G Fahs

C Ghigo

J F L Beloin

G Duval

Francius

J F L Duval

Dynamic modulation of fimbrial extension and FimH-mannose binding force on live bacteria under pH changes: a molecular atomic force microscopy analysis

Keywords: Single molecule force spectroscopy, type 1 fimbriae, unwinding, AFM, bacteria : equivalent contribution

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

Introduction

Early adhesion events with environmental surfaces or host tissues and subsequent interactions between bacteria initiate biofilm formation and are considered, in the case of pathogenic bacteria, as key steps of colonization/infection processes. Therefore, the investigation of molecular factors together with the mechanisms governing these early adhesion events has emerged as a critical point to understand the infection process and consequently to potentially develop alternative strategies to antibiotics in order to prevent bacterial adhesion to surfaces.

The adhesive bonds between bacteria and surfaces or between bacteria are subjected to tensile mechanical forces that possibly depend on parameters such as solution pH, osmolarity or temperature. A successful surface colonization reflects the ability of the bacteria to withstand natural detachment forces and environmental perturbations once anchored at the target surface. A relevant example of successful colonizers of human mucosa is E. coli. These bacteria are natural inhabitants of the gastro-intestinal tract and they may behave both as commensal and pathogenic entities. As pathogens, they can be at the origin of intra-intestinal diseases like diarrhea caused by enterohaemmoragic or enteroaggregative E. coli (EHEC and EAEC respectively), or extra-intestinal diseases, e.g. urinary tract infections caused by uropathogenic E. coli (UPEC). In the gastro-intestinal and urinary tracts, E. coli is exposed to shear forces imposed by mechanical host defenses (mucus and urine flows) and to changes in pH. This suggests that the mechanical and chemical properties of E. coli cell wall are key factors that make it possible for the bacteria to sustain competing shear forces and drastic changes in environmental conditions and Bacterial adhesion is mainly mediated by specific non-covalent bonds between tethered ligands and receptors. [START_REF] Camesano | Measuring bacterial adhesion at environmental interfaces with single-cell and single-molecule techniques[END_REF] The adhesion further generally involves proteinaceous molecules or macromolecular complexes referred to as adhesins, i.e. monomeric or polymeric polymers forming fimbrial or afimbrial structures brought to the bacterial surface by various secretion systems. [START_REF] Kline | Bacterial Adhesins in Host-Microbe Interactions[END_REF][START_REF] Proft | Pili in Gram-negative and Gram-positive bacteria - structure, assembly and their role in disease[END_REF] In E. coli, the two major classes of adhesins are (i) fimbrial adhesins transported by the chaperone-usher pathway, and (ii) afimbrial adhesins transported by type V secretion pathway. Prototypical chaperone-usher fimbriae of E. coli are P pili and type 1 fimbriae, which are commonly associated to UPEC and infections in the upper and the lower urinary tract, respectively. [START_REF] Wiles | Origins and virulence mechanisms of uropathogenic Escherichia coli[END_REF] The structure of these hairy surface appendages are very similar, but differ in terms of biomechanical properties. [START_REF] Andersson | The biomechanical properties of E-coli Pili for urinary tract attachment reflect the host environment[END_REF] Type 1 fimbriae are rod-shaped filaments consisting of FimA repeating subunits terminated by a specific adhesin called FimH that binds to -Dmannosylated proteins, [START_REF] Wu | In vitro binding of type 1-fimbriated Escherichia coli to uroplakins Ia and Ib: relation to urinary tract infections[END_REF][START_REF] Klemm | Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae[END_REF] while P pili consist of PapA subunits terminated by the PapG adhesin whose variants bind to different glycolipids. [START_REF] Mu | Structure and assembly of P-pili: A protruding hinge region used for assembly of a bacterial adhesion filament[END_REF][START_REF] Lane | Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney[END_REF] Since the last decade, atomic force microscopy (AFM) has emerged as a powerful tool for probing microbial surfaces and interfacial phenomena, in particular specific and non-specific interactions at the nanoscale. [START_REF] Dufrêne | Using nanotechniques to explore microbial surfaces[END_REF][START_REF] Dufrêne | Atomic force microscopy and chemical force microscopy of microbial cells[END_REF][START_REF] Francius | Detection, localization, and conformational analysis of single polysaccharide molecules on live bacteria[END_REF][START_REF] Dupres | Microbial nanoscopy: a closer look at microbial cell surfaces[END_REF] Dynamic Force Spectroscopy (DFS) and Single-Molecule Force Spectroscopy (SMFS) are derivative AFM techniques that provide access to molecular dynamic processes, chemical or surface reactivity, macromolecular conformational features, and interactions between single molecules. [START_REF] Hinterdorfer | Detection and localization of single molecular recognition events using atomic force microscopy[END_REF][START_REF] Alsteens | Force-induced formation and propagation of adhesion nanodomains in living fungal cells[END_REF] Recent mechanical measurements were performed on P pili and type 1 fimbriae using Force Measuring Optical Tweezers (FMOT). It was reported that both pili are highly flexible and they may be viewed as dynamic structures with spring-like properties. [START_REF] Andersson | The biomechanical properties of E-coli Pili for urinary tract attachment reflect the host environment[END_REF][START_REF] Bjornham | Physical properties of the specific PapG-galabiose binding in E-coli P pili-mediated adhesion[END_REF][START_REF] Andersson | Dynamic force spectroscopy of E coli P pili[END_REF][18] Indeed, when bacteria were subjected to hydrodynamic constrains, these structures maintain bacterial attachment to host cells or surfaces by reducing the impact of flushes and associated mechanical stress [START_REF] Andersson | The biomechanical properties of E-coli Pili for urinary tract attachment reflect the host environment[END_REF][START_REF] Castelain | Fast uncoiling kinetics of F1C pili expressed by uropathogenic Escherichia coli are revealed on a single pilus level using force-measuring optical tweezers[END_REF] following an unwinding of the pointed out that the length of P-pili and type 1 fimbriae could increase by a factor 5 following such unwinding process under high shear stress conditions. [START_REF] Bullitt | Structural polymorphism of bacterial adhesion Pili[END_REF][START_REF] Andersson | A Structural Basis for Sustained Bacterial Adhesion: Biomechanical Properties of CFA/I Pili[END_REF][START_REF] Li | Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli[END_REF] The forced unraveling of type 1 fimbriae is reversible, with helical rewinding taking place for shear forces of magnitude ~ 60 pN. [START_REF] Andersson | Dynamic force spectroscopy of E coli P pili[END_REF][START_REF] Yakovenko | FimH forms catch bonds that are enhanced by mechanical force due to allosteric regulation[END_REF] Although the dynamic elongation properties of pili have been investigated by AFM, optical tweezers or DFS, no studies has focused so far on the role of environmental factors like external pH.

In view of the various pH exposure conditions possibly encountered by E. coli in its natural environment, we report here a systematic investigation of the impact of pH on the dynamic elongation of type 1 fimbriae and on the adhesion force between such structures and sugarcoated surfaces. For that purpose, AFM-tips were functionalized with mannose to probe the specific interactions with FimH, the lectin ending part of fimbrial rods. We recorded retraction SMFS force curves at different pH values in the range 3.

Materials and Methods

Bacterial strains.

The E. coli K-12 strains used in this study are listed and described in previous works. [START_REF] Polyakov | Automated Force Volume Image Processing for Biological Samples[END_REF][START_REF] Francius | Bacterial Surface Appendages Strongly Impact Nanomechanical and Electrokinetic Properties of Escherichia coli Cells Subjected to Osmotic Stress[END_REF] These isogenic strains were constructed from Escherichia coli MG1655 (E. coli genetic stock center CGSC#6300). All strains used in this study contain the gfpmut3 gene linked to the bla ampicillin resistance gene (amp R , 100 µg/mL) that makes them fluorescent, and a deletion of the fliE to fliR genes replaced by the cat chloramphenicol resistance gene (cm R , 25 µg/mL).

Our reference strain (E2152) has been additionally deleted for both the fim operon encoding fimA-H::zeo, zeo R 50 µg/mL) and agn43 flu::km, km R 100 µg/mL).

The constitutive production of type 1 fimbriae was ensured by insertion of a constitutive promoter in front of the fim operon (strain with PcLfim, E2146). [START_REF] Francius | Bacterial Surface Appendages Strongly Impact Nanomechanical and Electrokinetic Properties of Escherichia coli Cells Subjected to Osmotic Stress[END_REF][START_REF] Da Re | Tight modulation of Escherichia coli bacterial biofilm formation through controlled expression of adhesion factors[END_REF][START_REF] Korea | coli K-12 possesses multiple cryptic but functional chaperone-usher fimbriae with distinct surface specificities[END_REF] Growth conditions and sample preparation for AFM experiments.

Bacteria were pre-grown overnight at 37°C under agitation (150 rpm) in M63B1 minimal medium supplemented with 0.4% glucose (M63B1glu) and with the appropriate antibiotics for the proper selection of the strain of interest. The next day, fresh M63B1glu medium was inoculated with the overnight culture to an OD 600 of c.a. 0.05 and cultivated under the same conditions until the biomass reached an OD 600 of 0.5-0.6.

Then, 2 mL of the bacterial suspension were removed and placed on a PEI-silicon wafer slide for 30 minutes. The samples were extensively rinsed with PBS solution to remove M63B1glu and placed directly into the AFM closed fluid-cell with 2 mL of PBS solution at the desired pH in the range 3 to 9.

Electrophoretic mobility measurements.

We performed electrophoretic mobility (EPM) measurements on bacteria in a quartz suprasil cell at 24°C (Zetaphoremeter IV, CAD Instrumentations, Les Essarts le Roi, France).

Displacements of bacterial cells were followed from the reflection by bacteria of a laser beam tracked with a charge-coupled device camera. Using image analysis software, recorded images were processed in real time to calculate the electrophoretic mobilities from the migration motion of bacteria subjected to a constant direct-current electric field (800V/m).

Different cycles were recorded to carry out 100 measurements of bacterial mobility in M63B1 minimal medium supplemented with 0.4% glucose (M63B1glu). Additionally, the reproducibility of the experiments was addressed by repeating the electrokinetic measurements with at least three different freshly-prepared bacterial suspensions.

Measurement of E. coli type 1 fimbriae-mediated yeast agglutination.

We assessed the capacity of type 1 fimbriae to agglutinate yeast cells following a protocol previously described. [START_REF] Hasman | Antigen 43 and type 1 fimbriae determine colony morphology of Escherichia coli K-12[END_REF] from Sigma-Aldrich (Sigma Aldrich, Saint-Quentin Fallavier, France) and used as received.

PEG-acetal linkers were purchased from Hermann Gruber group (Institute of Biophysics, University of Linz, Austria).

AFM-tips were functionalized with mannosamine using a procedure previously described [START_REF] Francius | Detection, localization, and conformational analysis of single polysaccharide molecules on live bacteria[END_REF][START_REF] Francius | Stretching polysaccharides on live cells using single molecule force spectroscopy[END_REF] for the detection and stretching of type Biological replicates from independent bacterial growth culture, and performed at pH 7 for the 6 pulling rates were close to the triplicates with less than 10% deviation (data not shown).

The uncertainties given in this work were calculated from the technical triplicates only.

In SMFS experiments, the adhesins located on the gold-surfaces and on the biological samples are stretched upon removal of the chemically-modified AFM tip away from the surface (Figure S1 in Supporting Information). The obtained force versus distance curves are then analyzed using WLC (Worm Like Chain) model. This model is most suitable and most frequently used to describe the extension of polypeptides. Within the framework of this theory, the extension z of the macromolecule is related to the retraction force F adh via

4 1 1 4 2 adh c c p B L z L z l T k z F ( 1
)
where the persistence length l p directly reflects the chain stiffness, L c is the total contour length of the macromolecule and k B is the Boltzmann constant.

The number of monomers in the polypeptidic chains is provided by

p c l L N (2)
All the FVI were automatically analyzed with a Matlab algorithm described elsewhere. [START_REF] Polyakov | Automated Force Volume Image Processing for Biological Samples[END_REF] It is emphasized that force-distance curves measurements were performed on freshly prepared PEI-coated glass slice and Concanavalin A-grafted gold surface in order to (successfully) verify the binding specifity of the AFM tip.

Results and discussion

Morphological analysis of cells constitutively expressing type 1 fimbriae.

To gain insight into the cell surface morphology of E. coli K-12 producing type-1 fimbriae, live bacterial cells were grown at 37°C and electrostatically immobilized onto a PEI-coated silicone substrate and gently dehydrated before imaging by AFM (Figure 1). The AFM images clearly showed that the cells constitutively expressing type 1 fimbriae (E2146) exhibited a 2 to 2.5 µm rod-like shape decorated with 10 nm diameter filamentous surface structures of length 50 to 500 nm. These characteristics well correspond to those reported for type 1 fimbriae, i.e. rod-shaped and helical structure of total contour length 1 to 10 µm and diameter 6 to 20 nm. [START_REF] Mol | Molecular and structural aspects of fimbriae biosynthesis and assembly in Escherichia coli[END_REF][START_REF] Hahn | Exploring the 3D molecular architecture of Escherichia coli type 1 pili[END_REF] Adhesion images also evidenced a pronounced disparity in terms of length of the surface appendages as some are partially retracted and others are completely collapsed along the contour of the cell wall. These observations underpin some flexibility of type 1 fimbriae structures, probably depending on their physiological state, in line with previous conclusions. [START_REF] Andersson | The biomechanical properties of E-coli Pili for urinary tract attachment reflect the host environment[END_REF][START_REF] Bjornham | Physical properties of the specific PapG-galabiose binding in E-coli P pili-mediated adhesion[END_REF][START_REF] Andersson | Dynamic force spectroscopy of E coli P pili[END_REF][18] Impact of pH and pulling rate on FimH-mannose interaction.

We performed single-molecule and dynamic force spectroscopy measurements (SMFS and DFS) on type 1 fimbriae anchored at the wall of living cells using mannose functionalized cantilevers (Figure S1) in order to (i) quantify the adhesion force between type 1 fimbriae and AFM-tip functionalized with mannosyl residues, (ii) estimate the impact of pH on the conformational properties of the type 1 fimbriae structure as derived from analysis of the retraction force curves using eq 1 described in the Materials and Methods section, and (iii) investigate the dynamic behaviour of type 1 fimbriae submitted to increasing pulling rates, which somehow mimics the effect of external shear forces the cells experience when attached to host-tissue surfaces. Indeed, previous study evidence that between the bladder and the kidney, urine flow can reach up to 30,000 µm/s. [START_REF] Vogel | Modelling of urine flow in an ureteral bolus[END_REF] Typical force curves and corresponding statistic distribution of the adhesion forces obtained at pH 7.4 for pulling rates of 2 and 20 µm/s are reported in Figure 2. A typical elongation forcecurve of type 1 fimbriae is generally composed of three distinct regimes as reported in literature. [START_REF] Andersson | Dynamic force spectroscopy of E coli P pili[END_REF][START_REF] Forero | Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds[END_REF] In the first regime, the force increases in absolute value upon retraction of the AFM tip, which corresponds to the stretching of the fimbriae structure until a force plateau is reached. This plateau is symptomatic of a tensed helical structure where the FimA units that predominantly constitute the fimbrial filament retain their conformation. In the second regime, the interactions between successive FimA units are broken at constant force, i.e. the fimbrial filament is unfolded and there is unwinding of the FimA units constituting the fimbrial rod. In a last regime the force gradually increases (in absolute value) and abruptly decreases to zero. This drop is related to the rupture between the FimH lectin-ending part of the fimbrial structure and mannose grafted on the tip. Regardless the value of the retraction speed of the AFM tip, we systematically measured for the last detected adhesion event a force plateau of about 62 11 pN. The occurrence of single and multiple adhesive events per force curve reflects the simultaneous detection, unfolding and rupture of one or several fimbriae captured by the tip. We underlined that the contact depth between the cell wall surface and the functionalized AFM-tip was fixed to 20 nm, which avoids the attachment of too many For both pulling rates aforementioned, we found similar retraction force curves characterized by the three characteristic regimes discussed above. However, different multimodal distributions for the rupture force (or FimH-mannose bond rupture force) can be identified (Tables 1 and2). In details, the first adhesion force in this distribution (Figures 2c,2d) significantly decreases from 95 25 to 70 19 pN while increasing the pulling rate by a factor 10, and the second rupture force value decreases from 169 21 to 141 43 pN for similar changes in pulling rates. This 16% to 26% decrease in rupture force may be related to the decrease of the interaction time between the FimH lectin domain and the mannosyl residue grafted on the AFM-tip upon increase of the pulling rate.

Furthermore, close inspection of Figure 2 reveals two concomitant phenomena when the pulling rate is increased by a factor 10. First, the largest rupture force detected in the multimodal distribution increases from 244 pN up to 281 pN with increasing the pulling rate, while the last rupture distance decreases from about 3.5 m to 2 m (see next section for discussion of this feature).

The qualitative dependence of the first two rupture forces on pulling rate is not in agreement with the so-the literature [START_REF] Yakovenko | FimH forms catch bonds that are enhanced by mechanical force due to allosteric regulation[END_REF][START_REF] Thomas | Biophysics of catch bonds[END_REF] for FimH adhesins.

According to such an effect, the adhesion of FimH onto glucose-coated surfaces is favored with increasing flow rates or, equivalently, as the adhesive bonds are strengthened by tensile mechanical forces. In this study, AFM measurements were carried out with pulling rates that are 10 to 100 times larger than those i that our experiments are probably performed outside the range of pulling rate magnitude that leads to increased adhesion with increasing pulling force. Despite these discrepancies, the adhesion forces corresponding to the interaction between AFM tip and a given fimbriae as measured here are comparable in magnitude to those reported in literature. [START_REF] Forero | Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds[END_REF] These results

indicate that the mechanical response of type 1 fimbriae may depend on flow rates according to processes that are different than those leading to the catch bond effect. In particular, Figure 2 suggests that the strength of FimH-mannose adhesion depends on the interplay between the characteristic time scales pertaining to FimH-mannose interaction and to the retraction dynamics of the mannose residues from the FimH ending units. In the next section, we further discuss the dependence of the last rupture distance on pulling rate.

Modeling the experimental stretching of type 1 fimbriae using worm-like-chain model.

The retraction force curves depicted in Figures 2a,2b were interpreted using the Worm like Chain (WLC) model (eq 1). Following this strategy, we derived two key-parameters pertaining to the structural properties of the type 1 fimbriae:

-the contour length L c defined as the length of the fully extended linear polypeptidic chain.

-the persistence length l p that reflects the intrinsic flexibility or rigidity of the polypeptidic chain.

A careful analysis of these L c and l p parameters enables to identify the structural or conformational changes of the fimbriae-mannose chain during its extension. The statistic distribution of l p and L c values obtained at pH 7.4 with 2 and 20 µm/s pulling rates are reported in Figure 3. For both conditions of pulling rate tested in this work, similar bimodal distributions of L p are found with values 0.05 0.03 nm and 0.21 0.07 nm (Tables 1 and2), which is in close agreement with data derived from WLC modeling of complex proteins like myosin [START_REF] Root | Coiled-Coil Nanomechanics and Uncoiling and Unfolding of -Helices of Myosin[END_REF] . The lowest l p value further matches the typical pitch distance for a -helix while the second value adequately compares to the typical pitch distance for an -helix [START_REF] Kajava | Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins[END_REF][START_REF] Yoder | New domain motif - The structure of pectate lyase-C, a secreted plant virulence factor[END_REF] or -helix rise per amino acids. [START_REF] Ainavarapu | Contour length and refolding rate of a small protein controlled by engineered disulfide bonds[END_REF][START_REF] Grater | Dissecting entropic coiling and poor solvent effects in protein collapse[END_REF][START_REF] Tobi | Distance-dependent, pair potential for protein folding: Results from linear optimization[END_REF] This result is in good agreement with conformation of the fimbrial subunits that are characterized by -sheet for FimH (Ig-like domains) whereas the stacking of FimA leads to formation of -helix structure. Altogether, the above results indicate that the obtained persistence length that reflects the very flexibility of the FimH adhesin, is not affected with increasing pulling rates at fixed solution pH. This conclusion is further supported by the systematic analysis of force-separation distance curves recorded at a fixed pH value in the range 3 to 9 and different pulling rates 2, 5, 10 and 20 µm/s (Figure S2 in Supporting Information).

The distribution in total contour length L c at pH 7.4 is broad (Figure 3c), starting from 0.2 µm up to 10 µm at a pulling rate of 2 µm/s. When the pulling rate is increased to 20 µm/s (Figure 3d), a narrower distribution is found with L c values in the range 0.2 µm to 2 µm and a weak distribution in the range of 2 µm to 6 µm, which is in line with literature. [START_REF] Rheinlaender | Contour and persistence length of Corynebacterium diphtheriae pili by atomic force microscopy[END_REF] In order to further rationalize this dependence of L c on pulling rate as derived from force curve modeling, we report in Figure 4 the pH- and pulling rate-mediated variations of the maximal elongation of type 1 fimbriae prior to the last rupture event detected in the force curves. The reader is referred to Figure S3 for typical force curves data collected at various pH values and 2 µm/s pulling rate, and to Figure S4 for corresponding statistical distribution in maximal elongation length (Table S1). For pulling rates of 0.5 to 20 µm/s, the maximal extension of the fimbriae obviously depends on solution pH (Figure 4). In details, the maximal extension increases with increasing pH and reaches a pronounced maximum at pH 7.4 (pH of PBS buffer with no addition of acid or base) before decreasing to values lower than 0.5 µm with further increasing pH. The largest value of the maximum extension amplitude is 8 µm for pulling rates lower than 5 µm/s and it gradually decreases to 3 µm with increasing pulling rate from 5 to 20 µm/s. Overall, the results displayed in Figure 4 suggest that the interactions between the FimA units in the fimbrial rod are strongly affected by changes in solution pH and in pulling rate. In particular, the collapse of the fimbrial structure length with increasing pulling rate is likely related to the marked dynamic nature of the FimA-FimA units interactions within the fimbrial rod, as detailed in Puorger et al. [START_REF] Puorger | Structure, Folding and Stability of FimA, the Main Structural Subunit of Type 1 Pili from Uropathogenic Escherichia coli Strains[END_REF] This dependence of the FimA-FimA units interactions on pulling rate also results in a decrease of the last rupture force at fixed pH value, as shown in Figures 2c and2d.

The understanding of the above features pertaining to the maximal elongation of the fimbrial structure (Figure 4) deserves additional comments. Several studies have evidenced that a decrease in rupture force with increasing pulling rate is related to changes in dynamics of fimbrial unwinding. These changes are governed by weak layer-to-layer interactions (of e.g.

hydrophobic nature) between subunits on adjacent helical structures, and by the nearly horizontal orientation of Fim protein subunits (FimA, FimG and FimF) with respect to the filament axis. [START_REF] Andersson | A Structural Basis for Sustained Bacterial Adhesion: Biomechanical Properties of CFA/I Pili[END_REF][START_REF] Li | Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli[END_REF][START_REF] Andersson | Physical properties of biopolymers assessed by optical tweezers: Analysis of folding and refolding of bacterial pili[END_REF] The dependence of CFA/I (colonization factor antigen I) pili rupture force on pulling rate was observed for pulling rate larger than a critical value (1.4 µm/s) referred to as the corner velocity. [START_REF] Andersson | A Structural Basis for Sustained Bacterial Adhesion: Biomechanical Properties of CFA/I Pili[END_REF] Our results are consistent with pulling rate dependentrupture force and elongation as reported for CFA/P/I pili [START_REF] Castelain | Fast uncoiling kinetics of F1C pili expressed by uropathogenic Escherichia coli are revealed on a single pilus level using force-measuring optical tweezers[END_REF][START_REF] Li | Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli[END_REF][START_REF] Miller | The mechanical properties of E. coli type 1 pili measured by atomic force microscopy techniques[END_REF] , and they additionally evidence a strong influence of solution pH on the extent of fimbrial unwinding, as schematically illustrated in Figure S5 and Figure S6.

The impact of pH and medium salinity on the elongation of pili was previously reported for P pili [START_REF] Andersson | Physical properties of biopolymers assessed by optical tweezers: Analysis of folding and refolding of bacterial pili[END_REF] without solid mechanistic explanation for the observed trends, and such data are not available for type 1 pili. We believe that fimbrial unwinding becomes incomplete for pulling rates larger than the corner velocity because retraction dynamics is too fast to allow a sufficient time delay to undo the connections (hydrogen bonds, salt bridges and hydrophobic contacts) between FimA units. [START_REF] Klinth | The Influence of pH on the Specific Adhesion of P Piliated Escherichia coli[END_REF][START_REF] Aprikian | The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor[END_REF][START_REF] Bosshard | Protein stabilization by salt bridges: concepts, experimental approaches and clarification of some misunderstandings[END_REF] It is also expected that a decrease of the solution pH leads to stronger hydrogen bonds inside the fimbrial rod therefore reducing the fimbrial unwinding.

This reduction in elongation is also observed under alkaline conditions where negative charges issued from deprotonated carboxylic groups likely increase the degree of connectivity between FimA units, which in fine results in decreased unwinding amplitude.

Dependence of FimH-mannose binding force on pulling rate and pH

In Figure 5, the modulations of the average FimH-mannose rupture force is displayed as a function of solution pH for various values of pulling rate: 0.5, 1, 2, 5, 10 and 20 µm/s. It is recalled that the last rupture force corresponds to the strength of the FimH-mannose bond. For the lowest pulling rate tested, the adhesion force basically remains independent of pH and equals 60-80 pN. With increasing pulling rate up to 5 µm/s, a pronounced maximum in the binding force is observed at pH 7.4 and its magnitude reaches ~150-200 pN at 5 µm/s. This maximum vanishes for experiments carried out at 10 and 20 µm/s pulling rates, and the binding force then reaches a pH-independent value of ~65-100 pN under such conditions.

Overall, the results of Figure 5 indicate that the FimH-mannose binding force strongly depends on solution pH for some critical values of the pulling rate (here 5 µm/s). For all other values tested in this work, the FimH-mannose binding force is minimal, quasi pH-independent (50-75 pN) and nearly independent of the retraction rate.

The pH value corresponding to pronounced maximum in binding force for a pulling rate of 5 µm/s, identifies to the pH marking a maximum unwinding distance of the fimbrial rod (Figure 4). Besides, we observed that the persistence length reflecting the FimH-mannose flexibility is independent of pH and pulling rate (Figure 3a,b and Figure S2), which suggests that the conformation of the FimH adhesin remains identical regardless the values of the stretching rate and the solution pH. Altogether, these elements indicate that the variations of the maximal fimbriae elongation distance with pH and pulling rate (Figure 4) is related to the only modification of the molecular interactions or connections between FimA units within the fimbrial rod.

the pH range 5 to 9 for pulling rates lower than 10 µm/s. This suggests that beyond a critical value of the pulling rate (i.e. 10 µm/s), the FimH-mannose binding is attenuated following dynamic effects, i.e. the bond between the fimbriae lectin ending part and the mannosyl residue has no longer time to fully settle. Overall, two important processes concomitantly take place at neutral pH when unwinding fimbria: a weakening of the FimA-FimA interaction forces (Figure 4) and an increased FimH-mannose interaction (Figure 5). The former effect was suggested in Rangel et al. [START_REF] Rangel | Observation of Bacterial Type I Pili Extension and Contraction under Fluid Flow[END_REF] on piliated bacteria subjected to various flow rates. This study indicated that the mechanical and functional properties of the pili, in particular, their extensibility, may reduce tension upon alignment of their structure along the flow direction.

In turn, this uncoiling of the pili modifies the force to be applied for detaching the terminal adhesion from the surface [START_REF] Rangel | Observation of Bacterial Type I Pili Extension and Contraction under Fluid Flow[END_REF] , which is pretty much in line with the results of Figures 4 and5.

Effect of pH on electrokinetics of bacterial cells.

In order to better understand the effect of pH on the cell wall properties of E. coli, we performed electrophoretic mobility measurements in M63B1glu medium over the pH range 3 to 9 for the strain E2146 (constitutively expressing type 1 fimbriae).

As detailed elsewhere, [START_REF] Francius | Bacterial Surface Appendages Strongly Impact Nanomechanical and Electrokinetic Properties of Escherichia coli Cells Subjected to Osmotic Stress[END_REF] these macroscopic measurements allow the evaluation of key electrostatic and structural properties averaged over the entire soft surface structure carried by the cell. As such, electrokinetics cannot be exploited to extract molecular details pertaining to the only FimH-mannose interaction but it is however relevant for understanding how the surface charge density of the bacterial cell wall structure is modified by pH and thus how the FimA-FimA interactions may potentially be involved in the fimbrial unwinding process. The electrophoretic mobility results were systematically compared to those obtained for the reference strain E2152 that does not produce type 1 fimbriae and does not agglutinate yeast cells regardless the pH medium (see next section). The dependence of the electrophoretic mobility of the bacterial strains E2152 and E2146 are reported in Figure 6a. For all strains, is negative, as expected for biological particles, [START_REF] Duval | Progress in electrohydrodynamics of soft microbial particle interphases[END_REF] and it increases in absolute value with increasing pH following the deprotonation of ionogenic groups distributed throughout the cell wall structures. The mobility of E2152 cells reaches a plateau value of about 1.5 10 -8 m 2 .s - 1 .V -1 for pH values in the range 6 to 8. The electrophoretic mobility of E2146 cells significantly differ from that of E2152. In details, the mobility increases in magnitude with pH, reaches a constant value in the pH range 6 to 7.5 and increases again for pH larger than 8.

In addition, under fixed pH condition, the mobility of E2146 strain is lower in magnitude as compared to that of the bacterial reference strain E2152 except at pH >8.5 where mobilities of both strains are similar. This trend is explained by an increased friction force exerted by the loose fimbrial appendage structure on the electroosmotic flow, [START_REF] Duval | Progress in electrohydrodynamics of soft microbial particle interphases[END_REF] thereby reducing the mobility of E2146 as compared to that of E2152 (Figure 6). With regard to E2146 strain, the increase in mobility values with pH basically corresponds to an increase of the negative charge density within the fimbrial surface structure surrounding the cell wall.

The differential mobility µ reported in Figure 6b was evaluated from the difference between the electrophoretic mobilities of E2146 and of the reference bacteria (E2152) determined under similar pH condition. Because of the strain constructions, µ may be related to the sole electrohydrodynamic features pertaining to the fimbrial structure. In details, µ exhibits a non monotonous behaviour with varying pH. It decreased by a factor 4 between pH 3 and 5.5, the latter pH value corresponding to the FimA isoelectric point (denoted as pI(FimA). Then, we observed a local maximum in µ at pH 7. With further increasing pH, µ tended to zero and the µ value at pH 7.5 (i.e. at the FimH isoelectric point (pI(FimH)) is identical to that at pH 5.5, i.e. at pI(FimA) . The fact that the µ tends to zero for pH larger than pI(FimA) and pI(FimH) means that the fully expressed charge of the fimbriae layer at the cell surface under such pH conditions counteracts the friction force exerted by the cell surface appendages on the electroosmotic flow so that, in turn, the mobility of E2146 strain is identical to that of the reference strain E2152 at high pH. The presence of a maximum in µ at pH 7 likely originates from the combination of two processes: first the important increase of the charges on the FimA units (at pH values around pI(FimA)) leads to an increase of the local repulsive electrostatic interactions between adjacent fimbriae. This will in turn likely induce their extension (swelling) even though the latter is expected to be limited under the salinity conditions of the experiments. Nevertheless, even such a slight extension of the fimbrial structure can dramatically impact cell electrophoretic mobility. [START_REF] Rangel | Observation of Bacterial Type I Pili Extension and Contraction under Fluid Flow[END_REF] In fine, the mobility of E2146 is reduced due to the larger friction exerted by the structure. Once the FimH charge is expressed the fimbrial filaments being already stretched, the electrophoretic mobility will increase again in absolute value and eventually return to the level of that measured for E2152.

Macroscopic measurements of bacterial surface structure properties, as obtained from electrokinetics, therefore support the importance of the electrostatic interactions within the fimbrial structure and, in particular, the key role played by the solution pH on their extension, as evidenced by the detailed molecular AFM analysis. The maximum in µ commented above and observed in the pH window 5 to 8 well matches that obtained for the maximal extension of fimbriae (Figure 4), thus revealing a role played by electrostatics in the nonmonotonous behavior measured for the fimbriae flexibility under the action of external forces.

Correlation between AFM analysis and bacterial yeast agglutination.

In order to evaluate the relevance -at a macroscopic scale- of the observed impact of solution pH on the adhesion force between FimH and mannose (Figure 5), we performed yeast agglutination assays (Figure 7). By virtue of interaction between FimH and mannosylated proteins located at the surface of yeast cells, E. coli expressing type 1 fimbriae has the capacity to interact simultaneously with several yeast cells, thus leading to yeast agglutination. Using this assay, the level of yeast agglutination can be directly correlated to the efficient binding between FimH and mannose. Interestingly, we obtained an optimum in yeast agglutination at pH 5, 6 and 7 while weaker agglutination was detected at pH 3, 8 and 9 (Figure 7). This result strongly supports the dependence of the FimH-mannose binding force on pH at pulling rates lower than 5 µm/s as obtained from AFM analysis. It further confirms that neutral pH conditions lead to maximum interaction between FimH and mannose under moderate pulling rates that are in line with Brownian encounter/collision between bacteria and yeasts.

A correlation may be also further drawn between our molecular FimH-mannose interaction measurements and the adhesion behavior of E. coli under physiological conditions when present in the gastro-intestinal and urinary tracts of human. Indeed, E. coli is not known as an efficient colonizer of the stomach where the pH is very acidic, which is in line with the low adhesion force measured in our study under such pH condition and with a reduced expression of type 1 fimbriae at such extreme pH value. [START_REF] Schwan | Osmolarity and pH growth conditions regulate fim gene transcription and type 1 pilus expression in uropathogenic Escherichia coli[END_REF] It is further accepted that type 1 fimbriae efficiently bind to colonic and ileal enterocytes 51 thereby suggesting a type 1 fimbriaemediated colonization of mammals gut. Accordingly, E. coli K1 and APEC type 1 fimbriae have been shown to be necessary for mouse and chicken intestinal colonization, respectively. [START_REF] La Ragione | The role of fimbriae and flagella in the adherence of avian strains of Escherichia coli O78 : K80 to tissue culture cells and tracheal and gut explants[END_REF][START_REF] Martindale | Genetic analysis of Escherichia coli K1 gastrointestinal colonization[END_REF] For humans, the pH in the gut is 6 in the duodenum part, 7.4 in the ileum, 5.7 in the caecum and 6.7 in the rectum [START_REF] Fallingborg | Intraluminal pH of the human gastrointestinal tract[END_REF][START_REF] Nugent | Intestinal luminal pH in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs[END_REF] , i.e. the pH value is compatible with that reported here where there is a maximum in adhesion of type 1 fimbriae. Additionally, in the urinary tract, pH of urine is generally larger than 5.0 [START_REF] Asscher | Urine as a medium for bacterial growth[END_REF][START_REF] Waters | Community study of urinary pH and osmolarity[END_REF] and it thus again corresponds to optimal condition for strong type 1 fimbriae-mediated cell adhesion and colonization of the bladder.

Conclusions

The results reported in this work demonstrate that type 1 fimbriae are dynamic structures whose flexibility and binding force to mannosylated surfaces can be modulated with changing external shear forces and solution pH conditions. Using single-molecule force and dynamic force spectroscopies, we found that this modulation is mostly governed by both FimA-FimA and FimH-mannose interactions. The combination of these interactions was evidenced from the analysis of the maximal fimbriae extension determined with using live bacterial cells investigated under various pH conditions. We further found that fimbrial unwinding that reflects the strengths of the FimA-FimA interactions was dramatically decreased by a factor 5 and 25 for pulling rates larger than a given corner velocity and for pH values outside the range 5 to 8, respectively. Furthermore, dynamic force spectroscopy analyses performed with AFM probes specific to FimH revealed the occurrence of a minimal FimH-mannose binding force of about 50-75 pN that is quasi-independent of the applied pulling force and the pH conditions. In addition, we observed that for increasing pulling rate up to 5 µm/s, the FimHmannose interaction force is maximal at pH 7.4 with a magnitude of ~150-200 pN. Finally, beyond a critical value of the pulling rate of about 10 µm/s, this maximum in FimH-mannose interaction force does only within the pH range 5 to 9 and for pulling rates lower than 10 µm/s.

The results derived from molecular AFM analysis are supported by the macroscopic results obtained fromyeast agglutination assays and electrophoretic mobility measurements performed on bacteria over-expressing type 1 fimbriae under different pH conditions. In particular, we evidenced an optimum of yeast agglutination in the pH range 5 to 7, in line with the maximum detected by AFM for the binding force between FimH and mannose at neutral pH under moderate applied pulling rate. Moreover, electrokinetic measurements Tables Table 1. Values for the rupture forces corresponding to the main modes of the statistical distribution given in Figure 2c. Corresponding conformational parameters (persistence and contour lengths, l p and L c , respectively) as derived from WLC model (eq 1). Data were extracted from a 5 m 5 m Force Volume Image (FVI) consisting of 1024 force curves recorded on several bacteria at pH 7 and for a pulling rate of 2 µm/s. Yeast agglutination assay for E2146 performed over the pH range 3 to 9. The strain E2152 is deleted for type 1 fimbriae fim operon and it thus does not agglutinate yeast cells.

2 to 9 . 2 .

 92 Data were analyzed on the basis of Worm Like Chain (WLC) model in order to quantify the impact of pH on the conformational properties of type 1 fimbriae. In addition, dynamic force spectroscopy (DFS) measurements were performed to analyze the elongation of type 1 fimbriae under various pH conditions over a large range of tip retraction speeds, thus exploring the effect of adverse shear force experienced by cells when attached to target surfaces. DFS and SMFS experiments were further correlated to macroscopic type 1 fimbriaedependent yeast agglutination assays with E. coli. Overall, the study highlights that fimbrial unwinding strongly depends on pH and is mediated by the corresponding modulation of the FimA-FimA interactions all along the fimbriae structure. In addition, the molecular interactions between FimH and mannose are significantly impacted both by retraction speed and solution pH. This in turn could probably modify the ability of the bacteria to efficiently colonize host surfaces.

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.

Figure 7 .

 7 Figure 7.

Figure S1 .

 S1 Figure S1.

Figure S2 .

 S2 Figure S2.

Figure 1

 1 Figure 1.

  pH

  Figure 5.

  pH

  Figure 6.

  pH

  Figure 7.

  Figure S1.

  Figure S3.

  Figure S5.

Figure S6.

  Figure S6.

Chemicals and AFM-tips functionalization.

	Briefly, 5 mL of bacteria and yeast cultures grown to stationary phase
	in LB or YPD (Yeast extract Peptone-Dextrose) respectively, were diluted to reach an optical
	density at 600 nm (OD 600nm ) of 1.0. Bacteria and yeast cells were washed with phosphate
	saline buffer (PBS) 1X and resuspended in PBS1X with pH 3 to 9. Bacteria were then mixed
	with yeast (OD 600nm 1:1) and placed in a 96-well microtiter plate and agglutination was then
	assessed after 10 min settling. Agglutination titers were expressed as the lowest bacterial
	concentration leading to yeast agglutination.
	Mannosamine (C 6 H 11 O 5 -NH 2 ), ethanolamine (C 2 H 7 NO), dimethylsulfoxyde (DMSO),
	triethylamine (C 6 H 15 N), sodium cyanoborohydride (NaCNBH 3 ) and PBS tabs were purchased

AFM imaging, Single-Molecule Force Spectroscopy (SMFS) and Dynamic Force Spectroscopy (DFS) measurements.

	AFM images were recorded with FastScan AFM (Bruker AXS, Palaiseau, France) and force-
	distance curves with a MFP3D-BIO instrument (Asylum Research Technology, Atomic Force
	F&E GmbH, Mannheim, Germany). Silicon nitride and gold coated cantilevers of conical
	shape were purchased from Bruker (MLCT and NPG, Bruker AXS, Palaiseau, France) with
	spring constants of 10-12 pN/nm and 120-150 pN, respectively. Experiments were performed
	in PBS at different pH values from 3 to 9 and at room temperature.
	Adhesion forces, conformational characteristics and extension of the adhesins were measured
	by recording Force-Volume Images (FVI) consisting of a grid of 32-by-32 force curves
	obtained upon approach and subsequent retraction of the tip (SMFS experiments) and for
	pulling rates of magnitude 0.5-20 µm/s (DFS experiments). Force measurements were
	performed in triplicate for each condition examined over an area of 5 µm 5 µm after
	locating single bacteria via the Olympus IX 71 inverted microscope that supports the AFM.
	1 fimbriae. Briefly, silicon nitride tips (MLCT, Bruker
	Nano AXS, Palaiseau, France) were first modified with amino groups that further reacted
	with PEG linkers carrying benzaldehyde functions. The latter were then directly attached to
	mannosamine through terminal NH 2 group.

Table 2 .

 2 Values for the rupture forces corresponding to the main modes of the statistical distribution

	given in Figure 2d. Corresponding conformational parameters (persistence and contour
	lengths, l p and L c , respectively) as derived from WLC model (eq 1). Data were extracted from
	a 5 m 5 m Force Volume Image (FVI) consisting of 1024 force curves recorded on several
	bacteria at pH 7 and for a pulling rate of 20 µm/s.

Table S1 .

 S1 Average maximal fimbriae unwinding calculated from the last rupture distance on the force curves showing adhesive events. Each average value was derived from a Force Volume Image

	of 1024 force curves recorded on single bacteria at pH 3, 5, 7 and 8 for a pulling rate of 5
	µm/s.
		Maximal fimbriae elongation in nm (v = 2 µm/s)
	pH = 3	pH = 5	pH = 7	pH = 8
	387 213	1634 1082	8487 1294	607 411
	1966 304	--	--	1838 284

Acknowledgement C.S. is supported by a MENE A.C., C.B and J-M.G. are supported by the Institut Pasteur and the French Government's Investissement d'Avenir program, Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant n°ANR-10-LABX-62-IBEID).

strongly point out the importance of pH conditions for understanding and explaining bacterial adhesion under various and changing environmental conditions.

Figure S3.

Statistical distributions of the persistence length for retraction rates of 2, 5, 10 and 20 µm/s at pH 3, 5, 6, 7 and 8. Values were obtained from analysis of Force Volume Images of 1024 force curves recorded on single bacteria.