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Adhesin Contribution to Nanomechanical Properties of the Virulent Bordetella pertussis Envelope L. Arnal,† D. O. Serra,†,⊥ N. Cattelan,† M. F. Castez,‡ L. Vázquez,§ R. C. Salvarezza,‡ O. M. Yantorno,*,† and M. E. Vela*,‡ †

Facultad de Ciencias Exactas, Centro de Investigación y Desarrollo de Fermentaciones Industriales (CINDEFI-CONICET-CCT La Plata), UNLP. 50 No. 227, 1900 La Plata, Argentina ‡ Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Universidad Nacional de La Plata − CONICET, Sucursal 4 Casilla de Correo 16; 1900 La Plata, Argentina § Instituto de Ciencia de Materiales de Madrid (CSIC), C\Sor Juana Inés de la Cruz 3, 28049 Madrid, Spain S Supporting Information *

ABSTRACT: Adherence to a biological surface allows bacteria to colonize and persist within the host and represents an essential first step in the pathogenesis of most bacterial diseases. Consequently, the physicochemical properties of the outer membrane in bacteria play a key role for attachment to surfaces and therefore for biofilm formation. Bordetella pertussis is a Gram-negative bacterium that colonizes the respiratory tract of humans, producing whooping cough or pertussis, a highly infectious disease. B. pertussis uses various adhesins exposed on its surface to promote cell-surface and cell−cell interactions. The most dominant adhesin function is displayed by filamentous hemagglutinin (FHA). B. pertussis Tohama I wild-type (Vir+) strain and two defective mutants, an avirulent (Vir−) and a FHA-deficient (FHA−) B. pertussis strains were studied by AFM under physiological conditions to evaluate how the presence or absence of adhesins affects the mechanical properties of the B. pertussis cell surface. Quantitative information on the nanomechanical properties of the bacterial envelope was obtained by AFM force-volume analysis. These studies suggested that the presence of virulence factors is correlated with an increase in the average membrane rigidity, which is largely influenced by the presence of FHA. Moreover, for this system we built a nanoscale stiffness map that reveals an inhomogeneous spatial distribution of Young modulus as well as the presence of rigid nanodomains on the cell surface.

1. INTRODUCTION Bacterial adhesion to a surface has been described as the balance of attractive and repulsive physicochemical interactions between bacteria and the target substrate. The adhesive nature of bacteria is due to various outer membrane components such as pili, flagella, proteins, and lipopolysaccharides. Adherence to a biological surface and the subsequent cell growth on it allows bacteria to colonize and persist within the host and represents an essential first step in the pathogenesis of many bacterial diseases. Consequently, the physicochemical properties of the outer membrane in bacteria play a key role for attachment to surfaces and therefore for biofilm formation. Changes in the composition of the outermost layer of a bacterium may thus strongly influence its interactions with other bacteria and with target host tissues or abiotic surfaces. Despite technical advances in the study of physicochemical properties of bacteria, still little is known about these properties at the single cell level. In recent years, atomic force microscopy (AFM) imaging and force spectroscopy have emerged as powerful tools for highresolution imaging with nanometer resolution under physio© 2012 American Chemical Society

logical conditions to address key questions in molecular microbiology that could not be answered by other techniques.1−5 In force spectroscopy, force−distance curves can be recorded either at single well-defined locations of the (x, y) plane or at multiple locations to yield a so-called “force-volume image”. In that way, spatially resolved maps of sample properties and molecular interactions can be obtained.6−8 In fact, AFM has been used to image subcellular structures, to measure ligand−receptor interaction forces, and to extract physicochemical information of macromolecules such as proteins and nucleic acids.1,3,9−12 In bacteria, AFM has been used to determine the elasticity of different Gram-negative and Gram-positive bacteria as well as dormant and germinative spores.13−17 All these features make AFM an excellent tool to explore the physicochemical heterogeneity of the external surface of bacterial cells with unprecedented resolution. Received: February 24, 2012 Revised: April 15, 2012 Published: April 19, 2012 7461

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(Sigma) were added, respectively, to the culture media. Liquid cultures were performed by inoculating bacteria into 100 mL Erlenmeyer flasks containing 30 mL of Stainer-Scholte (SS) broth, adjusting the optical density at 650 nm (OD650) to 0.15, and incubating the flasks at 37 °C under shaking conditions (160 rpm). Cells of the three strains were harvested at middle-exponential phase (centrifugation at 6000g for 5 min) and washed twice with phosphate-buffered saline (PBS) buffer. 2.2. AFM Sample Preparation. Bacteria were electrostatically immobilized onto polyethyleneimine (PEI)-precoated glass slides (Sigma) following previous procedures.38,39 Briefly, glass slides were washed twice with 96% (v/v) ethanol and Milli-Q water and then incubated overnight at 4 °C with a 0.1% (w/v) aqueous PEI solution. After incubation, PEI-coated slides were rinsed twice with Milli-Q water. Bacteria were immobilized by depositing 50 μL of a bacterial suspension at an OD650 of 1.0 over the PEI-coated slides. Bacteria were allowed to attach to the substrate for 30 min. Afterward, the slides were washed twice with water to remove nonadhered cells before AFM imaging. 2.3. AFM Imaging and Force Volume Measurements. The characterization of nanomechanical properties of B. pertussis cells was performed through a force−volume image analysis (FV) using a MultiMode Scanning Probe Microscope (Veeco, Santa Barbara, CA) equipped with a Nanoscope V controller. Briefly, each glass slide containing the immobilized bacteria was attached to a steel sample puck (Veeco, Santa Barbara, CA) using a small piece of adhesive tape and immediately transferred into the AFM liquid chamber. Fifty microliters of PBS buffer was added to the AFM fluid cell on each glass slide to keep the bacteria hydrated during the course of the experiment. All the measurements were performed using contactsharpened silicon nitride probes (NP-10, Veeco) with a tip radius of 20−60 nm. The spring constants of the cantilevers (Ks) were measured using the thermal tune method, and its characteristic values were between 0.12 and 0.35 N/m. Clean, flat muscovite mica surfaces (SPI V-1 grade) were used as rigid substrates for photodetector sensitivity calibration. Images of bacteria cells were recorded in contact mode at the minimum applied force compatible with stable imaging conditions, and the arrays of 32 × 32 force curves were acquired using the force volume routine at a scan rate of 1 Hz. Note that different scan rates within the range 0.1−2 Hz produced similar results. For force volume imaging (FV), a limit for the highest applicable force load of 15 nN was set to preserve both the tip and the bacterium status. 2.4. Analysis of Force Curves. Determination of Young's Modulus (E) and the Bacterial Spring Constant (Kb). The nanoindentation analysis of the force curves was performed using inhouse developed software, following the procedure described in previous works.40−42 The zero-force height (z0; y0) value of each force curve was manually defined by detecting the point at which the curve begins to lift off from the noncontact baseline.8 All curves showed a nonlinear behavior at low loading forces, which described the mechanical behavior of the cell envelope, and a linear behavior at higher loading forces, which was related to turgor pressure of the cells.33 Two different mechanical properties, Young’s modulus (E) and bacterial spring constant (Kb), were determined from each section of the curve.42,43 To determine the bacterial Young’s modulus, the force versus Zdisplacement curves were transformed into force (F) versus indentation depth (δ) curves by calculating the difference at constant loading force between the respective Z displacement measured on bacterial surface and the Z-displacement measured on a hard surface, mica (see Figure 2). Young's modulus was determined by fitting the nonlinear portion of the resulting curves with a proper Hertz model that considers the geometry of the tip. The tip shape can be generally modeled by two geometries, a conical or a paraboloid indenter.41 In our experiments, the best fit for the F vs δ curves was for a conical shape. The Hertz model used follows the equation:15,44

Bordetella pertussis is a Gram-negative bacterium that colonizes the respiratory tract of humans producing whooping cough or pertussis, a highly infectious disease that remains endemic despite widespread and highly sustained vaccination coverage.18,19 B. pertussis continues to be a serious public health problem worldwide. Recent studies strengthen the hypothesis that B. pertussis may utilize biofilms as a means to persist and circulate between the human host.20−23 A biofilm is a community-based mode of growth of microorganisms which critically depends on their ability to establish bacteria−substrate and bacteria−bacteria interactions. B. pertussis uses various adhesin factors exposed on its surface to promote these interactions. Among these factors, the most dominant adhesin function is displayed by the filamentous hemagglutinin (FHA), a 220 KDa surface-associated protein, which is a critical virulence factor for initiation of infection.24−26 This protein is also released to the environment during the growth of virulent bacteria.27 In a wild-type virulent phase (Vir+), B. pertussis expresses FHA together with other characteristic surface-exposed adhesins (fimbriae, pertactin) and toxins.28 The expression of virulence factors such as FHA is controlled by the BvgAS signal transduction system through a highly regulated program of gene expression in response to environmental stimuli.29,30 When the BvgAS system is inactive, B. pertussis does not express these surface adhesins and toxins, and thus cells are in an avirulent phase (Vir−). We and others recently showed that the avirulent phase of B. pertussis is unable to form biofilms and that the single loss of FHA (FHA−) renders the bacterium deficient in biofilm formation.25,26,31,32 Thus, while previous studies have shown that the absence of FHA or of all BvgAS-regulated surface adhesins and toxins affects the ability of B. pertussis cells to form biofilm, nothing is known about how these virulence factors affect B. pertussis physical surface properties at the single cell level. It has been reported that the presence of surface appendages strongly impact nanomechanical properties of bacteria33 and that their expression changes during adhesion and biofilm development.34,35 In this work, quantitative information on B. pertussis elasticity (e.g., Young’s modulus (E)) was obtained from the force (F) vs indentation (δ) curves. B. pertussis Tohama I wild-type strain and two derivative defective mutants, an avirulent (Vir−) and a FHA-deficient (FHA−) strain, were studied to evaluate how the presence or absence of adhesins affects the mechanical properties of the B. pertussis cell surface. Interestingly, our studies demonstrated that the expression of virulence factors, particularly the surface-associated protein FHA, correlated with an increase in the membrane rigidity. Moreover, from force− distance curves we built a nanoscale stiffness map that reveals an inhomogeneous spatial distribution of Young's modulus as well as the presence of rigid nanodomains on the cell surface.

2. MATERIALS AND METHODS 2.1. Bacterial Strains and Growth Conditions. B. pertussis Tohama I, a wild-type (Vir+) strain (8132 collection of Pasteur Institute), B. pertussis 537, a Tohama I derivative avirulent Bvg− phase locked strain (Vir−),36 and B. pertussis GR4, a Tohama I derivative mutant lacking the expression of FHA (FHA−)37 were used throughout this study. Stock cultures were grown on Bordet-Gengou agar (BGA; Difco Laboratories, Detroit, MI) plates supplemented with 1% w/v Bactopeptone (Difco) and 15% v/v defibrinated sheep blood (Instituto Biológico, La Plata, Argentina) at 37 °C for 72 h and then subcultured for 48 h. In the case of the Vir− and FHA− strains, 40 μg mL−1 kanamicin (Sigma, St. Louis, MO) and 50 μg mL−1 streptomycin

F= 7462

2E tan α 2 δ π(1 − ν 2)

(1) dx.doi.org/10.1021/la300811m | Langmuir 2012, 28, 7461−7469

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Figure 1. 40 × 40 μm2 AFM images of Vir− (a), FHA− (b), and wild type Vir+ cells (c) on PEI-modified glass in buffer PBS. (d) 0.8 × 0.8 μm2 force volume image of a single Vir+ bacterium. with F being the loading force, α the half opening angle of the conical indenter (53°; based in geometrical characteristics of the tip and SEM observations), E the Young’s modulus, and ν the Poisson's ratio (assumed to be 0.5 for cells).45,46 The bacterial spring constant (Kb) was determined from the slope, s, of the linear portion of each F vs Z-displacement curve according to the following equation:43

Kb = K s

s 1−s

we present two maps of the bacterial spring constant in relation to the corresponding E values of the same zone. 2.6. Statistical Analysis. Average values of Young’s modulus and of the bacterial spring constant were calculated for each independent culture of each bacterial strain. These average values were compared using a one-way ANOVA factor test. The same test was used to determined differences in the length of bacteria. When significant differences were observed, a posthoc Turkey test was performed to compare data pairs.

(2)

3. RESULTS AND DISCUSSION

where Ks is the cantilever’s spring constant. As a first step, we have made single force curves on 60 different cells of each strain that were chosen at random. For the single cell analysis, six cells from three independent cultures for the three strains were examined. Between 100 and 200 curves were recorded for each single cell. All curves were used to calculate E and Kb. In each case, the distribution of Young’s modulus was presented as histograms. To characterize the statistical behavior of Young’s modulus and taking into account that the total number of events was not equal for each individual bacterium, they were normalized by dividing them by the number of events corresponding to the most probable value of the histogram. 2.5. Cell Surface Mapping. Young’s modulus values were correlated with their X-Y coordinates in a spatial region of the force−volume image of the bacterial cell and were plotted as a surface elasticity map. Values between 0.0 and 0.5 MPa were presented in the map in a cyan-blue scale, whereas values between 0.5 and 1 MPa were presented in a pink-red scale. In Supporting Information (Figure S2),

3.1. Bacterial Cell Images of Wild-Type and Defective Mutant Strains of B. pertussis. AFM allows imaging in liquid media, which constitutes one of the main advantages over conventional structural research techniques. AFM images of Vir−, FHA−, and B. pertussis Vir+ cells attached to the substrate surface are presented in Figure 1a−c. The images show small aggregates and single cells spread out over the surface. All bacteria showed an expected cocobacillar shape, consistent with typical topology of B. pertussis. No significant differences were observed in the cell length for the three strains analyzed (average value 0.9 ± 0.2 μm). Neither was it possible to distinguish any surface appendage or fine structure differences among them in the topographic images, as expected when imaging in contact-mode in liquids, due to the interactions existing between the sample and the probe.47 7463


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displacement curves (Figure 3). We obtained an average value of 55 ± 23 nm for wild-type Vir+ strain whereas 120 ± 19 nm was found for FHA− and 137 ± 29 nm for Vir− strains. The significant increase in the indentation depth of the last two strains could be correlated with their lower protein densities in the outer membrane. Therefore, in the external envelope of the wild-type Vir+ strain, the AFM tip interacts with a larger number of proteins, having more rigid constituents10,48−51 which lead to a shallower indentation of the AFM probe than that observed in the other two strains. 3.3. Spring Constant, Young Modulus Determination, and Cellular Elasticity Map. The nanomechanical properties of the cellular envelope for the different strains employed in this study were explored by single force curves performed on 60 single cells of each strain. Vir+ showed a significantly higher average value (0.44 ± 0.23 MPa) than FHA− (0.18 ± 0.09 MPa) and Vir− (0.11 ± 0.06 MPa) strains (Turkey, p < 0.05) (Figure 3a). Moreover, the higher standard deviation for E values in Vir+ would reflect either the heterogeneity of the elasticity of individual cells or the heterogeneity of E values in different locations on the same cell surface. To elucidate the nature of this behavior at the individual cell level, we performed force volume imaging on six individual cells obtained from three independent cultures for each strain. Curves from the FV were recorded, converted into force vs indentation depth curves, and analyzed according to eqs 1 and 2. From the linear regime we determined bacterial spring constants which did not show significant differences between the three strains (ANOVA, p > 0.05). The average values ranged between 0.044 and 0.058 N/m, which were in agreement with those previously reported for other Gramnegative bacteria such as Escherichia coli (Kb = 0.04 N/m);44 Shewanella putrefaciens (Kb = 0.05 N/m)15 among others.33,52

Once individual cell images had been achieved, force volumes were performed in the same area as shown in Figure 1d. 3.2. Force−Distance Curves Analysis. To evaluate nanomechanical properties of B. pertussis cells, force−distance curves were recorded. Figure 2 shows representative force vs Z-

Figure 2. Force vs Z-piezo displacement measured on mica (black line), and representative curves for different bacterial strains: wild type Vir+ (blue), FHA− (magenta) and Vir− (brown). The tip indentation depth (δ) can be calculated from the difference in the piezo displacement at constant loading force for a deformable surface referring to a hard surface, mica in this system.

piezo displacement curves for the Vir+ strain and FHA− and Vir− mutants. In all cases, the force curves exhibit two regimes: a nonlinear behavior within the first 100 nm and a linear one at higher loading forces. These two regimes can be correlated with the structure of the bacterial envelope. The thickness of the external structures contributing to the elastic response can be measured from the distance between the zero height point (Z0) and the crossover in the force vs Z-piezo

Figure 3. (a) Average E values obtained from single force curves of 60 different cells for each one of the three strains. Loading force vs indentation depth (δ) (blue spots) obtained for (b) wild-type Vir+, (c) FHA−, and (d) Vir−. Curves were fitted with the Hertz model (red) and with a linear model (black) in the region of high loading forces to determine bacterial spring constants. 7464


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Figure 4. Statistic distribution of Young's modulus for B. pertussis cells. (a) Vir− strain, (b) FHA− strain, (c, d, e) Vir+ strain. Histogram representatives of six (a and b), three (c), one (d), and two (e) analyzed cells.

The similarity of the Kb values for the three strains is likely due to the equally nonstressful osmotic conditions for all measurements. In contrast, Young's moduli values that were determined from the nonlinear regime of the curves using eq 1 as shown in Figure 3b−d exhibited significant differences (ANOVA, p < 0.05) between the three strains. One further step consisted of investigating the Young's modulus distributions for individual bacterium. Figure 4a,b shows representative histograms of E values measured from the FV analysis for Vir− and FHA− mutants that exhibit a monomodal distribution around a most probable E value of ≈0.1 MPa and ≈0.22 MPa, respectively. Note that both distributions were fitted using a logNormal mathematical function, previously used for describing other biological relevant properties.53 Also note that these values are consistent with the corresponding ones obtained in Figure 3a from single force curves randomly measured on 60 different cells. While for FHA− and Vir− strains the histograms were reproducible for bacteria recovered from both the same and independent cultures, the wild-type Vir+ strain showed different histograms for different cells in the same culture conditions. The histogram displayed in Figure 4c is representative of the analysis of three Vir+ cells. It was possible to distinguish two different distributions of E values, one around a low mean value (0.2 MPa), and a second at higher values (around 0.6 MPa). This bimodal behavior was not observed in any of the other two

strains. Histograms shown in Figures 4d and 4e correspond also to E distributions obtained from Vir+ strain surface. Although in these cases it was not possible to differentiate two E populations, they still displayed broader distributions (up to 1 MPa) compared to those calculated for mutant strains (up to 0.5 MPa) and, consequently, higher average E values. These results show a clear difference between the envelope elasticity of the Vir+ strain and Vir− and FHA− mutant strains, the first one being much more rigid than the other ones. A similar effect was reported by Park and Abu-Lail for Listeria monocytogenes.54 The heterogeneity among histograms of different wild-type (Vir +) cells could be explained considering that cells were harvested at the middle exponential phase, when the population growth rate is maximum and individual cells can transit different stages associated with the division process; therefore, the amount of surface proteins is not equal. On the other hand, this effect was not observed for the two mutants because they lack either the most numerous adhesin in the surface (FHA) or even more proteins, which makes the outer membrane of the bacterium more homogeneous despite the growth stage. For bimodal histograms, each distribution could be fitted using two independent log-normal functions, although for low E values the fitting is poor due to the few experimental values recorded in that range. There are two striking features regarding the statistical distribution of Young's modulus in our measurements: (i) 7465


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Figure 5. Force image of one B. pertussis Vir+ cell (a), its corresponding E histogram (b), and its surface elasticity map (c).The same analysis for a different B. pertussis Vir+ cell (d).

mainly dominated by E values higher than 0.5 MPa (pink-red squares). This behavior was not observed for E maps of Vir− and FHA− (Figure S1, Supporting Information). The bimodal spatial distribution observed for Vir+ leads us to propose that the spatial location of FHA is not random. It has been reported57 that in some Gram-negative bacterium, including B. pertussis, many autotransporters are expressed at the cell poles.57 Likewise, spatially resolved adhesion maps of individual adhesions on living bacteria have been created for heparin-binding hemagglutinin adhesin (HBHA) with heparinmodified tips revealing that the adhesion is localized in discrete regions of the cell surface58 The formation and propagation of cell-wall protein nanodomains were investigated by singlemolecule AFM.59 These results indicate that the adhesion function is coupled to the local assembly within adhesion nanodomains. It was also reported that the constituents of the S. salivarius cell surface would rearrange during adhesion until the bacterium is positioned in the energetically most favorable conformation. This interfacial rearrangement leads to an increasing number of contact points and enhances the adhesion strength between the bacterial cell and the target surface.60 Recently, for yeasts the induction of adhesins clusters into nanodomains after stimulation by extension forces using AFM modified with antibodies or through cell−cell interaction was reported.61 Taking all these precedents and our own results into account, we can infer that FHA could be preferentially

typical Young's modulus values in the range 0.2−0.3 MPa observed for the three strains, consistent with data published for other Gram-negative bacteria;15,42 (ii) high Young's modulus values (≈0.6−0.7 MPa) only observed for the wildtype (Vir+) strain. The principal differences between the surfaces of these strains refer to their outer membrane composition: the Vir+ strain presents different protein virulence factors, including FHA, fimbriae, pertactine, Vag8, and BrkA autotransporter.28,55,56 All these proteins are not expressed in the Vir− mutant strain. On the other hand, the surface of the FHA− mutant strain only lacks FHA adhesin. Because no significant differences between the E value distributions of the two mutants were found, these results hint that FHA is likely the component responsible for the increased rigidity of the Vir+ strain envelope. The FV capability allows us to build a map of the nanomechanical properties of single bacteria. For such purposes, for each spot in the topographic FV images, the corresponding force curve was localized and analyzed and its corresponding Young's modulus was calculated. Thus, a spatial map of the E values measured on the bacterium surface can be built as shown in Figure 5. Both maps (Figure 5b,d) clearly show that Young's moduli are not stochastically distributed in Vir+ cells. It is possible to distinguish two main areas on the bacterium surface: a first one predominantly characterized by E values lower than 0.5 MPa (blue squares), and a second one, 7466


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(2) Dupres, V.; Alsteens, D.; Andre, G.; Dufrêne, Y. F. Microbial nanoscopy: A closer look at microbial cell surfaces. Trends Microbiol. 2010, 18, 397−405. (3) Alsteens, D.; Dague, E.; Verbelen, C.; Andre, G.; Dupres, V.; Dufrêne, Y. F. Nanoscale imaging of microbial pathogens using atomic force microscopy. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2009, 1, 168−180. (4) Scheuring, S.; Dufrêne, Y. F. Atomic force microscopy: Probing the spatial organization, interactions and elasticity of microbial cell envelopes at molecular resolution. Mol. Microbiol. 2010, 75, 1327− 1336. (5) Hinterdorfer, P.; Dufrene, Y. F. Detection and localization of single molecular recognition events using atomic force microscopy. Nat. Methods 2006, 3, 347−355. (6) Dufrêne, Y.; Hinterdorfer, P. Recent progress in AFM molecular recognition studies. Pfluguers Arch. 2008, 456, 237−245. (7) Müller, D. J.; Dufrêne, Y. F. Force nanoscopy of living cells. Curr. Biol. 2011, 21, R212−R216. (8) Gaboriaud, F.; Dufrêne, Y. F. Atomic force microscopy of microbial cells: Application to nanomechanical properties, surface forces and molecular recognition forces. Colloids Surf., B 2007, 54, 10− 19. (9) Dufrêne, Y. F. Atomic force microscopy: A powerful molecular toolkit in nanoproteomics. Proteomics 2009, 9, 5400−5405. (10) Parra, A.; Casero, E.; Lorenzo, E.; Pariente, F.; Vázquez, L. Nanomechanical Properties of Globular Proteins: Lactate Oxidase. Langmuir 2007, 23, 2747−2754. (11) Ho, D.; Zimmermann, J. L.; Dehmelt, F. A.; Steinbach, U.; Erdmann, M.; Severin, P.; Falter, K.; Gaub, H. E. Force-Driven Separation of Short Double-Stranded DNA. Biophys. J. 2009, 97, 3158−3167. (12) Kurland, N. E.; Drira, Z.; Yadavalli, V. K. Measurement of nanomechanical properties of biomolecules using atomic force microscopy. Micron 2012, 43, 116−128. (13) Vadillo-Rodriguez, V.; Beveridge, T. J.; Dutcher, J. R. Surface Viscoelasticity of Individual Gram-Negative Bacterial Cells Measured Using Atomic Force Microscopy. J. Bacteriol. 2008, 190, 4225−4232. (14) Andreeva, N.; Bassi, D.; Cappa, F.; Cocconcelli, P. S.; Parmigiani, F.; Ferrini, G. Nanomechanical analysis of Clostridium tyrobutyricum spores. Micron 2010, 41, 945−952. (15) Gaboriaud, F.; Bailet, S.; Dague, E.; Jorand, F. Surface Structure and Nanomechanical Properties of Shewanella putrefaciens Bacteria at Two pH values (4 and 10) Determined by Atomic Force Microscopy. J. Bacteriol. 2005, 187, 3864−3868. (16) Vadillo-Rodriguez, V.; Schooling, S. R.; Dutcher, J. R. In Situ Characterization of Differences in the Viscoelastic Response of Individual Gram-Negative and Gram-Positive Bacterial Cells. J. Bacteriol. 2009, 191, 5518−5525. (17) Hoh, J. H.; Schoenenberger, C. A. Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy. J. Cell Sci. 1994, 107, 1105−1114. (18) Mooi, F. R.; van Loo, I. H. M.; King, A. Adaptation of Bordetella pertussis to Vaccination: A Cause for Its Reemergence? Emerg. Infect. Dis. 2001, 7, 526−528. (19) Hellenbrand, W.; Beier, D.; Jensen, E.; Littmann, M.; Meyer, C.; Oppermann, H.; Wirsing von Konig, C.-H.; Reiter, S. The epidemiology of pertussis in Germany: past and present. BMC Infect. Diseases. 2009, 9, 22. (20) Conover, M. S.; Sloan, G. P.; Love, C. F.; Sukumar, N.; Deora, R. The Bps polysaccharide of Bordetella pertussis promotes colonization and biofilm formation in the nose by functioning as an adhesin. Mol. Microbiol. 2010, 77, 1439−1455. (21) Sloan, G. P.; Love, C. F.; Sukumar, N.; Mishra, M.; Deora, R. The Bordetella Bps Polysaccharide Is Critical for Biofilm Development in the Mouse Respiratory Tract. J. Bacteriol. 2007, 189, 8270−8276. (22) Serra, D.; Bosch, A.; Russo, D.; Rodríguez, M.; Zorreguieta, Á .; Schmitt, J.; Naumann, D.; Yantorno, O. Continuous nondestructive monitoring of Bordetella pertussis biofilms by Fourier transform

located in nanodomains on the cell surface, generating adhesion clusters that represent a clear advantage for its in vivo function. FHA plays a key role during adhesion and colonization of epithelial cells of the respiratory tract and is crucial for the development not only of biofilms in vivo but also for intracellular invasion.25,26,62−64 The fact that they could be located in nanodomains instead of spreading over the surface could contribute to a highly efficient interaction with host tissues and consequently to infection. Nevertheless, to confirm the localization of FHA, AFM experiments with specifically functionalized tips should be performed.

4. CONCLUSIONS In this work a characterization at the nanoscale level of mechanical properties of the B. pertussis cell has been performed for the first time. From our force−distance curves, it is possible to conclude that B. pertussis Tohama I virulent (Vir +) strain displayed a more heterogeneous surface in terms of elasticity, showing a broader distribution of Young's modulus values and including more rigid domains than those exhibited by both of its avirulent counterparts, Vir− and a FHA− defective strain. The rigid domains (E > 0.5 MPa) found in Vir + strain could be related to the cell-surface-associated FHA appendages. Although the pathogenic role of FHA has been extensively studied, to our knowledge it has not been reported that the presence of FHA on the cell surface enhances the cell membrane rigidity. Forthcoming molecular recognition experiments should confirm the presence of FHA in the rigid nanodomains, a fact that could explain their role in a more efficient adhesion of B. pertussis to target cells.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(M.E.V.) E-mail: [email protected]; http://nano. quimica.unlp.edu.ar/; tel: +54 221 4257430; fax: +54 221 4254642. (O.M.Y.) E-mail: [email protected]; tel/ fax: +54 221 4833794. Present Address ⊥

Institut für Biologie − Mikrobiologie, FB Biologie, Chemie and Pharmazie Freie Universität Berlin; Königin-Luise-Strasse, 12-16, 14195 Berlin, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge support from ANPCyT (Argentina, PICT2010-2554, PICT-CNPQ 08-0019), CONICET (Argentina, PIP 0362), Comunidad Autónoma de Madrid (Project No. S2009/PPQ-1642, AVANSENS), and Ministerio de Economiá y Competitividad (Spain, FIS2009-12964-C05-04). L.A. and N.C. received fellowships from CONICET. M.E.V. is researcher from CIC, Bs. As., Argentina.

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REFERENCES

(1) Müller, D. J.; Dufrêne, Y. F. Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nat. Nano 2008, 3, 261−269. 7467


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