (Fig 3F-H, and Table 1). The Tle1 catalytic domain interacts with the two α-helices (581-619) of one VgrG (chain A) and at the level of the β-prism with the two other VgrG monomers (chains B and C) (Fig 3F-H). The lid domain of Tle1 (aa 335-405) interacts only with chains B and C at the level of the anti-parallel β-sheet (Fig 3F-H). Finally, the finger domain of Tle1 (aa 3-33) and the loop of the catalytic domain (aa 225-230) between helix 8 and strand 5 interact with the TTR domain of VgrG (aa 778-841) (Fig 3C-E). The TTR domain is globular: it contains 2 sheets composed of 3 and 2 anti-parallel β-strands respectively (Fig 3C-E). The finger domain of Tle1 complements the strands of the TTR domain to form a β-barrel (Fig 3C-E). The surface of interaction of VgrG with Tle1 is 1160 Å 2 and has a ∆G of -12.2 kcal/M. The TTR-Tle1 interaction is mediated by several hydrogen

  Fig 5B shows that Tle1 H was inhibited by S VgrG in a dose-dependent manner, but not with the unrelated lysozyme protein. As a control, we purified S VgrG 1-490, a C-terminal truncated variant of VgrG deleted of the gp5 needle-like domain which mediates most of the contacts with Tle1 H (Fig 5C). In contrast with S VgrG, incubation of Tle1 H with S VgrG 1-490 did not cause Tle1 H inhibition (Fig 5B).

  template picker selected 1.94 million particles that were extracted with a box-size of 350 pix. After several rounds of 2D classification in cryosparc (Fig EV2A), a heterogeneous ab initio reconstruction with 2 classes, and a heterogeneous refinement (2 classes), 468,438 particles were selected for homogeneous refinement. An initial unmasked refinement using the ab initio model from the crysoparc ab initio class, gave us a resolution of 2.7 Å with no applied symmetry (Fig EV2B-D). A masked local refinement around the physiological complex yielded a final resolution of 2.6 Å (Fig EV2E). The density corresponding to the Tle1 effector was refined locally with cryosparc to a resolution of 2.5 Å.

  The model was shaken by 0.5 Å and the FSC map-to-model was calculated with one Half map (FSC work ). This refined model was then used to calculate the FSC map to model with the other Half map (FSC free ) (Appendix Fig S8A-C).The cross-correlation between each amino acid in the model and map and the Molprobity score (Chen et al, 2010) were calculated using phenix.real_space_refine (Afonine et al, 2018) (Appendix Fig S9, Table

  FIGURE LEGENDS

Figure 2 .Figure 3 .

 23 Figure 2. Figure 3 Cryo-EM density and pseudoatomic model of the VgrG needle (491-834) bound to Tle1. A-C. Autosharpened and masked cryo-EM density (Level 0.728) of the physiological complex in different orientations D-F. Ribbon diagram of the VgrG and Tle1 pseudoatomic structure, coloured according to the chain and in different orientations G. Full VgrG structure composed of the experimentally-obtained spike (red) and the homology model of the base (dark red) H. Structure of the VgrG spike at different planes

Figure 4 .

 4 Figure 4. Supplementary Figure 10. Comparison of the active site of Tle1. A-B. Surface representation and cross section of Tle1 from P. aeruginosa (PDB 4O5P) (A) and EAEC -this study (B). The active site is shown in red. The substrate entry channel is indicated when present.

Figure 5 .

 5 Figure 5. Inhibition of Tle1 by VgrG. A. Specific phospholipase A 1 (PLA1) activity measurements of Tle1 H , S VgrG-Tle1 H complex, and isolated S VgrG. Thermomyces lanuginosus lipase (TLL) was used as positive standard for PLA1 activity. Mean values and standard deviation from at least three independent assays are shown. Statistical analysis relative to the Tle1 activity is indicated. ***P<0.001 (P=0,0001998), unpaired two-sample Fisher-Pitman permutation test. B. VgrG inhibition of Tle1 H . Specific phospholipase A 1 (PLA1) activity measurements of purified Tle1 H in the presence of 1:10 molar ratio of lysozyme from chicken egg white (Lyso) and increasing molar ratio (0, 1, 5, and 10) of S VgrG or S VgrG 1-490 . Inhibition experiments with S VgrG were repeated three times with independent protein preparations, each measured in triplicate. Inhibition experiments with S VgrG 1-490 were repeated twice with two independent protein preparations, each measured in triplicate. Mean values and standard deviation are shown. Statistical analysis relative to the Tle1 activity is indicated. ns, non-significant (P>0.05), **P<0.01, ***P<0.001, unpaired two-sample Fisher-Pitman permutation test. (P values: Lyso, 0.7006; VgrG 1:1, 0.004946; VgrG 1:5, 1.448e-07; VgrG1:10, 1.448e-07;VgrG 1-490 1:1, 0.5627; VgrG 1-490 1:5, 0.2977; VgrG 1-490 1:10, 0.002631).

Table 1 . Interacting amino acids between VgrG and Tle1 as determined by PISA software

 1

		VgrG-Tle	VgrG	Tle-TTR
		(EMD-10218)	(EMD-10219)	(EMD-10225)
		(PDB 6SJL)	(PDB 6SK0)	(PDB 6SKI)
	Data collection and processing
	Magnification	165,000X	165,000X	165,000X
	Voltage (kV)	300	300	300
	Electron exposure (e-/Å 2 )	53.94	53.94	53.94
	Defocus range (μm)	0.5 to 2	0.5 to 2	0.5 to 2
	Pixel size (Å)	0.85	0.85	0.85
	Spot size	9	9	9
	Symmetry imposed	C1	C1	C1
	Number of images	11,670	11,670	11,670
	Initial particle images (no.)	1,942,084	1,942,084	1,942,084
	Final particle images (no.)	468,438	468,438	468,438
	Map resolution (Å)	2.6 (2.9)	2.26 (2.7)	2.55 (2.8)
	FSC threshold	0.143 (0.5)	0.143 (0.5)	0.143 (0.5)
	Map resolution range (Å)	2.2-15	2.15-2.48	2.25-7
	Binning	1.28	1.28	1.28
		Refinement
	Initial model used (PDB code)	N/A	N/A	N/A
	Model resolution (Å)	2.6 (2.7)	2.1 (2.2)	2.6 (2.7)
	FSC threshold	0.143 (0.5)	0.143 (0.5)	0.143 (0.5)
	Map sharpening B factor (Å 2 )	84.99	66.07	75.21
	Model composition:
	Number of chains	6	3	2
	Non-hydrogen atoms	19460	5890	4434
	Protein residues	2537	801	569
	Ligands	0	0	0
	B factors (Å 2 ):
	Protein (min/max)	25.27/157.57	34.83/63.05	40.72/92.41
	Ligand	N/A	N/A	N/A
	R.m.s. deviations:
	Bond lengths (Å)	0.013	0.008	0.004
	Bond angles (˚)	0.855	0.726	0.713
	Validation:
	MolProbity score	2.02	1.70	1.55
	Clashscore	12.96	9.23	8.64
	Poor rotamers (%)	0.05	0.97	1.29
	Ramachandran plot:
	Favoured (%)	94.06	96.69	98.38
	Allowed (%)	5.74	3.31	1.62
	Disallowed (%)	0.20	0	0

Table 2 . Cryo-EM data acquisition and analysis parameters 890

 2

Acknowledgements

We thank the members of the Cascales, Lloubès, Bouveret and Sturgis groups for discussions, Artemis Kosta for EM assistance, Moly Ba, Isabelle Bringer, Annick Brun and Olivier Uderso for technical assistance. This work has benefitted from the facilities and expertise of the Biophysical and Structural Chemistry platform (BPCS) at IECB, CNRS UMS3033, Inserm US001, Bordeaux University, in particular we would like to thank Armel Bezault. The authors acknowledge the use of resources of the cryo-electron microscopy service platform at the EMBL Heidelberg and the support of Felix Weis for data collection. The access to the facility has been supported by iNEXT, grant number 653706, funded by the Horizon 2020 programme of the European Commission. This work was supported by the Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université and grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006-02 and ANR-18-CE15-0013-01). NF was supported by the ANR-14-CE14-0006-02 grant. The work of ED is supported by the Institut National de la Santé et de la Recherche Médicale (INSERM). The work of MR is supported by the Institut Pasteur, the CNRS and the Projet d'Investissement d'Avenir BIP:BIP (ANR-10-BINF-03-13).

Data availability

Electron microscopy maps of VgrG-Tle1, VgrG and Tle1-TTR complexes have been deposited in the Electron Microscopy Databank (EMDB) and the PDB under accession codes EMD-10218 (PDB 6SJL), EMD-10219 (PDB 6SK0) and EMD-10225 (PDB 6SKI) respectively.

Conflict of Interests

The authors declare that they have no conflict of interest. B. Cross-section of the active site entrance channel of Tle1, coloured according to its charge, as calculated with the APBS server.

EXPANDED VIEW FIGURE LEGENDS