2.1.

Instrumentation

The optical tweezers system used is constructed around an inverted microscope (Olympus IX70) with a high numerical aperture oil-immersion objective (Zeiss CP-Achromat $100\times \mathrm{NA}=1.25$ ). An argon ion laser-pumped continuous-wave titanium-sapphire laser, operating at a wavelength of $810\mathrm{nm}$ (which is a wavelength where the bacteria exhibit low absorption of light¹⁶), was employed for optical trapping. The laser beam was directed into the microscope by an optical arrangement consisting of a number of lenses and mirrors, and focused by a high numerical aperture objective. A stepper-motor controlled gimbal mounted mirror allows steering of the beam so that its focus can be moved in the lateral $\left(x\text{-}y\right)$ direction in the focal plane without loss of trapping power. The focal point can also be moved along the depth (in the $z$ direction) by changing the divergence of the beam by means of translation of one of the lenses that forms an afocal lens system. A complete description of the optical system for controlling the laser beam that forms the optical trap as well as the modifications made to the microscope to realize the OT system is found in Refs. 17, 18.

2.2.

Force Measurement

The displacement of a small object (in this case a spherical bead with a diameter of $3.2\mu \mathrm{m}$ ) held in the optical trap constitutes a measure of the force that is exerted on the object. To monitor the displacement, a weak probe laser beam (HeNe) is conveyed into the microscope, using a similar optical arrangement as that of the trapping beam. The probe beam intensity was significantly lower than that of the trapping beam to minimize additional forces on the trapped bead. Furthermore, the probe beam is focused a short distance below the trapped bead, whereby the trapped bead acts as a lens and refracts the beam, together with the microscope condenser, in such a way that a distinct spot is produced in the far field. A position sensitive detector (PSD) is used to monitor the position of this spot, which is directly proportional to small lateral movements of the trapped bead. The position voltage signals are amplified, digitalized, and sampled by a data acquisition card in a computer. Figure 1 illustrates the optical trap and the position detection principle. Reference 18 presents a detailed description of the force measurement system.

Fig. 1

An illustration of the measurement setup is shown. A bacterium is mounted on a large polystyrene bead. The trapping beam, marked T, has a small Leb-coated polystyrene bead in its focus. The thinner of the laser beams, marked P, is the probe laser, which eventually marks a spot on the position sensitive device (PSD) revealing the position of the trapped bead.

2.3.

Force Calibration Procedures

To convert the voltage signals received from the PSD to a force, an accurate calibration of the entire force measuring system has to be performed. The calibration was performed in two steps, in which the first step relates the PSD signal to the position of the trapped bead. The second step relates a given position in the trap to the force the optical trap exerts on the bead. Both calibration parameters are determined before each measurement to achieve as accurate assessments as possible.¹⁸

The first calibration step is done by moving the optical trap (focal point) in the lateral plane by means of the stepper-motor controlled mirror in the optical arrangement. A $3.2\text{-}\mu \mathrm{m}$ bead is held by the optical trap during the movement, resulting in a shift of the position of the probe beam on the detector. The optical trap with the bead is moved in a stepwise manner a precise distance $(\pm 0.40\mu \mathrm{m})$ around the zero position to verify that the voltage-versus-position response is linear for bead displacements in this interval. Finally, the detector response factor V/nm) is determined by linear regression within the linear region of the voltage-versus-position curve.

To transform the position of the bead into the corresponding force, the second calibration step is needed. This calibration step is based on the Brownian motion (the random thermal movement) a small particle makes in a liquid. The bead is trapped by the OT, and the minute movements the bead makes when exposed to the combined effects of the forces of the trap and the thermal motion of the molecules in the liquid are recorded. This data, in combination with the well-known properties of Brownian motion in the absence of a trap, make it possible to deduce the required force constant of the trap in units of pN/nm through a power spectrum. The force constant of the optical trap in this work was $\sim 0.2\mathrm{pN}∕\mathrm{nm}$ .

The Brownian motion calibration procedure was, in turn, validated by means of the Stoke’s drag force technique.¹⁹ The resulting force constants obtained by the two calibration techniques were found to agree within a few percent.

3.1.

Bacterium

The H. pylori used in the measurements were the 17875Leb strain and the 17875babA1∷kanbabA2∷cam (denoted the BabA mutant), described in Ref. 15. Bacteria were grown for $40\text{to}45\mathrm{h}$ on Brucella agar medium supplemented with 10% bovine blood and 1% Iso Vitox (Becton Dickinson, Franklin Lakes, NJ) at $37°\mathrm{C}$ , under 10% $\mathrm{C}{\mathrm{O}}_2$ and 5% ${\mathrm{O}}_2$ .

3.2.

Large Beads

The large functionalized polystyrene beads with carboxyl surface groups (Interfacial Dynamics Corporation, Portland, Oregon) had a diameter of $9.6\mu \mathrm{m}$ and were immobilized on the coverslip and served as a wall onto which a single H. pylori bacterium was mounted. The large beads were attached to the coverslip surface by means of a heating procedure. This procedure was accomplished by keeping the beads suspended in PBS in an oven at $\sim 100°\mathrm{C}$ a few minutes until the liquid had evaporated and the beads were fixed to the glass. The large carboxyl beads were functionalized to allow attachment of bacteria. This was done by the use of two-step protocol.²⁰ In the first step, beads were washed once in $0.025\text{-}\mathrm{M}$ MES buffer, pH 6.0 by centrifugation at $2500\mathrm{rpm}$ for $15\min$ and resuspended to $20\text{-}\mathrm{mg}∕\mathrm{ml}$ solids. The second step was performed after the beads have been attached to the coverslip. A droplet $\left(25\mu \mathrm{l}\right)$ of the activation buffer composed of $1\mathrm{ml}$ ( $0.1\text{-}\mathrm{M}$ MES, $0.5\text{-}\mathrm{M}$ NaCl, pH 5.0), $0.6\text{-}\mathrm{mg}$ EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] and $1.2\text{-}\mathrm{mg}$ N-hydroxy-succinimide (NHS) was added to the coverslip and incubated at room temperature for $15\text{to}25\min$ . Next, the activation buffer was removed and $250\text{-}\mu \mathrm{l}$ MES buffer was added to maintain the low pH and prevent the coverslip from drying. The bacteria were attached to the beads immediately before preparing the sample for measuring. The measurements were performed in a PBS solution, which means that the pH was increased from 5 to 7.4 in the liquid surrounding the beads during preparation of the sample. The increased pH destabilized the reactive amine intermediate, and bacteria were allowed to bind covalently to the activated beads.

3.3.

Leb Beads

The small carboxyl polystyrene beads (Interfacial Dynamics Corporation), with a diameter of $3.2\mu \mathrm{m}$ , were coated with Leb human serum albumin (HSA) conjugate (IsoSep AB, Tullinge, Sweden). Carboxy-functionalized polystyrene beads ( $1\mathrm{mL}$ , $40\mathrm{mg}∕\mathrm{mL}$ , prod. Nr. 7-3000, $3.2\text{-}\mu \mathrm{m}$ , Interfacial Dynamics Corporation) were washed twice by suspending them in MES buffer ( $4\mathrm{mL}$ , $25\mathrm{mM}$ , pH 5.2), centrifuging, and decanting the mother liquor. The beads were then suspended in MES buffer $\left(2\mathrm{mL}\right)$ . Leb-HSA conjugate ( $0.25\mathrm{mg}$ in $94\text{-}\mu \mathrm{L}$ MES buffer, prod. Nr. 61/08, Isosept AB) and N-(3-dimethylaminopropyl)- ${\mathrm{N}}^{\prime }$ -ethylcarbodiimide ( $5\mathrm{mg}$ in $63\text{-}\mu \mathrm{L}$ MES buffer) was added to a portion of the bead suspension $\left(0.157\mathrm{mL}\right)$ . The mixture was shook overnight, then washed four times by centrifugation, decanted of mother liquor, and resuspended in PBS buffer (pH 7.2). Finally, the beads were suspended in PBS $\left(300\mu \mathrm{L}\right)$ and glycine $\left(0.3\mathrm{mg}\right)$ and $\mathrm{Na}{\mathrm{N}}_3$ $\left(0.3\mathrm{mg}\right)$ were added. The H-1 antigen is monofucosylated and defines the blood group O-phenotype. The difucosylated Lewis b antigen is formed by addition of a second fucose residue.

3.4.

Sample Synthesis

The sample mixture was prepared by applying a small portion of bacteria, suspended in a droplet of $25\text{-}\mu \mathrm{l}$ PBS, onto a coverslip with attached large beads. Next, $5\mu \mathrm{l}$ of PBS solution containing Leb beads was added. All PBS solutions contained 1% bovine serum albumin (BSA, Sigma, Saint Louis, Missouri) to minimize the influence of unspecific bindings between the Leb bead and the bacterial surface. The sample mixture was placed in a chamber consisting of two glass coverslips fixed to each side of an aluminum slide holder. The chamber was completely sealed to prevent evaporation during the experiment and to allow for the use of an oil immersion condenser. A piezo stage, mounted on the microscope table, held the sample chamber and allowed precise translation of the sample at a controlled speed relative to the microscope objective.

4.1.

Beads and Bacteria Mounting

A free-floating bacterium was trapped and mounted, through covalent binding, onto the large bead using the OT. Low laser power ( $\sim 100\mathrm{mW}$ measured at the entrance of the microscope objective) was used to minimize the effects of heating the bacterium by the light. A small Leb-coated polystyrene bead was trapped with increased laser power $(\sim 600\mathrm{mW})$ . The force calibration of the trap was then performed by the procedures described in Sec. 2(C).

4.2.

Finding a Start Position

Before an actual force measurement could be performed, it was necessary to locate a good starting position for the Leb bead relative to the bacterium. First, the bead was moved to a starting position in close proximity to the bacterium, but still at such a distance from the bacterial surface that no binding could take place. The coverslip, with the attached large bead and bacterium, was then moved away from the Leb bead with a speed of $0.5\mu \mathrm{m}∕\mathrm{s}$ . At this first measurement cycle, there was no force detected, since no bacterial attachment had been established. When the coverslip had moved $5\mu \mathrm{m}$ it was returned to a new starting position located $0.1\mu \mathrm{m}$ closer to the Leb bead than the previous starting position, whereby a new measurement cycle was performed. This iterative procedure continued until contact occurred and an adhesion force appeared.

4.3.

Measurement Method

A measurement cycle from a starting position that gave rise to bacterial adhesion is illustrated in Fig. 2 . The Leb bead was in this case first gently pressed onto the bacterium, whereby the bead was displaced slightly from the center of the optical trap [Fig. 2(a)]. The contact between the adhesins and the receptors allowed specific bindings to take place. After $5\mathrm{s}$ , the bacterium was retracted from the Leb bead in the trap by a movement of the coverslip with the large bead and the bacterium. The binding force pulled the Leb bead away from the center of the trap [Fig. 2(b)]. The force between the bacterium and the bead increased until the binding suddenly ruptured [Fig. 2(c)]. The force at rupture is referred to as the de-adhesion force. Repeated measurements from the same starting position were performed to investigate the repeatability of the binding.

Fig. 2

(a), (b), and (c) illustrate the measurement procedure in three chronological steps. (a) The bead is pressed onto the bacterium and adhesion occurs. (b) The ligand is bound to the receptors and forces the bead from the focus of the trapping beam as the distance between the trap and the bacteria increases. (c) The bead returns to the center of the trap as the force from the laser trap exceeds the maximum adhesion force that the receptor-ligand binding can support.

4.4.

Data Acquisition and Presentation

The entire measurement procedure was computer controlled by a custom-made LabView® program. Position data of the trapped Leb bead were collected with a sample rate of $1000\text{sample}∕\mathrm{s}$ while moving the coverslip. The position data was converted to forces by means of the calibration constants from the force calibration procedure. Although the movement of the coverslip was the entity that was externally controlled in the experiments, it is most often the distance between the bacterium and the Leb bead that is the entity of primary interest. These two entities differ by an amount given by the displacement of the Leb bead from the center of the trap. The software allows the output force data to be displayed either versus time or versus the bacterium-to-bead distance. For analysis purposes, the latter presentation form is more convenient, as the slope of the force curves in this form directly gives information of the elastic behavior of the bacterium.

5.1.

Typical Force Measurement

Figure 3 displays force data from a measurement on an individual bacterium of the 17875Leb strain. The graph can be seen as consisting of three characteristic regions. The first region, labeled (a) in the graph, consists of a negative force. This arises when the large bead, with the bacterium, is pressed onto the Leb bead, pushing the latter out of the center of the trap. The negative force data demonstrate that a close contact between the bacterium and Leb bead has been established. The second region, labeled (b) in the graph, consists of a linear force-versus-distance dependence, which demonstrates adhesion and originates from the stress response of the biological system under elongation. The force increases with elongation until the binding ruptures, whereby the Leb bead detaches from the bacterium and returns instantaneously to the center of the optical trap. The third part of the data, labeled (c), which constitutes of a zero force region, indicates that the binding has ruptured. The three regions observed in Fig. 3 correspond to the events described in Figs. 2(a), 2(b), 2(c).

Fig. 3

A measurement curve starts at negative force when the bead is pressed onto the bacterium (a). The force increases linearly as the bead is pulled away (b) and then suddenly drops to zero as the binding breaks, whereby the bead immediately returns to the center of the laser trap (c).

There are no data points between regions 2 and 3. This gap appears as a consequence of an immediate increase of the Leb bead-bacterium distance as the binding breaks. The maximum value of region (b) is considered to be an assessment of the de-adhesion force in the system under prevailing conditions. For the particular measurement shown in Fig. 3, the de-adhesion force amounted to $80\mathrm{pN}$ .

5.2.

Exclusion of Bead-to-Bead Interaction

It is not trivial, because of diffraction effects in the microscope, to ensure that the bacterium is mounted at the same height as the Leb bead. If a measurement is performed with a slight difference in height between the two, direct contact may occur between the large mounting bead and the Leb bead, which might cause unspecific bead-to-bead interaction. Since this type of interaction does not provide any information about the specific receptor binding, it was necessary to find means to exclude them from the bacterium-to-bead interaction.

It was found that the force response from the two types of interaction was of similar magnitude, wherefore the exclusion could not be done based on the magnitude of the force. On the other hand, it was found that they could be distinguished from each other based on a difference in their elongation dependence. Since a bacterium to some extent is elastic, measurements involving a bacterium will give rise to force curves with lower slopes than measurements made on direct bead-bead interactions. An example of a force curve for a bacterium-to-bead interaction is given by curve A in Fig. 4 . A corresponding measurement with a direct bead-to-bead interaction is shown in curve B in Fig. 4. As can be seen from the figure, the bead-to-bead interaction shows a response that has a significantly larger slope than the bacterium-to-bead interaction. Based on this type of analysis, all measurements with a slope indicating a bead-to-bead interaction were excluded from further analysis.

Fig. 4

Curve A shows a typical bacterium-bead interaction, whereas curve B is a result of a measurement performed without bacterium, thus only bead-bead interaction. The slope of the force curve reveals the source of the force.

5.3.

Verification of Specific BabA-Leb Binding

To verify the existence of a specific BabA-Leb binding, the maximum de-adhesion forces in each series for the 17875Leb and for the BabA mutant were compared. The reason for using the maximum forces as a mean for comparison is the observation that the binding force in a particular series of measurements tended to decrease with the number of measurement on a given bacterium, possibly indicating a destruction of the adhesins after repeated interactions. To not be affected by any such possible destruction, the maximum de-adhesion force in each series was therefore identified. These maximum de-adhesion forces (one for each series, thus one for each bacterium) are presented in a force distribution diagram shown in Fig. 5 with the series with 17875Leb bacteria given by square markers and those of the BabA mutant represented by diamond-shaped markers. It was found that the average of the maximum forces for the 17875Leb strain was $97\mathrm{pN}$ , with a majority of the maximum forces in the $80\text{to}110\text{-}\mathrm{pN}$ interval, whereas the average of the maximum forces for the BabA mutant was significantly smaller, $26\mathrm{pN}$ , with only one series (in fact, only one data point in one series) above $45\mathrm{pN}$ . This difference clearly indicates the existence of a specific BabA-Leb binding, which is in agreement with previous findings.² However, the data do not only verify the existence of a specific BabA-Leb binding, they also indicate that some minor unspecific bacterium-to-bead interactions are present in the system. These unspecific forces are, however, markedly weaker than the specific BabA-Leb binding.

Fig. 5

Comparison between de-adhesion forces measured on 17875Leb (square markers) and the BabA mutant (diamonds). The maximum forces in each series are presented.

5.4.

Identification and Assessment of Specific BabA-Leb Binding Forces

The identification and assessment of specific BabA-Leb binding forces is based on the distribution of forces. The data from the eight series of measurements from the 17875Leb strain are displayed in terms of a histogram in panel A in Fig. 6 , while the data from the nine series from the BabA mutant are shown in panel B. The intervals have been chosen as multiples of $12.5\mathrm{pN}$ , with a width of $\pm 6.25\mathrm{pN}$ , for reasons discussed later. The data from the 17875Leb strain (panel A) shows a pronounced high-low staggering. The histogram shows that a significant fraction of all measurements (46 measurements, 75%) gave rise to forces that appear in the even intervals (marked with filled bars), whereas a substantially smaller fraction of all measurements (15 measurements, 25%) appeared in the odd intervals (unfilled bars). This distribution of forces suggest the existence of an elemental force in the system, $F_{\mathrm{Leb}}$ . The even intervals represent multiple bindings with a force of $N{F}_{\mathrm{Leb}}$ , with $N$ being an integer, whereas the odd intervals correspond to intermediate force intervals, centered around $(N-0.5)F_{\mathrm{Leb}}$ .

Fig. 6

Histograms of the distribution of de-adhesion forces. The intervals are centered at multiples of $12.5\mathrm{pN}$ with a width of $\pm 6.25\mathrm{pN}$ . (a) and (b) correspond to the 17875Leb and the BabA-mutant strains, respectively.

The elemental force used in Fig. 6 was found by maximizing the appearance of high-low staggering. Figure 7 shows the fraction of measurements that belongs to the even intervals (i.e., those consisting of multiples of an elemental force $N{F}_{\mathrm{Leb}}$ ) as a function of elemental force $F_{\mathrm{Leb}}$ . As can be seen in the figure, a maximum of the high-low staggering appears for a $F_{\mathrm{Leb}}$ value of $25\mathrm{pN}$ . Taking the half-width-half-maximum (HWHM) of this curve as the uncertainty, it is possible to assess the elemental force in the BabA-Leb system to $25\pm 1.5\mathrm{pN}$ . The data from the BabA mutant (Fig. 6 panel B) do not show the same high-low-staggering feature, which demonstrates the absence of an elemental force in the control system.

Fig. 7

The fraction of measurements on the 17875Leb strain that belongs to the even intervals of a histogram representation of the measurements data as a function of elemental force $F_{\mathrm{Leb}}$ . A maximum is observed at $25\mathrm{pN}$ .

5.5.

Bacterium Deformation

A bacterium expands or contracts along the same direction at which the force is applied. The resistance to an elongation is often referred to as stiffness. The stiffness can be directly obtained from the data as the slope of a force-versus-distance curve. The experiments showed that the stiffness of the bacterium was uncorrelated to the de-adhesion force. The stiffness was also found to be the same for the 17875Leb as well as the BabA mutant. This suggests that it does not depend on the adhesion mechanism, but that it rather is related to a deformation of the bacterium. The mean value of the stiffness of all measurements (along the direction perpendicular to the length axis) was found to be $\sim 280\mathrm{pN}∕\mu \mathrm{m}$ .

6.1.

Verification of Origin of Forces

Force measurements based on oligosaccharide-activated beads that interact with immobilized bacterial cells are challenged by unspecific bacterial binding as well as occasional direct contact between bead surfaces. In the force measurements reported here, the unspecific binding was reduced by efficient blocking of both beads and bacterial surfaces with albumin protein, which is a serum protein that is naturally nonglycosylated and will not take part in the BabA-Leb binding event. The pure bead-to-bead interaction could be readily identified and eliminated from the bacterium-to-bead interactions due to the lack of elasticity in the measurements (infinite slope of the force curve at the start). In addition, a BabA mutant, devoid of the BabA adhesin protein and thus lacking all Leb binding properties, was analyzed as a negative control. As was shown in Fig. 5, the maximum binding force of the BabA mutant $(\sim 26\mathrm{pN})$ was distinctly different from and weaker than that of the 17875Leb bacteria $(\sim 97\mathrm{pN})$ . This indicates that the BabA protein-Leb antigen binding is both specific and, furthermore, involves multivalent interactions that collectively adds to the avidity of the tight bacterial-host cell interaction revealed by the OT-force measurements. The results are also in qualitative agreement with previous studies by means of Scatchard analyses, in which Leb has been identified as the most important receptor that mediates the binding to the BabA protein present on the bacterial surface.²

6.2.

Verification of an Elementary Binding Force

The distribution of forces shown in Fig. 6(a) is strong evidence for an elemental force in the BabA-Leb system. If there was not an elemental force in the system, the number of measurements would be expected to distribute evenly in odd and even intervals, since their widths are all the same. However, it was found that 75% of all measurements ended up in even intervals, whereas 25% appeared in the odd. The large difference in population of the two groups of intervals can be investigated using standard binomial distribution theory. Assuming that each measurement has the same probability (50%) to appear in an odd and even interval results in a symmetric distribution function. Using this function, it is possible to assess the probability to measure such an uneven distribution of forces as 75% versus 25% to less than 0.5%. Due to this regularity in the distribution of forces and the low number of force measurements at $12.5\mathrm{pN}$ , it is possible to conclude, with a high certainty, that there exists an elemental force of $\sim 25\mathrm{pN}$ in the BabA-Leb system. Since there is no similar high-low staggering in the control system, it is furthermore reasonable to assume that this force corresponds to an individual BabA-Leb binding. Unspecific forces in combination with natural variations in the BabA-Leb binding are assumed to be responsible for the spread of forces into odd intervals.

Although the previous discussion is not an irrefutable proof, the distribution of forces displayed in Fig. 6 supports the assumption that there is a minimum specific elemental force of $25\pm 1.5\mathrm{pN}$ in the BabA-Leb system under the pertinent conditions (at a loading rate of $\sim 100\mathrm{pN}∕\mathrm{s}$ ). The absence of a high-low staggering in the control system provides evidence that the elementary force corresponds to the de-adhesion force associated with an individual BabA-Leb binding site.

6.3.

Assessment of the Number of Binding Sites

From the data shown in Fig. 6(a), and under the assumption that an individual BabA-Leb binding corresponds to $\sim 25\mathrm{pN}$ , it can further be concluded that BabA-Leb adhesion was, in a majority of the cases, mediated by a small number of bonds, in general, a few (1 to 4), and occasionally slightly more. These numbers seem at first sight to be unexpectedly low, since the number of binding sites in the contact area can be estimated to be significantly larger than this. It has previously been shown by Scatchard analysis that 17875Leb has an average of about 500 adhesins per bacterium.² It can further be estimated that about 15% of the total bacterium surface was in contact with the Leb bead under the pertinent experimental conditions (with a $20\text{-}\mathrm{pN}$ application force). From a pure geometrical consideration, it is possible to derive an expression for the fraction of the total area of the bacterium that is in contact with the Leb bead, $A_F$ , that reads

6.4.

Single Rupture

Almost no multiple peaks are found in the force curves. A multiple peak structure would be expected if the bonds that act in parallel would rupture one after another in succession. However, again because of the short length of the bonds, it is expected that when one or a few of several bonds have ruptured, the remaining bonds will directly be further elongated and exposed to such a high force (both from the increase in force due to the sudden increased bead-to-bacterium distance, but also from the dynamics of the movement) that they would immediately rupture. This can be seen as an immediate breakdown sequence appearing on a timescale below that of the measurements. Hence, it is not expected that multiple peak structures should be frequent in these types of experiments. This is also in agreement with the experimental findings.

6.5.

Elasticity

It is also of interest to investigate the origin of the measured stiffness $k_H\sim 280\mathrm{pN}∕\mu \mathrm{m}$ of the bacterium. In general, the force exerted on the bacterium results in a deformation that can have two origins: an elastic stretching of the cell wall (like the elongation of a mechanical spring) or an alteration of the geometrical shape of the bacterium. In both cases, a restoring force is produced.

In the work reported in Ref. 21, it is suggested that the stability of the cell wall of Gram-negative bacteria is associated with the peptidoglycan layer, which has a thickness of $\sim 3\mathrm{nm}$ . The stretching of the cell wall can be expressed in terms of Young’s modulus. This entity, denoted by $Y_H$ , can, for a 1-D material, be estimated from the relation

A bacterium also acts as a pressure chamber that will strive to maintain its original shape when exposed to deformations. The turgor pressure is the osmotic pressure difference between the inside of the bacterium and the surrounding medium. This pressure thus determines the resistance of the bacterium to an alteration of its geometrical shape. The turgor pressure is normally found to be in the range from 1 to 5 atmospheres.²¹ A pressure of $\sim 3$ atmospheres is enough to produce restoring forces in the order of $100\mathrm{pN}$ if the bacterium is deformed an amount of $\sim 0.3\mu \mathrm{m}$ , which is in agreement with the stiffness measured in these experiments $(k_H\sim 280\mathrm{pN}∕\mu \mathrm{m})$ . The conclusion is therefore that the turgor pressure gives rise to the restoring forces of the bacterium body as it is deformed from applied forces.

6.6.

Comparison with Other Specific Bacterial Adhesion Forces

Only a few results of specific bacterial adhesion forces seem to be reported in the literature. For example, in Ref. 11 the specific force required to break a single interaction between an E. coli pilus and its receptor (a mannose group) is estimated to $1.7\mathrm{pN}$ , a value that seems surprisingly low with respect to the thermal energy in the system. The thermal energy, given by the product of the Boltzmann’s constant and the absolute temperature $k_{B}T$ , is in the order of $4\mathrm{pN}\bullet \mathrm{nm}$ , which for a bond length in the interval of $0.2\text{to}0.5\mathrm{nm}$ corresponds to a thermal force in the range from $8\text{to}20\mathrm{pN}$ . Reference 12 reports on a more reasonable value of $18\mathrm{pN}$ for the force required to rupture a single, specific S. epidermidis bond with fibronectin. Based on general considerations regarding the thermal energy and bond lengths, it is reasonable to assume that specific bacterial adhesion should be mediated by a force that is larger than the thermal force. This is consistent with our BabA-Leb measurements that indicate a binding force of $25\mathrm{pN}$ .

6.7.

Future Work

A noncovalent bond between a ligand and a receptor can generally be described as a process driven by Brownian-thermal fluctuations, which can be characterized by an energy landscape. The energy landscape describing the BabA-Leb bond is so far unknown. Exposing the bond to an external force lowers the energy barriers and increases the likelihood of unbinding. Theory predicts that the de-adhesion force has a logarithmic dependence on the rate at which the force is applied.²² In this study we have only used one loading rate, $\sim 100\mathrm{pN}∕\mathrm{s}$ , and the forces measured are thus statistical reflections of the energy landscape for this rate. A spectrum of the average unbinding force as a function of loading rate would provide a means for characterization of the energy landscape of the BabA-Leb bond in some detail. The experimental setup and the biological assay used here provide a possibility to investigate a large number of features and entities in this biological system. This work covers only a first study in which specific adhesion forces and some elastic properties of H. pylori have been investigated. Further investigations are planned to cover issues such as the impetus of the contact time, loading rate, and pH of the solvent, which all are assumed to be important factors for a general characterization of the adhesion properties of H. pylori, but whose influence is yet unknown. Also different variations of the Leb conjugate will be investigated.