Terry Brissac

Guillain Mikaty

Guillaume Duménil

Mathieu Coureuil

Xavier Nassif
email: xavier.nassif@inserm.fr

Meningococcal minor pilin PilX is responsible for type IV pilus conformational changes associated with signalling to endothelial cells

Neisseria meningitidis (Nm) cross the blood brain barrier (BBB) following the activation of the β2-adrenergic receptor by the type IV pili (tfp). Two components of the type IV pili recruit the β2-adrenergic receptor the major pilin PilE and the minor pilin PilV. Here we report that a strain deleted in PilX, one of the three minor pilins, is defective in endothelial cells signalling. The signalling role of PilX was abolished when pili were not retractable.

Purified PilX was unable to recruit the β2 adrenergic receptor, thus suggesting that PilX was playing an indirect role in endothelial cell signalling. Considering the recent finding that type IV pili can transition into a new conformation (Biais, N.,

Introduction

Meningeal colonization by Neisseria meningitidis (Nm) is a consequence of the bacterial adhesion to brain endothelial cells [START_REF] Mairey | Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier[END_REF][START_REF] Pron | Interaction of Neisseria maningitidis with the components of the blood-brain barrier correlates with an increased expression of PilC[END_REF]. The initial adhesion of only few diplococci, followed by bacterial division and growth leads to the formation of microcolonies on the apical surface of the cells. This process is mediated by type IV pili (tfp) which promote the initial adhesion [START_REF] Kallstrom | Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria[END_REF][START_REF] Kirchner | CD46-independent binding of neisserial type IV pili and the major pilus adhesin, PilC, to human epithelial cells[END_REF][START_REF] Nassif | Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells[END_REF][START_REF] Rudel | Neisseria PilC protein identified as type-4 pilus tip-located adhesin[END_REF][START_REF] Virji | The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells[END_REF] and the bacteria-bacteria interactions which are required for the formation of bacterial aggregates [START_REF] Merz | Interactions of pathogenic neisseriae with epithelial cell membranes[END_REF]. Subsequent to the formation of these microcolonies, tfp elicit the organization of specific ''honeycomb''-shaped molecular complexes underneath bacterial colonies, referred to as ''cortical plaques.'' Cortical plaques result from the recruitment of molecular linkers, such as ezrin and moesin, adhesion molecules, membrane receptors, and polymerized cortical actin [START_REF] Doulet | Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures[END_REF][START_REF] Merz | Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells[END_REF]. The formation of the cortical plaque is associated with the recruitment of intercellular junction molecules at the site of bacterial-host cell interaction, thus leading to the depletion of junction proteins at the cellcell interface, the opening of intercellular junctions, and the subsequent crossing of the BBB [START_REF] Coureuil | Meningococcal Type IV Pili Recruit the Polarity Complex to Cross the Brain Endothelium[END_REF]. In endothelial cells, the formation of the cortical plaque is due to a direct interaction between tfp and the β2-adrenergic receptor [START_REF] Coureuil | Meningococcus Hijacks a beta2-Adrenoceptor/beta-Arrestin Pathway to Cross Brain Microvasculature Endothelium[END_REF].

Type IV pili are formed of a major subunit, the major pilin, PilE. PilE is not only a structural component but is also a signalling protein able to directly interact with the β2adrenergic receptor [START_REF] Coureuil | Meningococcus Hijacks a beta2-Adrenoceptor/beta-Arrestin Pathway to Cross Brain Microvasculature Endothelium[END_REF]. The major pilin subunit is subject to antigenic variation [START_REF] Seifert | Genetic mechanisms of bacterial antigenic variation[END_REF]. Some pilin variants have been associated with an ability to promote higher adhesiveness than others.

The high adhesive phenotype was linked to the ability of these variants to form bundles of pili and to allow bacterial aggregation, thus increasing interbacterial interactions [START_REF] Nassif | Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells[END_REF]. In addition, three minor pilins, designated PilV, PilX and ComP, are present in the fiber at low levels [START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF][START_REF] Winther-Larsen | Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence[END_REF][START_REF] Wolfgang | The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation[END_REF]. These minor pilins are involved in specific functions supported by tfp. ComP is essential for the natural transformation of the bacteria [START_REF] Wolfgang | The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation[END_REF]. PilV, participates in the induction of signalling since mutants not producing PilV were defective in endothelial cell signalling [START_REF] Mikaty | Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress[END_REF] and recently PilV has been shown to interact directly with the β2-adrenergic receptor, as PilE [START_REF] Coureuil | Meningococcus Hijacks a beta2-Adrenoceptor/beta-Arrestin Pathway to Cross Brain Microvasculature Endothelium[END_REF]. PilX is necessary for the aggregative properties of tfp [START_REF] Helaine | PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells[END_REF]. Bacteria-bacteria interactions due to the aggregative property of PilX are responsible for an increased number of bacteria capable of interacting with cells [START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF]. This property is related to a specific protruding region of the PilX subunit that connects pili from two different bacteria. Another major property of tfp is their ability to retract. Following pilus-mediated adhesion pili have been shown to retract [START_REF] Pujol | The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion[END_REF]. The cytoplasmic PilT ATPase is responsible for retraction of the fiber. In a pilT mutant, PilX is no longer required to form bacterial aggregates, this lead to the hypothesis that the protruding region of PilX links two anti-parallel pili and prevents the fibers to slide upon pilus retraction. In addition to these components other proteins located in the bacterial membranes or periplams are important for pilus biogenesis (3). Among these the PilC proteins play a role which is still enigmatic. Two pilC alleles that encode for two paralogous proteins PilC1 and PilC2 have been described [START_REF] Nassif | Roles of pilin and PilC in adhesion of Neisseria meningitidis to human epithelial and endothelial cells[END_REF]. PilC-null strains show impaired pilus expression. In addition, PilC1 is required for adhesion and PilC1-/PilC2+ strain are unable to adhere to endothelial cells. On the other hand, PilC2, which is expressed independently of PilC1 possesses these adhesiveness properties specifically on some cell types but not on endothelial cells [START_REF] Kirchner | CD46-independent binding of neisserial type IV pili and the major pilus adhesin, PilC, to human epithelial cells[END_REF][START_REF] Morand | Neisseria meningitidis differentially controls host cell motility through PilC1 and PilC2 components of type IV Pili[END_REF][START_REF] Morand | The adhesive property of the type IV pilus-associated component PilC1 of pathogenic Neisseria is supported by the conformational structure of the N-terminal part of the molecule[END_REF][START_REF] Nassif | Roles of pilin and PilC in adhesion of Neisseria meningitidis to human epithelial and endothelial cells[END_REF].

It has recently been shown that mechanical force can extend the repertoire of tfp structures by revealing hidden epitopes of PilE previously buried in the tfp fiber [START_REF] Biais | Force-dependent polymorphism in type IV pili reveals hidden epitopes[END_REF][START_REF] Craig | Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions[END_REF]. Indeed, when subject to force, tfp will transition into a new conformation. The new structure is longer and narrower than resting tfp. Upon release of the force, tfp regain their original form [START_REF] Biais | Force-dependent polymorphism in type IV pili reveals hidden epitopes[END_REF]. SM1 monoclonal antibody (mAb) recognizes the conserved epitope EYYLN in the pilin monomer [START_REF] Virji | Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp[END_REF]. The SM1 epitope is predicted to be buried in the fiber, and in the resting unstretched state the SM1 mAb recognizes by electron microscopy only the exposed end of the fiber [START_REF] Biais | Force-dependent polymorphism in type IV pili reveals hidden epitopes[END_REF][START_REF] Craig | Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin[END_REF][START_REF] Forest | Type-4 pilus-structure: outside to inside and top to bottom--a minireview[END_REF][START_REF] Parge | Structure of the fibre-forming protein pilin at 2.6 A resolution[END_REF]. On the other hand, in the elongated form of the pilus, SM1 mAb binds to the length of the stretched fiber [START_REF] Biais | Force-dependent polymorphism in type IV pili reveals hidden epitopes[END_REF]. Thus force-induced stretch exposes residues in the tfp that are normally hidden. The role in vivo of this newly described characteristic of tfp is unknown.

During the course of a systematic analysis of the role of tfp components or tfp associated proteins in meningococcal signalling, we found that a strain deleted for PilX was defective in endothelial cells signalling. Unlike the major pilin PilE and the minor pilin PiV, PilX does not interact directly with the β2-adrenergic receptor. We subsequently hypothesized that PilX could be necessary for structural change within the fiber. Based on the labeling of the fiber by the SM1 mAb, we show that following pilus-mediated adhesion the SM1 epitope was exposed on the length of the fiber, wheras bacterial aggregation was not sufficient for this epitope to be exposed. Moreover, the binding of the SM1 mAb in adhesive bacteria along the pili required PilX. These data suggest that upon bacterial adhesion the fiber was elongated and that this structural change required PilX. We subsequently propose that the minor pilin PilX following bacterial adhesion is necessary for the structural modification of the fiber that allows bacterial cell signalling.

Material and Methods

Bacterial strains and infection conditions

A piliated and noncapsulated Opa+ derivative of the serogroup C meningococcal strain 8013, designated 2C43 siaD-Opa+, was used throughout the study [START_REF] Coureuil | Meningococcal Type IV Pili Recruit the Polarity Complex to Cross the Brain Endothelium[END_REF][START_REF] Nassif | Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells[END_REF]. This noncapsulated derivative was engineered by introduction of cat resistance cassette into the siaD gene. All the mutants and derivatives used in this work were isogenic derivatives of meningococcal C strain 2C43 siaD-Opa+ and are listed in Table 1.

N. meningitidis strains were grown on GCB agar plates (Difco) containing Kellog's supplements and supplemented with appropriate antibiotics in a moist atmosphere containing 5% CO 2 at 37°C.

On the day of infection, a suspension of the bacteria from an overnight culture on GCB agar plate was adjusted to OD 600Nm =0.05 and incubated for 2h at 37°C in a pre-warmed cell culture medium. Cells were infected with bacteria at a multiplicity of infection (MOI) of 100 for 30 minutes to allow N. meningitidis adhesion and then washed with cell culture medium and maintained in appropriate fresh medium for 1 or 2 hours.

Cell culture

Human Umbilical Vein Endothelial Cells (HUVECs; Promo-cell) were used between passages 1 and 8 and grown in Endo-SFM supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories GmbH), 0.5 IU/mL of heparin and 40 mg/mL of endothelial cell growth supplement (Sigma-Aldrich). Human Embryonic Kidney 293 (HEK-293) cells were grown in DMEM medium supplemented with 10% Fetal Calf Serum. Cells were grown at 37°C in a humidified incubator under 5% CO 2 . For immunofluorescence assays, HUVECs and HEK293 cells were grown on 12-mm diameter glass coverslips coated with human fibronectin (10 μg/mL, Life-Technologies) or Poly-L-lysine (0,01%, Sigma-Aldrich), respectively. HEK-293 cells were transfected using Lipofectamine 2000 (Life-Technologies) following manufacturer instructions.

Antibodies and Chemicals

The following antibodies were used for immunofluorescence labeling or enzymelinked immunosorbent assay (ELISA): anti-ezrin rabbit polyclonal antibody (generously provided by Dr. P Mangeat CNRS, UMR5539, Montpellier, France); anti-pilE mouse monoclonal antibody (clone 20D9) [START_REF] Pujol | The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion[END_REF]; anti-pilE mouse monoclonal antibody (clone SM1) (generously provided by Dr. Mumtaz Virji). Goat secondary antibodies were used for immunofluorescence labeling or ELISA: anti-mouse IgG (H+L) coupled to Horse Radish Peroxidase (Jackson ImmunoResearch Laboratories) and anti-rabbit IgG (H+L) or anti-mouse IgG (H+L) coupled to Alexa-488 or Alexa-546 (Life-Technologies). 4',6-diamidino-2phenylindole (DAPI) was purchased from Life-Technologies. Isopropyl β-D-1thiogalactopyranoside (IPTG) was purchased from Promega and used at a concentration of 1mM.

Analysis of piliation

Analysis of piliation was performed using a whole-cell ELISA, as already described [START_REF] Helaine | PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells[END_REF]. Briefly, bacteria were grown 2h in RPMI/BSA1% from an OD 600nm =0.5 and after washes were resuspended in PBS at an OD 600nm =0.1. Serial twofold dilutions were coated in the wells of a microtitre plate. The plates were incubated without covers at 56°C for 1 hour. Coated plates were washed several times with PBS-0.1% Tween 80 (washing solution), then the anti-pilE mouse monoclonal antibody 20D9 diluted in washing solution was added to the plates and incubated 1 h at 37°C. After several washes a peroxidase-linked anti-rabbit IgG was added to the wells for 1h. Finally, after several washes, the staining was revealed using TMB (3,3',5,5'-tetramethylbenzidine) substrate and STOP solution following the manufacturer's instructions (Cell Signalling Technology).

Immunofluorescence experiments

Infected cells were fixed for 20 min in PBS 4% PFA. For Ezrin immuno-staining only, cells were permeabilized for 5 min in PBS containing 0.1% Triton X-100. Cells were incubated with primary antibodies in PBS containing 0.3% BSA or 0.1% Gelatin for 1 hr.

After 3 washes with the same buffer, DAPI was added to Alexa-conjugated secondary antibodies for 1 hr. After additional washes, coverslips were mounted in Mowiol (Citifluor Ltd).

N. meningitidis aggregates were coated on glass coverslips by centrifugation after a 2 hours liquid culture in HUVEC culture medium (3000 rpm, 10 min), fixed using 4% PFA and processed for immunostaining.

Image acquisition was performed on an inverted microscope or a laser-scanning confocal microscope (Leica SP5). Confocal images were collected and processed using the Leica Application Suite AF lite software (Leica).

Quantitative Analysis of Ezrin (and other Proteins) Recruitment under Bacterial Colonies

The recruitment efficiency was estimated by determining the proportion of colonies that efficiently recruit the protein of interest in a ''honeycomb shape'' just under the colonies.

At least 50 colonies were scrutinized per coverslip. Each experiment was performed in duplicate and repeated several times (at least 3). Data were examined for significance using Student's t test.

Expression and Purification of MBP-Pilin Recombinant Proteins

Recombinant MBP-pilin fusion proteins were produced as described before [START_REF] Coureuil | Meningococcus Hijacks a beta2-Adrenoceptor/beta-Arrestin Pathway to Cross Brain Microvasculature Endothelium[END_REF][START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF].

Briefly, fragments of pilE, pilV, comP and pilX genes lacking the region coding for the first 28 amino acid residues were amplified by PCR and subcloned in pMAL-p2X vector (New England Biolabs). The resulting plasmids, pMAL-pilE, pMAL-pilX, pMAL-pilV and pMAL-comP contain in frame the malE gene, which encodes the maltose-binding protein (MBP), followed by the Factor Xa protease recognition site and the truncated pilin coding regions. These plasmids were transformed in the PAP5198 E. coli strain (generously provided by Dr.

Olivera Francetic, Institut Pasteur, Paris, France), deleted for the periplasmic enzymes: OmpT Prt and DegP. The fusion proteins were purified on amylose resin (New England Biolabs).

Coating of Staphylococcus aureus with MBP-Pilin Fusion Proteins and infection

S. aureus ATCC 25923 expressing specific receptors for the Fc domain of IgG immunoglobulins were used for this experiment as described before [START_REF] Coureuil | Meningococcus Hijacks a beta2-Adrenoceptor/beta-Arrestin Pathway to Cross Brain Microvasculature Endothelium[END_REF]. Briefly, about 10 8 bacteria were incubated with the anti-MBP rabbit polyclonal antibody for 20 min at room temperature. Bacteria were then centrifuged at 4000 rpm for 5 min, washed with warm LB 3 times, incubated with 2.5 µg of each recombinant fusion protein for 20 min at room temperature. After several washes using DMEM supplemented with 10% Fetal Calf Serum, bacteria were incubated 30min with HEK293 cells previously transfected with plasmid encoding the YFP-tagged human β2-adrenergic receptor (β2AR-YFP) (1). Cells were then fixed using 4% PFA in PBS and analyzed by immunofluorescence assay for β2AR-YFP recruitment underneath S. aureus colonies.

Results

The minor pilin PilX is required for type IV pilus-mediated signalling of meningococcal microcolonies to endothelial cells.

In an attempt to identify all pilus components, beside PilE and PilV, involved in meningococcal signalling, we tested systematically the ability of strains carrying a mutation in identified tfp components or tfp-associated proteins to signal to endothelial cells. Considering that some of these mutants are non piliated (Table2) or incapable of pilus-mediated adhesion, such as the PilC1 defective derivative, all the mutations were introduced into a strain expressing an Opa protein, a secondary adhesin. However in order to be able to promote adhesion, an Opa+ expressing strain has to be non capsulated [START_REF] Virji | Meningococcal Opa and Opc proteins: their role in colonization and invasion of human epithelial and endothelial cells[END_REF], a non capsulated background was therefore used by introducing a siaD mutation [START_REF] Coureuil | Meningococcal Type IV Pili Recruit the Polarity Complex to Cross the Brain Endothelium[END_REF]. A noncapsulated Opa+ strain is indeed able to adhere to eukaryotic cells expressing CEACAMs (Carcinoembryonic Antigen-related Cell Adhesion Molecules) independently of tfp expression [START_REF] Coureuil | Meningococcal Type IV Pili Recruit the Polarity Complex to Cross the Brain Endothelium[END_REF][START_REF] Virji | The Ndomain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae[END_REF]. Therefore, all experiments described in this work were performed in a SiaD-, Opa+ background. As previously shown, such a piliated strain is not affected in its ability to signal to cells [START_REF] Coureuil | Meningococcal Type IV Pili Recruit the Polarity Complex to Cross the Brain Endothelium[END_REF].

Results of these experiments are reported Table 2 and Figure 1. As expected, none of the non piliated mutants (pilE, pilD, pilQ, pilH, pilI, pilJ, and pilK mutants [START_REF] Geoffroy | Large-scale analysis of the meningococcus genome by gene disruption: resistance to complementmediated lysis[END_REF]) was able to recruit the cortical plaque, thus confirming that pili are required for meningococcal formation of the cortical plaque. However this cannot rule out the implication of one of these components in signalling. The lack of piliation may prevent the evaluation of the role of some components in signalling independently of their role in pilus biogenesis, such as the major pilin PilE.

The adhesive properties of tfp to endothelial cells have been shown to require the expression of PilC1, an outer membrane protein associated with tfp [START_REF] Nassif | Roles of pilin and PilC in adhesion of Neisseria meningitidis to human epithelial and endothelial cells[END_REF]. Indeed, pilC1 mutant are piliated if they express PilC2 but are unable to adhere to HUVECs. In a SiaD-, Opa+ background both pilC1 or pilC2 mutant formed microcolonies. Both strains are able to recruit ezrin and actin (Table 2 and Figure 1). The fact that the pilC1 mutant was capable of signalling to cells even though unable to promote pilus-mediated adhesion confirms that type IV pilus mediated-signalling and -adhesion are two independent processes mediated by the same bacterial attribute (4).

Consistent with previous results [START_REF] Mikaty | Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress[END_REF], the pilV mutant expressed tfp, but recruited the cortical plaque very poorly (Table1). Surprisingly a pilX mutant had a dramatic signalling defect (Table2 Figure 1 and2) and was unable to induce cortical plaque recruitment (Figure 2). The introduction into the chromosome of a pilX allele under the control of an IPTG inducible promoter restored the capability to induce signalling upon addition of IPTG (Figure 2), thus excluding that the signalling defect was due to a polar effect of the mutation. The pilX mutant adhered to cells via Opa proteins and, due to the absence of bacteria-bacteria interactions, formed microcolonies of smaller size than those of the parental strain. It should be pointed out that none of the microcolonies made by the pilX mutant, regardless of their size, was capable of signaling to endothelial cells. The parental strain expressed a high adhesive pilin variant, thus being responsible for large colonies adhering to the apical surface of the endothelial cells. We subsequently repeated this experiment using a derivative expressing a low adhesive variant, defective in bacterial aggregation (i.e. expressing the SA pilin variant [START_REF] Nassif | Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells[END_REF]). The SA pilin variant is responsible for few bacteria-bacteria interactions and hence induced the formation of small microcolonies on the surface of apical cells [START_REF] Nassif | Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells[END_REF]. As shown Figure 2, adhesive bacteria of this derivative recruited the cortical plaque in a manner similar to that of the strain expressing a high adhesive pilin variant. Altogether, these results demonstrate that the lack of signalling observed with the pilX mutant in not due to the small size of the microcolonies interacting with the endothelial cells.

The PilT protein is responsible for tfp retraction. PilT dependant retraction has been shown to enhance Nm signalling to epithelial cells, probably by a mechano-transduction mechanism [START_REF] Lee | PilT is required for PI(3,4,5)P3-mediated crosstalk between Neisseria gonorrhoeae and epithelial cells[END_REF][START_REF] Merz | Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells[END_REF]. Interestingly, on endothelial cells, a retraction deficient isolate was able to induce the formation of cortical plaques (Table 1 and Figure 1 and3). Considering that a pilT mutation has been shown to compensate some phenotypical defects in strains carrying a mutation in tfp components or tfp-associated components [START_REF] Helaine | PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells[END_REF], a pilT mutation was introduced into the pilX mutant. The signalling ability of the resulting strains was then tested.

As shown Figure 1 and 2, the pilT mutation partially restored the cortical plaque recruitment defect of the pilX mutant.

Altogether, these results clearly confirmed that type IV pili are required for the formation of the cortical plaque and that the minor pilin PilX was playing a major role in the recruitment of the cortical plaque but was dispensable in a non retractable strain.

PilX does not recruit the β2-adrenergic receptor.

As already mentioned, PilE and PilV are involved in the recruitment of the signalling receptor, the β2-adrenergic receptor. Indeed, recombinant PilV and PilE proteins when fused with the maltose binding protein (MBP) and bound to live staphylococci via anti MBP antibodies are able to induce the recruitment of the the β2-adrenergic receptor in a model of HEK293 cells transfected with both the β-arrestin and the β2-adrenergic receptor; (see experimental procedures section and ( 4)). In order to assess a possible interaction of PilX with the adrenergic receptor, we used a similar strategy and produced recombinant PilX fused with the maltose binding protein (MBP). HEK cells overexpressing the β-arrestin and the β2adrenergic receptor tagged with YFP were infected with Staphylococcus aureus coated with anti-MBP antibody and the MBP-Pilin fusion proteins. The ability of these proteins to recruit the β2-adrenergic receptor in these transfected HEK293 cells was subsequently tested. Data are reported in Figure 4. In accordance with our previous work (4), MBP-PilV and MBP-PilE but not MBP-ComP recruits the β2-adrenergic receptor. On the other hand, MBP-PilX was unable to recruit the receptor. This result is not consistent with a direct interaction of PilX with the β2-adrenergic receptor, thus suggesting that PilX may have an indirect interaction effect on tfp-induced signalling.

Upon bacterial adhesion, PilX is needed to reveal the previously hidden SM1 epitope.

As already mentioned, tfp, when subjected to force, will transition into a new conformation [START_REF] Biais | Force-dependent polymorphism in type IV pili reveals hidden epitopes[END_REF]. The new structure is longer and narrower than the original structure. Upon release of the force, the tfp fiber regains its original form, indicating a reversible transition.

These force-induced conformations expose hidden epitopes, such as those recognized by the SM1 monoclonal antibody. This epitope is not exposed in fibers of bacteria grown in broth culture [START_REF] Biais | Force-dependent polymorphism in type IV pili reveals hidden epitopes[END_REF][START_REF] Craig | Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions[END_REF]. The above data suggesting that PilX does not play a role via a direct interaction with the signalling receptor raised the question of the mechanism by which PilX may enhance pilus-mediated signalling to endothelial cells. Previous reports have proposed that surfaceexposed motifs in PilX subunits stabilize bacterial aggregates against the disruptive force of pilus retraction [START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF]. We subsequently postulated that upon bacterial interaction with cells, the forces generated by pilus retraction may stretch the fiber, thus exposing epitopes previously buried in the tfp fiber,.

The strain used in this study carries the SM1 epitope [START_REF] Virji | Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp[END_REF]. We subsequently addressed the availability of this epitope in isogenic ΔpilX or ΔpilX/ΔpilT meningococcal strains adhering or not onto endothelial cells. As already mentioned, since a pilX mutant is non adhesive, all strains used were non capsulated (SiaD-), Opa+ derivatives of strain 2C43. We first looked at the expression of the SM1 epitope by adhesive bacteria. As shown Figure 5A in monolayers infected with a strain expressing wild type pili, the SM1 antibody-stained pili were seen as filamentous structures among bacterial aggregates in the great majority of the microcolonies. In addition a SM1 staining was seen not only in large microcolonies of adhering bacteria, but also when only a few diplococci were adhering to the cells (Figure 5A, left panel), thus excluding the possibility that this staining was a consequence of the bacterial aggregation in large microcolonies. 80 to 90% of the microcolonies on the surface of the cells are associated with the formation of a cortical plaque, and only bacterial colonies in which a SM1 staining was observed were associated with the formation of a cortical plaque (Figure 5B). On the other hand, pili of the PilX-derivative were never stained by the SM1 monoclonal antibody (Figure 5A), even though adhesive PilX-bacteria are heavily piliated as shown in Figure 3 [START_REF] Helaine | PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells[END_REF]. A western blot performed using purified PilX, ruled out the possibility that the effect observed was due to recognition of the PilX molecule by the SM1 monoclonal antibody (data not shown). A strong SM1 staining of pili was observed in microcolonies of pilT-strains expressing non retractable pili (Figure 5A-5B). However, consistent with the above data (Figure 2), the SM1 labelling was stronger in the PilT-/PilX+ strain than in the PilT-/PilX-strain. It should be pointed out that a mutation in another minor pilin such as PilV even though important for meningococcal cell signalling (20) to endothelial cells did not affect the SM1 binding when bacteria were interacting with the cells (data not shown). These results clearly showed that the SM1 epitope is exposed in wild type pili upon bacterial adhesion and not in PilX-strain but are present in non retractable (PilT-) pili regardless of the presence of PilX.

We then assessed the expression of the SM1 epitope in non adhering bacteria grown in broth. As shown Figure 5C, wild type pili were not stained by SM1 antibody, even though these bacteria were forming large bacterial aggregates, thus confirming that the exposure of the SM1 epitope is not a result of the ability of the bacteria to form aggregates. This is consistent with previous results indicating that the SM1 epitope is hidden when bacteria are grown in broth [START_REF] Biais | Force-dependent polymorphism in type IV pili reveals hidden epitopes[END_REF][START_REF] Craig | Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions[END_REF]. On the other hand, in a strain expressing non retractable tfp, a clear staining of pili by the SM1 monoclonal antibody was observed inside the bacterial aggregates. These data demonstrated that, for broth grown bacteria, the SM1 epitope is not accessible to the antibody in wild type pili but is exposed in non retractable (PilT-) pili.

Altogether these data demonstrate that in wild type pili, upon bacterial adhesion onto cells, the minor pilin PilX allows the expression of hidden epitopes such as the SM1 epitope which are not exposed on pili in liquid culture growth, thus demonstrating that upon bacterial adhesion conformational changes occurs in the fiber.

The D-region of PilX is required for pilus-mediated signalling and is responsible for the PilX induced conformational change.

The structure of PilX has previously been reported [START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF][START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF] and shows a classical pilin structure with a C-terminal region containing two disulfide bonds delineating the D region which forms a hook exposed on the surface of the fiber (6) (Fig 6A). In a previous work, it has been shown that this protruding D region is responsible for bacterial aggregation by enhancing bacterial interaction [START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF]. Considering the above reported results, we aimed at testing the hypothesis that both the tfp-mediated signalling and the PilX-induced conformational changes observed upon bacterial interaction with host cells were also linked to the presence of the hook formed by the D region. We subsequently assessed the ability of a PilX protein deleted in the D region to signal to cells and to allow the binding of the SM1 monoclonal antibody upon bacterial cell interaction. The strains used for these experiments were pilX derivatives of non capsulated (SiaD-) Opa+ strains, complemented in trans either with a full length pilX allele (ΔpilX::pilX) or a pilX allele lacking residues 127 to 138 of the D-region (ΔpilX::pilX 127-138 ) (Fig 6A). These strains have previously been reported [START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF] and this deletion of PilX does not prevent the protein's localisation in the fiber. The data are reported in Fig 6B and6C. They clearly show that the ability of tfp to signal to cells required the presence of the hook formed by the D region of PilX. Moreover, this region is necessary for the pilus conformational change following bacterial adhesion to host cells which allows the binding of the SM1 antibody to its epitope.

Altogether these data demonstrate that the ability of meningococcal tfp to signal to host cells correlate with changes in the fiber structure induced by the exposed D region of the PilX protein.

Discussion

Type IV pili are essential for meningococcal pathogenesis. In invasive capsulated bacteria, they promote bacterial adhesion to endothelial cells and signalling. The latter is a consequence of the interaction of pili components with the N-terminal portion of the β2adrenergic receptor. Two components of the bacterial pili have been implicated as interacting with the extra cellular N-terminal portion of the β2-adrenergic receptor, the major pilin PilE and a minor pilin component PilV [START_REF] Coureuil | Meningococcus Hijacks a beta2-Adrenoceptor/beta-Arrestin Pathway to Cross Brain Microvasculature Endothelium[END_REF]. How these two components interact with the receptor remain to be understood, especially whether they act cooperatively or independently is unknown.

In this work a third component of the pili has been identified as playing a role in pilus mediated signalling, the minor pilin PilX. This component has previously been identified as required for efficient bacterial adhesion and aggregation. However PilX could not be identified as an adhesin and it is believed that the bacteria-bacteria interactions due to the aggregative property of PilX are responsible for the increase number of bacteria capable of interacting with cells [START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF]. This property is related to a specific protruding region of the PilX subunit that is believed to be able to connect pili from two different bacteria upon pilus retraction preventing them to slide. Here, we identify PilX as a pilus component required for tfp-mediated signalling. As for adhesion and aggregation, this effect of PilX is abolished in a PilT deficient strain. Unlike the major pilin PilE and the minor pilin PilV, purified PilX does not promote recruitment of the β2-adrenergic receptor, thus making it unlikely that the function of PilX in signalling is via a direct interaction with the signalling receptor. We subsequently hypothesized that PilX may be responsible for conformational changes which allow some pilus epitopes to be exposed. Our data clearly demonstrate that the SM1 epitope expressed by wild type pili is not accessible to monoclonal antibodies in bacteria grown in liquid cultures whereas it is clearly labelled once bacteria interact with cells. These results are consistent with a recent report showing that neisserial tfp can undergo a reversible forceinduced transition into a longer and narrower structure than the resting state, and that the labelling of the SM1 epitope along the length of the pili is made possible by this elongation of the fiber [START_REF] Biais | Force-dependent polymorphism in type IV pili reveals hidden epitopes[END_REF]. Altogether our data suggest that upon meningococcal-cell interaction pili are elongated, and that PilX is required for this structural change. In addition this function of PilX is supported by the D region of the molecule that forms a hook exposed at the surface of the fiber. This conformational change is not due to bacterial aggregation, since bacterial aggregates grown in broth are not labelled by the SM1 epitope (Fig 5C) and SM1 labelling is observed when only a few diplococcic adhere to cells (Fig 5A). These results suggest that the force necessary for this conformational change is coming from pilus retraction following bacterial adhesion. PilX, via its protruding region, by preventing pili sliding, may be responsible for the elongation of the fiber upon interaction with cells, thus exposing previously hidden epitopes in the pili. The fact that the SM1 epitope is spontaneously exposed in the fiber of PilT-pili suggests that the structure of non retractable pili is not that of the resting pilus but that of an elongated one. This hypothesis is consistent with a previous report showing that pili tend to retract spontaneously [START_REF] Morand | Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins[END_REF] and may explain why non retractable pili are able to signal to cells in the absence of PilX.

The absence of recruitment of the β2-adrenergic receptor by purified PilX, the fact that strain expressing non retractable pili do not require PilX bacterial cell signalling, and the association between the ability of the fibers to be stained by the SM1 epitope and to signal to cells support the hypothesis that the epitopes responsible for bacterial signalling are capable of interacting with the β2-adrenergic receptor only in specific conformations of the quaternary structure of the fiber. aggregates were obtained after a 2h liquid culture in HUVEC culture medium. SM1 epitope is visualized by immunofluorescence using the anti-SM1 monoclonal antibody (in green). DNA was stained using dapi (in blue). Non specific staining (*). SM1 stained pili (Arrowhead).

Figure 1 .

 1 Figure1. Neisseria meningitidis pilX mutant strain is defective in ezrin recruitment. HUVEC cells were infected with a piliated non-capsulated strain of Neisseria meningitidis (wt) and its pilE, pilX, pilC1, pilT and pilX/pilT derivatives. The formation of the cortical plaque was assessed by the recruitment of ezrin. Ezrin was immunostained (in green) and DNA was stained using dapi (in blue). Nucleus (N); Microcolonies (Arrowhead). Scale bars=5µM. All bacteria are Opa+ and noncapsulated.

Figure 2 .Figure 3 .

 23 Figure2. pilT mutation partially restores signalling defect of the pilX mutant. HUVEC cells were infected with a piliated non-capsulated strain of Neisseria meningitidis (wt), its pilE, pilX, pilT and pilX/pilT derivatives and a strain expressing the pilin variant SA. The strain ΔpilX::pilX is a PilX mutant which has been complemented in trans with a pilX allele under the control of an IPTG inducible promotor. It should be pointed out that this strain promotes even in the absence of IPTG a low level of ezrin recruitment due to a low activity of the promotor in the absence of IPTG. Ezrin recruitment was revealed by immunostaining. The ezrin recruitment index was estimated by determining the proportion of colonies that efficiently recruit ezrin under the colonies and expressed as normalized mean values (±SEM) of three independent experiments in duplicate.a student t test (p < 0,001) shows statistical significance when the ezrin recruitement was compared to that of the strain expressing wild type pili

Figure 4 .

 4 Figure 4. PilX is not able to recruit the β2 adrenergic receptor. HEK cells over expressing the β2 -adrenergic receptor tagged with YFP were infected with Staphylococcus aureus coated with anti-MBP antibody and MBP-Pilin fusion proteins as described in the materials and methods sections. Receptor recruitment was counted in all cells in contact with bacterial aggregates and expressed as normalized mean values (±SEM) of three independent experiments in duplicate. a student t test (p < 0,001).

Figure 5 .

 5 Figure 5. SM1 epitope is exposed upon bacterial adhesion to endothelial cells or in a pilTbackground. HUVEC cells were infected with a piliated non-capsulated strain of Neisseria meningitidis (wt) and its pilT and pilX and pilX/T derivatives. (A) SM1 epitope is visualized by immunofluorescence using the anti-SM1 monoclonal antibody (in green). DNA was stained using dapi (in blue). SM1 stained pili (Arrowhead). (B) HUVEC cells were infected with a piliated non-encapsulated strain of Neisseria meningitidis (wt) and its pilT derivative. SM1 epitope and ezrin were immunostained (in green and in red, respectively). DNA was stained using dapi. SM1 stained pili (Red arrowhead). Microcolonies without SM1 stained pili (White arrowhead). (A, B, C) Nuclei (N); Scale bars=10µM (C) Neisseria meningitidis

Acknowledgments

We thank Dr. Colin Tinsley for a careful reading of the manuscript. We thank Dr.

Mumtaz Virji for sending us the monoclonal antibody directed against the SM1 epitope. We thank M. Garfa-Traore and N. Goudin of the Necker Institute imaging facility for their technical support and the Imagine foundation for the Leica SP5 microscope funding. The laboratory of XN is supported by INSERM, Université Paris Descartes, a grant from "La Fondation pour la Recherche Médicale" and ANR grant # ANR-AAP-2009-06-26.

This study a Cm: Chloramphenicol (6 µg.mL -1 ), Kn: Kanamycin (100 µg.mL -1 ), Em: Erythromycin (2 µg.mL -1 ) b pilX genes under control of IPTG inducible plac promoter (lacI q OP)