Single Molecule Force Spectroscopy Reveals Two- Domain Binding

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Single Molecule Force Spectroscopy Reveals Two-Domain Binding Mode of Pilus-1 Tip Protein RrgA of Streptococcus Pneumoniae to Fibronectin Tanja D. Becke, Stefan Ness, Raimund Gürster, Arndt F. Schilling, Anne-Marie di Guilmi, Stefanie Sudhop, Markus Hilleringmann, and Hauke Clausen-Schaumann ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b07247 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Single Molecule Force Spectroscopy Reveals TwoDomain Binding Mode of Pilus-1 Tip Protein RrgA of Streptococcus Pneumoniae to Fibronectin Tanja D. Becke# † § &, Stefan Ness§, Raimund Gürster§, Arndt F. Schilling# +, Anne-Marie di Guilmi°, Stefanie Sudhop† &, Markus Hilleringmann§ *, and Hauke Clausen-Schaumann& † * #

Department for Plastic Surgery and Hand Surgery, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany

†

Center for Applied Tissue Engineering and Regenerative Medicine, Munich University of Applied Sciences, 80335 Munich, Germany

§

FG Protein Biochemistry & Cellular Microbiology, Munich University of Applied Sciences, 80335 Munich, Germany &

Center for NanoScience, Ludwig-Maximilians-Universität München, 80799 Munich, Germany

+

Klinik für Unfallchirurgie, Orthopädie und Plastische Chirurgie, University Medical Center Göttingen, 37075 Göttingen, Germany ° DRF/IRCM/SIGRR/LRIG, 92265 Fontenay-aux-roses Cedex, France

* Address correspondence to: [email protected], [email protected] 1 ACS Paragon Plus Environment

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Keywords: streptococcus pneumoniae, gram-positive bacteria, pili, rrgA, fibronectin, single molecule force spectroscopy, AFM

Abstract For host cell adhesion and invasion, surface piliation procures benefits for bacteria. A detailed investigation of how pili adhere to host cells is therefore a key aspect in understanding their role during infection. Streptococcus pneumoniae TIGR 4, a clinical relevant serotype 4 strain, is capable of expressing pilus-1 with terminal RrgA, an adhesin interacting with host extracellular matrix (ECM) proteins. We used single molecule force spectroscopy to investigate the binding of full length RrgA and single RrgA domains to fibronectin. Our results show that full length RrgA and its´ terminal domains D3 and D4 bind to fibronectin with forces of 51.6 (full length), 52.8 (D3), and 46.2 pN (D4) at force-loading rates of around 1500 pN/s. Selective saturation of D3 and D4 binding sites on fibronectin showed that both domains can interact simultaneously with fibronectin, revealing a two-domain binding mechanism for the pilus-1 tip protein. The high off-rates and the corresponding short lifetime of the RrgA Fn bond (τ = 0.26 s) may enable piliated pneumococci to form and maintain a transient contact to fibronectin-containing host surfaces and thereby to efficiently scan the surface for specific receptors promoting host cell adhesion and invasion. These molecular properties could be essential for Streptococcus pneumoniae pili to mediate initial contact to the host cells and shared with other piliated Gram-positive bacteria - favour host invasion.

Streptococcus pneumoniae is one of the most important etiologic agents of respiratory tract infections and invasive diseases, such as otitis media, community acquired pneumonia, septicemia and meningitis.1 The Gram-positive bacterium is able to express long, polymeric protein appendages, so called pili, which are anchored to the cell wall and can reach through the polysaccharide capsule and/or host surface barriers (e.g. mucosa).1-4 Pili have been associated with biofilm formation, cell aggregation and adhesion to host tissue.3, 5-7 Pilus-1 of 2 ACS Paragon Plus Environment

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S. pneumoniae decorates up to 30 % of the pneumococcal serotypes 8 and has been associated with bacterial virulence, its ability to adhere to host cell surface receptors and the development of drug resistance.3, 9-12 The more than 1 µm long, multimeric structure consists of a RrgC cell wall anchor protein, multiple RrgB backbone subunits and a terminal RrgA adhesin, which are covalently linked by sortase-mediated isopeptide bonds (Fig. 1a).13, 14 RrgA harbours four domains (D1, D2, D3 and D4) which are connected via flexible linker sequences (Fig. 1b). It shows sequence and structure similarities to other Gram-positive pilins, such as GBS52 and GBS104 from Streptococcus agalactiae, or SPY0125, from Streptococcus pyogenes.15-18 Domains D1 and D4 show high similarity to IgG domains 16, 18 and D2 to the collagen-binding adhesin Cna from S. aureus.16, 19 The fold of D3 resembles that of the A3 domain of human von Willebrand factor, concealing an integrin metal ion-dependent adhesion site (MIDAS) motif, and forms a large cradle shaped surface of basic character.16 It has been shown that RrgA can interact with fibronectin (Fn), which is present in the connective tissue matrix 6, 20 and which becomes particularly accessible after degradation of the mucosal epithelial layer during infection and is therefore a target for a large number of bacterial adhesins.21, 22 However, a detailed understanding of the interaction of RrgA and its four domains D1 - D4 with Fn is still lacking, and until today no isolated RrgA fragment was found to retain its capability to adhere to epithelial cells or any ECM components.23 In recent years atomic force microscopy (AFM) based force spectroscopy on the single molecule and single cell level has provided new insights into the binding mechanisms of numerous bacterial adhesins,24-27 such as, pilus proteins of Pseudomonas aeruginosa,28, 29 Neisseria gonorrhoeae,30 and Escherichia coli,31, 32 the large adhesion protein of Pseudomonas fluorescens,33 and adhesion molecules of Lactobacillus rhamnosus,5, 34 Lactobacillus reuteri,35 Streptococcus pyogenes,36 Streptococcus mutans,37 Streptococcus

agalactiae,38 and

Staphylococcus epidermis.39, 40 For Staphylococcus aureus, binding to host cell and ECM proteins, as well as biofilm formation via cell-cell interaction have been investigated.19, 41-51 3 ACS Paragon Plus Environment

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Some bonds, such as the SdrG-fibrinogen 40 or the Cna-collagen bond,47 withstand remarkably high forces, close to rupture forces of covalent bonds,52 which has been explained by ligandbinding induced conformational transitions in the adhesion molecules, such as the “dock, lock, and latch” mechanism for SdrG-fibrinogen or the “collagen hug” for Cna-collagen.53, 54 By applying a pre-stress to the bond between the E. coli pilus protein FimH and mannose, Yakovenko et al. elegantly demonstrated that mechanical stress converts this “catch bond” to a tight binding conformation, showing that the binding mechanism is mechanically controlled through allosteric regulation.55, 56 Other examples of bacterial adhesion proteins that strengthen in response to mechanical stress in a “catch-bond” like manner are the adhesion complex of the Ruminococcus flavefaciens cellulosome, and clumping factor B of Staphylococcus aureus when interacting with loricin.57, 58 Several studies have addressed the binding strengths of the Fn binding proteins FnBPa and FnBPb of S. aureus,41, 43-45, 48 because Fn is abundant not only in the ECM and in blood plasma, but is also found on implanted medical devices, such as cardiovascular prostheses. For strains isolated from patients with infected cardiovascular implants, bond strengths and lifetimes were significantly higher, compared to strains from patients with uninfected implants. This has been attributed to elevated expression levels of FnBPb and to polymorphisms in the high affinity Fn-binding regions FnBR-5 and FnBR-9 of FnBPa.48, 59 Higher binding forces of mutant polypeptides mimicking the tandem -zipper repeat FnBR-9 have confirmed this hypothesis.41, 42, 48 These findings are consistent with polymorphisms observed in the FnBR-9, FnBR-10, and FnBR-11 regions of FnBP in methicillin-resistant S. aureus strains isolated from patients with persistent bacteremia, which form stronger bonds to Fn than strains from patients with resolving bacteremia.45 Finally, elevated levels of the transcription factor SigB in S. aureus strains isolated from cystic fibrosis patients lead to increased expression of FnBPa and increased Fn-adhesion rates and Fn-binding forces.43

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In the present study, we have used atomic force microscopy (AFM) in the single molecule force spectroscopy (SMFS) mode, to explore the interaction forces and binding kinetics of S. pneumoniae TIGR 4 pilus-1 tip protein RrgA and its four domains D1 - D4 with Fn, in order to gain a better understanding of how the pilus-1 of S. pneumoniae interacts with and adheres to host cells. Our results show that full length RrgA and its terminal domains D3 and D4 bind to fibronectin, with different binding strengths. Both domains can interact with Fn independently or in concert, revealing a two-domain binding mechanism for the pneumococcal pilus-1 tip protein. The short bond lifetimes observed for full length, D3 and D4 binding to Fn indicate that RrgA mediated Fn - adhesion may constitute a transient, initial contact allowing the bacterium to further search host structures for more specific surface receptors which permit stronger attachment and, e.g. promoting bacterial virulence, as has been proposed e.g. for Lactobacillus rhamnosus.5

Figure 1. Scheme of S. pneumoniae TIGR 4 pilus-1 and adhesive tip pilin RrgA. (a) Left: Fine pili, which can be longer than 1 µm, protrude from the bacterial surface (Hilleringmann, M., et. al, EMBO J 2009). Right: The pilus-1 consists of multiple RrgB, with the adhesin RrgA at the distal and the cell wall anchored RrgC at its proximal end. (b) RrgA is composed of four domains, which are aligned much like pearls on a string; Domain D1 is shown in green (N5 ACS Paragon Plus Environment

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terminus); domain D2 in yellow, domain D3 in blue and domain D4 in red (C-terminus) (compare Izoré,T., et al., Structure 2010 and RCSB PDB: 2WW8).

Results and Discussion Interaction of Full Length RrgA with Fibronectin To investigate binding forces of RrgA to Fn, full length RrgA (RrgA Fl, supporting information Fig. S1, S2 and S3) was expressed in E. coli, purified and covalently attached to the AFM tip via a 5 kD silane-PEG- COOH linker (Fig. 2a). Human Fn was immobilized on a glass substrate using the same surface linker (see Material and Methods section for details). AFM imaging of Fn coated glass surfaces (Fig. 2b) confirmed a homogeneous distribution of Fn on the substrate surface. The dimeric structure of Fn can be recognized, and Fn seems to be compact with a height of 4 – 5 nm and a length of ~ 120 nm, which is consist with previous AFM data and reminiscent of the structure of Fn in solution.60 Figure 2c shows the distribution of rupture forces and rupture lengths of 1400 rupture events together with five representative force-distancecurves of RrgA Fl on Fn, recorded at a pulling velocity of 1000 nm s-1. After overcoming non-specific surface interactions between AFM tip and substrate, and stretching the PEG linkers (> 70 nm), 19 % of the force curves showed mainly single or double rupture events at rupture lengths between 70 and 400 nm, while in 81 % of the force curves, no specific interaction was observed. The corresponding force-extension traces were fitted using an extensible Worm like chain (eWLC) model.61 f(x) =

𝑘𝐵 𝑇 1 𝐿𝑃

𝑥

𝐹

𝑥

F

1

[4 ( 1- 𝐿 + Φ)-2 + 𝐿 - Φ - 4] 𝐶

𝐶

(Eq. 1)

where kB is the Boltzmann constant, T the temperature, LC the contour length, LP the persistence length and Φ the stretch modulus of the backbone of the molecule. The most probable rupture force between RrgA Fl and Fn was 51.6 ± 2 pN, which is in the typical force range of receptor-ligand interactions.5, 37 and well below the 100 – 300 pN reported 6 ACS Paragon Plus Environment

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for the interaction of single staphylococcal FnBPs with Fn at comparable pulling velocities. 39 The majority of rupture events occurred at a tip substrate separation of approximately 100 nm, which matches well with the length obtained by stretching the PEG spacers (~40 nm each), together with the RrgA Fl and Fn molecules. Unfolding of Fn or RrgA Fl is therefore rather unlikely, which is consistent with earlier AFM measurements, which report unfolding forces above 100 pN for single Fn modules.62, 63 In addition RrgA Fl is stabilized by two intramolecular isopeptide bonds which can sustain much higher forces than the ones observed in our experiments.16, 64, 65 The frequency of rupture events could be reduced by blocking the binding sites of RrgA on Fn with free RrgA Fl, or when performing control experiments with BSA or no protein immobilized on the PEG-coated substrate (Fig. 2d), indicating that the rupture events observed in Fig. 2c were indeed caused by specific RrgA Fl - Fn interactions. To obtain the kinetic parameters of the RrgA Fl - Fn interaction, the force-loading rate was varied by altering the retraction velocity of the z-piezo of the AFM between 250 and 4000 nm s-1. In Figure 2e the rupture forces of 676 rupture events are plotted vs. the logarithm of the force loading rate (grey circles). In addition, the most probable rupture forces for seven distinct force-loading rate intervals between 50 and 30.000 pN s-1 were determined by fitting Gaussian distributions to the rupture force histograms within these intervals (Fig. 2e, black squares, and supporting information Fig. S4). To extract the average bond lifetime at zero force from these data, we used the Bell-Evans model,66 which assumes that the activation barrier of the binding potential decreases linearly with force, leading to an exponential amplification of the dissociation kinetics with force and a logarithmic increase of the most probable bond rupture force fMP with the force-loading rate: 𝑓𝑀𝑃 =

𝑘𝐵 𝑇 𝛥𝑥

ln [𝑘

𝑟 𝛥𝑥 𝐵𝑇

𝑘𝑜𝑓𝑓

]

(Eq. 2)

Here kB is the Boltzmann constant, T the temperature, Δx the separation of the activation barrier from potential minimum, r the force-loading rate (df/dt) and koff the reaction rate constant 7 ACS Paragon Plus Environment

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without external force. Fitting the Bell Evans model (black curve in Fig. 2e) to the most probable rupture forces (black squares in Fig. 2e) renders a zero force off-rate koff for the RrgA Fl – Fn interaction of 3.91 ± 0.88 s-1 (Δx = 0.25 nm) which corresponds to a mean bond life time at zero force τ = 1/koff of 0.26 ± 0.06 s and indicates a rapid dissociation of RrgA Fl - Fn bonds. Comparably short bond lifetimes and similar Δx values have already been reported for other bacterial adhesion proteins, such as SpaC, the key adhesion protein of the Lactobacillus rhamnosus GG pilus or FnBP of S. epidermis and S. aureus.5, 39, 41, 48 It has been suggested, that such comparably weak bonds may enable bacteria to form an initial contact to host cells and at the same time provide sufficient mobility on the tissue surface to permit searching for stronger and more specific surface receptors, e.g. under shear conditions, as they are encountered in the nasopharynx and lung or on blood vessels.5, 34, 67 A similar interaction scheme has been observed for neutrophils, which roll along the lymphatic vessel wall, where L-selectin on the neutrophils surface rapidly binds to and detaches from peripheral lymph node addressin, which is displayed on the vessel wall (τ of 0.15 s).68 In this way, the immune cells are able to withstand moderate detachment force generated by shear stress and are able to search the vessel walls for specific inflammatory induced receptors, such as ICAM-1 and PECAM1.69, 70 Considering that S. pneumoniae triggers pneumonia particularly in individuals with preexisting diseases 71, 72 which evoke alterations in the ECM composition, such as increased fibronectin expression,73, 74 pilus-1 adhesion to Fn may play a similar role, constituting an additional bacterial fitness factor favoring the expansion and invasion of the bacterium into prestressed tissue. However, RrgA might also facilitate adhesion to host surfaces at physiological Fn amounts in the presence of a capsule, which is not possible for other Fn-binding molecules of S. pneumoniae.


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Figure 2. Single molecule force spectroscopy of RrgA Fl – fibronectin (Fn) interaction (a) RrgA Fl and Fn were covalently linked via a hetero-bifunctional PEG spacer to an AFM tip or a glass surface, respectively. (b) AFM image of functionalized glass surfaces with (left) and without (right) Fn coating. Arrows indicate individual Fn molecules. (c) Rupture force and corresponding rupture length histograms together with five representative force curves obtained from RrgA Fl – Fn interaction (n = 1400) at a retraction velocity of 1000 nm s-1. Red curves 9 ACS Paragon Plus Environment

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represent extensible worm-like chain fits. The histogram reveals a most probable rupture force fMP of 51.6 pN (Gauss fit, black line). Rupture lengths accumulate at about 100 nm. (d) Control experiments performed on PEG/Fn surfaces pre-incubated with RrgA Fl (top, n = 1200), on PEG/BSA surfaces (centre, n = 700) or on PEG-coated substrates without any protein immobilized (bottom, n = 700). (e) Semi-logarithmic representation of RrgA Fl – Fn rupture forces vs. force-loading rate at retraction velocities from 250 to 4000 nm s-1. Grey circles correspond to single rupture events, whereas black squares represent the most probable rupture forces for seven distinct force-loading rate intervals between 50 and 30.000 pN s-1, determined by fitting Gaussian distributions to the rupture force histograms within these intervals (see supporting information, Fig. S4). The black line represents a Bell fit to the seven fMP with the fit parameters Δx = 0.25 nm and koff = 3.91 s-1 (corresponding to τ = 0.26 s).

Interaction of individual Domains D1 – D4 with Fibronectin To address the question which domain of RrgA mediates the bond with Fn, we repeated the SMFS experiments with distinct RrgA domains. A structure-based approach taking RrgAs three-dimensional structure 16 (RCSB PDB: 2WW8) as a basis was applied to design respective RrgA domain constructs (see supporting information, Fig. S1). Domains D1 - D4 of high purity were generated (see Material and Methods and supporting information Fig. S2), checked for correct folding via Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (supporting information Fig. S3) and covalently attached to the AFM tip via the same PEG surface linker used for RrgA Fl. Figure 3 shows the binding frequencies of RrgA Fl as well as all four domains individually against Fn and BSA. The terminal domains, D3 and D4 adhere to Fn with frequencies of 19.7 % and 16.5 %, respectively (total number of evaluated force curves, n (D3 - Fn) = 1200; n (D4 - Fn) = 1000), which is in the same range as RrgA Fl (19.2 %) and significantly higher than on the BSA controls (4.9 % and 2.7 %) and on pure PEG surfaces (data 10 ACS Paragon Plus Environment

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not shown). D1 and D2 on the other hand show adhesion rates of only 3.8 % and 4.1 %, respectively, which is well below the value determined for RrgA Fl and only slightly above the 2.4 % and 2.9 % determined on the respective BSA controls. This indicates, that the adhesion of D3 and D4 to Fn is the result of a specific molecular recognition event, highlighting the special role of RrgAs´ terminal domains D3 and D4 for the adhesion of the pilus-1 to Fn, while the interaction of D1 and D2 with Fn is negligible. Structure based analysis and comparison with other bacterial systems suggest that domains D1 and D2 serve as stalk regions with additional specific functions in other host factor interactions. In addition, the intramolecular isopeptide bonds in D2 (and D4) are essential for RrgA Fl stability.16 Fig. 4a and 4b show rupture force and length distributions as well as representative forcedistance curves of D3 and D4, on Fn. For D3, the most probable rupture force fMP at 1000 nm s - 1 pulling velocity is 52.8 pN, which is close to fMP for the full length protein at the same conditions, while for D4 fMP is only 46.2 pN. For both, D3 and D4, most rupture events were observed at tip-sample separations around 100 nm and a smaller percentage at separations as far as 400 nm. As already observed for RrgA Fl on Fn, the rupture forces of both D3 and D4 on Fn increase nearly logarithmically with the force-loading rate (Fig. 4c), which is in accordance with the Bell model, indicating an almost linear decrease of the binding potential barrier with force. The dissociation rate constants and average bond lifetimes at zero force were again derived using the Bell model (Eq. 2) and are in the same range as for the RrgA Fl - Fn interaction. For D3 on Fn we obtain a dissociation rate constant at zero force koff of 6.72 s-1 which corresponds to a bond lifetime at zero force of τ = 0.15 s and a Δx of 0.19 nm. The average bond lifetime at zero force is thus slightly smaller than for RrgA Fl on Fn (τ = 0.26 s). With koff = 2.33 s-1 which corresponds to τ = 0.43 s the D4 domain exhibits a somewhat longer average bond lifetime as RrgA Fl and a Δx of 0.32 nm. Both D3 and D4, show fast detachment from Fn, which is characteristic for transient binding. Note that with τ = 0.29 s and


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∆x = 0.23 nm the average bond lifetime and bond length of the two domains D3 and D4 is close to the values observed for RrgA Fl (τ = 0.26 s and a Δx of 0.25 nm).

Figure 3. RrgA Fl and single domain interactions with fibronectin. AFM-tips coated with D3 and D4 adhere to Fn substrates with frequencies of 19.7 % and 16.5 %, respectively, which is in the same range as for RrgA Fl – Fn interaction (19.2 %). Adherence to BSA coated control surfaces was significantly reduced (4.9 % for D3 and 2.7 % for D4). D1 and D2 show adhesion rates of only 3.8 % and 4.1 %, respectively, which is well below the value determined for RrgA Fl and only slightly above the 2.4 % and 2.9 % determined on the respective BSA controls (error bars originate from n = 3 independent experiments with > 1000 analyzed force curves for each protein pair).


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Figure 4. Dynamic force spectroscopy of the RrgA D3 and D4 – fibronectin (Fn) interaction. (a, b) D3 or D4 domains were covalently attached to the AFM tip, FN was coated on glass. Rupture force and corresponding rupture length histograms derived from n = 1200 (D3 - Fn) or n = 1000 (D4 - Fn) force-distant curves are illustrated (left). The Gauss fits (black line in the rupture force histograms) render a most probable force fMP of 52.8 pN for D3 – Fn and of 46.2 pN for D4 – Fn. Rupture length histograms (middle), and five representative force curves (right), together with an extensible worm-like chain fit (red) are shown. Retraction speed: 1000 nm s-1. (c) RrgA D3 – Fn (blue) and RrgA D4 – Fn (red) rupture forces, obtained by varying the retraction velocity from 250 to 4000 nm s-1. Dots represent data gained from single rupture events, while the squares show the most probable rupture force for five (D3 - Fn) and six (D4 - Fn) distinct force-loading rate intervals between 50 and 30.000 pN s-1 and were 13 ACS Paragon Plus Environment

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determined by fitting Gaussian distributions to the rupture force histograms within these intervals. The blue line represents a Bell fit to the most probable forces of D3 - Fn, the red line to the most probable forces of D4 - Fn.

Simultaneous Binding of D3 and D4 to Fibronectin Finally, we investigated whether RrgAs´ terminal domains D3 and D4 can bind to Fn simultaneously, which could explain the double peaks observed in the force-distance curves of the RrgA Fl - Fn interaction (Fig. 2c). We blocked the binding sites for D3 or D4 on Fn by applying either free D4 or D3 molecules on the Fn substrate prior to the SMFS experiments with RrgA Fl attached to the AFM tip (Fig. 5a). The frequency of RrgA Fl adhesion events on the Fn substrate was reduced by approximately 50 %, from 19 % to ~12 % and ~9 % upon blocking Fn with D3 and D4, respectively (total number of analyzed force curves, n (Fn + D4) = 1400, n (Fn + D3) = 1300). These results clearly show that the two domains D3 and D4 bind to distinct binding sites along the Fn molecule and that the two sites are far enough apart, in order to permit binding to one site, if the other site is blocked by a bound domain.

The force curves showing double rupture events were reduced from ~7 % with RrgA Fl to ~3 % for Fn treated with D3 and to 2 % for Fn treated with D4, which indicates that the double ruptures observed in Figure 2c may indeed be caused by a stepwise detachment of the two domains from Fn. To further corroborate this assumption, we determined the length increment Δl between the two peaks for all force curves with double rupture events (see Fig. 5b – black arrows), and plotted the distribution in Fig. 5c. For RrgA Fl on Fn, there is a distinct peak in the Δl distribution around 15 – 20 nm (Fig. 5c, top), which is significantly reduced when blocking Fn with D3 or D4 and which disappears completely for single domain (D3 and D4) interactions (Fig. 5c, center and bottom). The observed Δl of 15 – 20 nm correlates rather well 14 ACS Paragon Plus Environment

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with the distance between domain D3 and D4 on native RrgA Fl of about 18 nm to 19 nm 13, 16 (see also Fig. 1a). This corroborates our assumption that the two terminal domains D3 and D4 can bind to Fn simultaneously, giving rise to the double ruptures observed in the forcedistancecurves of RrgA Fl on Fn (Fig. 2c and Fig. 5b). Note that binding of RrgA Fl to multiple Fn molecules, as it has been reported for S. aureus FnBPs 42, 59 is extremely unlikely in our study, because individual Fn molecules are typically separated by several hundred nm, as can be seen in Fig. 2b. However, in the case of higher Fn densities, multi-Fn binding cannot be ruled out. Nevertheless, unlike staphylococcal FnBPs, which have up to 11 largely unstructured Fn binding regions that can bind up to 6 Fn molecules via tandem -zipper repeats, RrgA comprises only four highly structured domains, making a similar binding mechanism as in staphylococcal FnBPs unlikely. Additional control experiments where Fn was linked to OPSS-PEG coated substrates via thiol groups, which leaves the N-terminal site of Fn accessible 63, 75 showed the same double ruptures with identical Δl (see supporting information Fig. S5), indicating that the bindings sites could be located near the Nterminal end of Fn, as for staphylococcal FnBPs. This however remains speculative, and the exact locations of the D3 and D4 binding sites on Fn have to be identified in future studies with e.g. defined Fn fragments. Unlike for other bacterial adhesins showing multi-domain binding to Fn, where the interaction strength increases with the number of interactions, 42, 44, 48, 76 in the case of the RrgA Fl - Fn interaction, adhesion force and bond lifetime seem to be unaffected by multidomain binding: In the SMFS experiments, the two domains unbind sequentially and independently and not in a concerted and cooperative manner. As already pointed out in the last section, the bond lifetime (τ) and bond length (Δx) of RrgA Fl seem to be average values of the individual domains D3 and D4 rather than the sum of both, as it would be the expected if both domains would act in concert and unbind in a cooperative manner. The independent binding of the domains D3 and D4 may increase the likelihood of binding to Fn and their simultaneous 15 ACS Paragon Plus Environment

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binding may extend the overall interaction time by enhancing the chance of rebinding of one domain while the other one remains attached. However, as rebinding effects are not readily accessible via SMFS, this assumption has to be validated in future experiments, e.g. with surface plasmon resonance spectroscopy or a quartz crystal microbalance setup.

Figure 5. Simultaneous binding of RrgA domains D3 and D4 to Fn (a) Frequency of RrgA Fl adhesion events on Fn was reduced from 19 % to ~ 12 % and ~ 9 % upon blocking Fn with 16 ACS Paragon Plus Environment

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D3 and D4, respectively. Force curves displaying double rupture peaks were mainly observed for the RrgA Fl - Fn interaction (7 %) and nearly disappeared after the saturation of binding sides on Fn with D4 (2 %) or D3 (3 %). Force curves with two rupture events rarely appeared in D3 - Fn and D4 - Fn adhesion (4 % each). Number of analyzed force curves: RrgA Fl – Fn, n = 1400;

D3 - Fn,

n = 1200;

D4 - Fn,

n = 1000;

RrgA Fl - (Fn + D4),

n = 1400;

RrgA Fl - (Fn + D3), n = 1300. (b) Representative force-distance curves with two rupture events obtained from RrgA Fl - Fn interaction. Arrowheads indicate the rupture peaks. (c) Distribution of the length increment Δl between the two rupture peaks. For RrgA Fl - Fn, Δl frequency is highest for distances from 15 – 20 nm. This accumulation is significantly reduced by blocking Fn with D3 or D4 (middle) and disappears in single domain (D3 or D4) - Fn interactions (bottom). Conclusion In the present study, we have shown that the pilus-1 tip protein RrgA of Streptococcus pneumoniae TIGR4 as well as its two terminal domains D3 and D4 bind to Fn. D3 and D4 bind independently to distinct binding sites on the Fn molecule. RrgA Fl binds to Fn either through one single domain or with two domains simultaneously, constituting a two domain binding mode and giving rise to double rupture events in the respective force-distancetraces. Average bond lifetimes in the absence of force are in the order of a few hundred milliseconds, and both adhesion force and bond lifetime are unaffected by dual-domain binding and unbinding, which seems to be a sequential rather than a cooperative process. The fast dissociation rate of the RrgA Fl - Fn bond enables the bacterium to rapidly detach and bind new receptor sites. We therefore assume that the pilus-1 tip protein RrgA, which protrudes from the protective capsule, enables the pneumococci to establish and maintain an initial, but transient adhesion, allowing the bacterium to remain near the host tissue surface, e.g. in shear force environments, and scan for additional and more specific binding sites promoting host infection (Fig. 6). Complementary 17 ACS Paragon Plus Environment

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studies on cellular level might provide additional information of the role of pilus-1 mediated pathogen-host interaction and may thereby help to devise future strategies to control and possibly inhibit bacterial adhesion to host cells and tissue and thereby prevent pneumococcal invasion.

Figure 6. Putative mechanism of S. pneumoniae - host adhesion mediated by pilus-1 RrgA - Fn binding. Exposed pilus-1 adhesin RrgA enables the pneumococci to establish and maintain an initial, but transient contact to fibronectin with short bond lifetimes (1). This allows the bacterium to remain near the host tissue surface, e.g. in shear force environments (2), and scan for specific host receptors, providing a more intimate attachment to the host cells and thereby promoting infection (3). The proposed scheme provides a mechanistic picture of initial pilus-mediated host interaction, based on a model proposed by Telford, JL., et al. (Nat Rev Microbiol. 2006), which is extended here by the ability of full length RrgA to bind to Fn either through single D3 or D4 domains or both domains simultaneously in a dual domain binding mode.


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Material and Methods Protein expression and purification For heterologous expression, genes encoding full length RrgA (clade I, locus tag: SP RS02280, PDB: 2WW8) and individual RrgA domains (D1, D2, D3 and D4; using a structurebased approach) (supporting information Fig. S2) were cloned into pETDuet™-1 expression vectors and transformed in chemically competent Escherichia coli (BL21-CodonPlus DE3RIL) applying standard protocols with 5 ng DNA and 100 µg ml-1 Ampicillin (Fluka) as respective selection marker. Protein expression was performed as described before 23 with minor modifications: Individual E. coli strains were cultivated in LB medium (37 °C) supplemented with 100 μg ml-1 ampicillin. Protein expression was induced at an optical density OD600 of around 1.0 by adding a final concentration of 0.5 mM IPTG (isopropyl-β-dthiogalactopyranoside, Amresco). To avoid inclusion body formation growth temperature during ON-induction was reduced (25 °C and 15 °C, for RrgA Fl and RrgA domains, respectively). Cells were harvested by centrifugation and the pellets were resuspended in 50 mM Tris HCl, 150 mM NaCl, pH 8.0 at 4 °C. Ultrasonic cell disruption (Bandelin Sonoplus HD 2070) was performed on ice applying an amplitude of 70 % and an interval of 0.5 s. for 2 x 10 min. Following another centrifugation step, the supernatant was sterile-filtrated and subjected to a metal chelate affinity chromatography (HisTrap HP 1 ml columns; Äkta avant 25 System, GE-Healthcare) exploiting N-Terminal 6 x His TAG sequences of respective RrgA constructs (supporting information Fig. S1). Polishing of protein containing fractions was performed using a Size-Exclusion-Chromatography step (Superdex 75 10/300 GL or Superdex 200 10/300 GL column for RrgA domains or RrgA Fl respectively with Äkta avant 25 System, GE-Healthcare). Purity of final protein samples was determined by standard SDS PAGE (13.5 % Acrylamide and MOPS Buffer, Sigma-Aldrich, RAPIDstainTM, G-Biosciences, supporting information Fig. S2). Respective protein concentration was measured at 280 nm (NanoDropTM One, Thermo ScientificTM). In addition to observed correct molecular weights of 19 ACS Paragon Plus Environment

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individual RrgA constructs (supporting information Fig. S2), western blot analysis using polyclonal anti-RrgA antibodies confirmed identity of the purified protein samples (data not shown). Proper protein folding was analyzed by Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR, Tensor 27, CONFOCHECK, Bio-ATR IITM, Bruker Optics, supporting information Fig. S3). Surface immobilization strategies Glass microscope slides and silicon nitride AFM cantilevers (MLCT, Bruker AXS S.A.S.) were cleaned and functionalized as described before. 52, 65, 77 Briefly, the AFM tips were irradiated with UV light (UV PenRay UVP, LLC, Upland, CA, USA; Mercury spectrum with the primary energy at 254 nm) for 90 min, and subsequently rinsed with ethanol (p.A., Carl Roth). Glass slides were cleaned in a 10 % solution of hydrochloric acid (Carl Roth) in doubly distilled in an ultrasonic bath for 90 min, followed by three times cleaning in purified water in the ultrasonic bath for 10 min. For surface silanization both, slides and cantilevers, were incubated for 90 min in a solution of 0.1 mg ml-1 COOH-PEG-Si(OC2H5)3 (MW 5000, Nanocs) dissolved in ethanol and doubly distilled water (w/w 95 %/5 %, pH 4.6). After rinsing the slides and cantilevers with ethanol, they were cured at 110 °C for 30 min and stored in a vacuum chamber for up to one week. Previously to AFM experiments the carboxyl group of the functionalized surfaces and cantilever tips were activated by incubating them in a solution of 42 mg ml-1 of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, Sigma-Aldrich), and 20 mg ml-1 of N-hydroxysuccinimide (NHS, Sigma-Aldrich) in standard phosphate buffered saline (PBS; 137 mM NaCl, 2,7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4).65, 77 Afterwards the slides and AFM tips were thoroughly rinsed with PBS. The activated AFM tips were incubated in a droplet of either 3 µM RrgA Fl, D1, D2, D3 or D4 for two hours. The surfaces of the glass slides were exposed to 0.6 mg ml-1 human fibronectin (Fn, human plasma, Sigma-Aldrich), 0.6 mg ml-1 bovine serum albumin (BSA, fraction V, PAA) or PBS containing no protein for 2 hours, respectively. All proteins were dissolved in PBS and the formation of a 20 ACS Paragon Plus Environment

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covalent bound between the EDC/NHS activated COOH group of the PEG molecule and the accessible NH2 groups on the protein surfaces took place at room temperature. Subsequently, the slides and AFM tips were rinsed thoroughly with PBS and unbound NHS groups were saturated by placing the probes in Tris-buffered saline (TBS; 50 mM Tris, 150 mM NaCl, pH 7.6) for 20 min. Surfaces for blocking experiments were subject to an additional incubation step with 3 µM RrgA Fl, D3, or D4 in PBS for 30 min at room temperature, followed by a PBS washing step. The AFM cantilever and the glass slides were stored in PBS and used the same day. Single molecule force measurements Single molecule force spectroscopy experiments were performed in PBS pH 7.4 at room temperature with a NanoWizard® I atomic force microscope (JPK Instruments) and silicon nitride AFM cantilevers (MLCT, Bruker) with a nominal force constant of 0.03 N m-1. The optical lever sensitivity and spring constant of the cantilever were determined by the thermal noise method.78 Force-distance curves were obtained with the Force RampDesignerTM applying a contact force of 250 pN, a contact time of 1 s and a retraction velocity of 1000 nm s-1. Experiments for every protein combination were carried out at least in triplicate and the force curves were obtained at three different positions on the substrates. For experiments with increasing force loads the retraction speed was varied between 250 and 4000 nm s-1. Force extension traces were analyzed with JPK data processing (JPK Instruments) and screened for force peaks occurring at rupture lengths above 70 nm, in order to sort out nonspecific interactions. In the case of multiple adhesion peaks, only the last event was considered. Force curves were analyzed using the extensible worm-like-chain model 61 to obtain rupture lengths and force loading rates for the respective protein combination. The kinetic parameters were determined using the Bell Evans model (see supporting information Fig. S4).66 Diagrams were illustrated and data fitted using Microsoft Excel 2016 and Wavemetrics IGOR Pro 6.3.


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Acknowledgements TB, SS and HCS acknowledge financial support through the research focus “Herstellung und biophysikalische Charakterisierung dreidimensionaler Gewebe – CANTER” of the Bavarian State Ministry for Science and Education, and Conny Hasselberg-Christoph and Martina Hörig for technical support.

Supporting Information Available Supporting information provides three figures which illustrate RrgA Fl and the constructs used for heterologous protein expression and respective characterization of the purified proteins including the secondary structure analysis. In addition, a detailed description and illustration of dynamic force spectroscopy data analysis and results of control experiments with Fn immobilized via its thiol groups are shown. This material is available free of charge via the Internet at http://pubs.acs.org.

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