biophysical approaches to study the dynamic
TRANSCRIPT
Research in Microbiology 159 (2008) 415e422www.elsevier.com/locate/resmic
Biophysical approaches to study the dynamicprocess of bacterial adhesion
Karen Otto*
Department of Molecular Biology, Umea University, 901 87 Umea, Sweden
Received 2 February 2008; accepted 14 April 2008
Available online 2 May 2008
Abstract
Recent applications of biophysical techniques to the study of adhesion and biofilm formation are playing an important role in broadening ourunderstanding of bacterial interactions. While non-invasive methods enable measurement of adhesion kinetics in real time, single-cellapproaches provide information about adhesion forces mediated by specific cell surface structures. Promising approaches are presented inthis review.� 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Biofilm formation; Bacterial adhesion; Adhesion kinetics; Quartz crystal microbalance; Surface plasmon resonance; Adhesive strength; Micromanip-
ulation; Optical tweezers; Atomic force microscopy; Inhibition of adhesion
1. Introduction
Microorganisms can live and proliferate as individual cellsor they can attach to surfaces, where they grow as highlyorganized multicellular communities. These communities arereferred to as biofilms and are now regarded as the predomi-nant mode of microbial life in nature and disease.
In the last decade, much progress has been made in eluci-dating the molecular mechanisms of bacterial adhesion andunderstanding the structure and composition of biofilms.These achievements are mainly due to the use of two majorapproaches: genetic screens for adhesion-deficient mutantsusing the microtiter dish assay [26] and application of confo-cal laser scanning microscopy combined with digital imageanalysis [21]. From these studies, we know that biofilms arefundamentally different from planktonic cells. They developon surfaces in a series of ordered steps, each of which requiresreprogramming of gene expression [35]. Among thesechanges, altered expression of cell envelope components isparticularly important because it directly affects the mode ofcellesurface contact.
* Tel.: þ46 90 7856752; fax: þ46 90 772630.
E-mail address: [email protected]
0923-2508/$ - see front matter � 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.resmic.2008.04.007
Although useful and informative, these techniques haveapparent limitations. They cannot provide information aboutcritical adhesion parameters such as kinetics or strength. Tofurther reveal the underlying mechanisms of adhesion, novelapproaches have been introduced into the field of biofilmresearch. Non-invasive methods have been applied thatmeasure the adhesion process in situ and in real time [6,13].Furthermore, it has become possible to measure adhesiveproperties on a single-cell level, which allows for carefulanalysis of molecular functions of cell surface structures [4].In this review, the principles and applications of promisingmethods that address qualitative parameters of bacterial adhe-sion are summarized.
2. Monitoring dynamic changesduring the adhesion process
When biofilms are disrupted and cells released into suspen-sion before they are studied, it is uncertain whether theremoved cells display the same phenotypic characteristics aswhen attached. Flow devices were designed to study adhesionunder well-defined hydrodynamic and mass transport condi-tions [3]. In combination with biosensors, adhesion can bestudied in situ and in real time without destroying the system.
416 K. Otto / Research in Microbiology 159 (2008) 415e422
2.1. Quartz crystal microbalance (QCM)
The QCM is an acoustic biosensor which senses masschanges in the nanogram range. It can be used to study a vari-ety of surface processes including bacterial adhesion [6]. Theprinciple of the QCM technique is based on piezoelectricity,i.e. coupling between a material’s mechanical and electricalbehaviors. By applying an alternating electric field, the crystalstarts to oscillate at its resonant frequency, which is dependenton the total oscillating mass. Adsorption of molecules causesan increase in the total oscillating mass, which can be moni-tored as a decrease in frequency (Fig. 1).
2.1.1. Conventional QCMA simple QCM experiment was set up to determine the ad-
hesion of Staphylococcus epidermidis to the protein fibronectin[32]. After fibronectin had been immobilized on the crystalsurface, the system was exposed to suspensions of bacteriaat concentrations ranging from 102 to 106 cells ml�1. Looselyadhering bacteria were washed off and a final frequency wasmeasured. Hereby, a linear relationship between the decreasein frequency and the logarithm of cell concentration wasfound. Extrapolation suggested a theoretical limit of thistechnique at approximately 15 cells ml�1.
Another QCM study monitored biofilm formation of Strep-tococcus mutans as a function of sucrose concentration [20].Crystals were coated with sterile saliva and S. mutans biofilmswere grown in the presence of different sucrose concentra-tions. After initial attachment under static conditions, biofilmgrowth was monitored for 10 h under flow with continuousmedium supply. Surface attachment of S. mutans was possiblewithout sucrose, but only under static conditions. Under flowconditions, the supplied sucrose concentration affected boththe kinetics of biofilm formation and the total biomass.Although only changes in frequency were measured, theauthors realized that QCM measurements could provide
UU
A
B
Fig. 1. Principle of the quartz crystal microbalance. (A) The quartz crystal is coate
electric field. The crystal is excited every second and the changes in frequency an
shear wave caused by oscillation of the crystal penetrates the liquid approximately
which can be measured as the dissipation factor (For interpretation of the reference
article.).
some clues to biofilm ‘‘strength’’. Cells appeared to be moresticky at high sucrose concentration and continued to growon the surface despite constant shear stress.
2.1.2. QCM with dissipation monitoring (QCM-D)Frequency shifts are proportional to the attached mass
only when the attached mass is thin, evenly distributed, rigidand tightly coupled to the surface, which is not the case forbiological samples. Non-rigid binding results in energy dissi-pation, which is recorded simultaneously by the QCM-D(Fig. 1) [37]. The versatility of interpreting QCM-D measure-ments was exemplified in a study on adhesion of fimbriatedand non-fimbriated Escherichia coli mutants as a functionof ionic strength [29]. For the experiment, cells weresuspended in buffer at a certain ionic strength and allowedto attach to the crystal for 30 min. By calculating the dissipa-tion shift per frequency shift (DD/Df ) for each time point ofthe experiment, a qualitative measure for the adhesionprocess was obtained, which enabled distinguishing betweenbinding properties for fimbriated and non-fimbriated cells.Non-fimbriated cells appeared to establish better surfacecontact with increasing ionic strength, whereas fimbriatedcells seemed to have flexible contact with the surface inde-pendently of ionic strength. After removing loosely boundcells, frequency and dissipation still changed, which showedthat firmly attached cells continue to undergo time-dependentsurface interactions, possibly due to structural reorganizationof the cells.
The QCM-D technique has been useful for identifyinga gene that does not influence attachment per se but thatmediates changes in the cellesurface contact following attach-ment [30]. While deletion of ompX weakened the cell surfacecontact of non-fimbriated cells, it improved cell surfacecontact of fimbriated E. coli cells. Subsequent phenotypiccharacterization of the mutant demonstrated that expressionof ompX interfered with the proper regulation of fimbriae
quartz crystal coatedwith gold electrodes
cells attachingto the surface
d with thin gold electrodes on both sides which are connected to an alternating
d energy dissipation are recorded. (B) At a resonant frequency of 5 MHz, the
250 nm. Adhesion influences the decay (red lines) of the oscillation amplitude,
s to colour in this figure legend, the reader is referred to the web version of this
θ
goldglass
flow channel
prism
lightsource
lightdetector
Fig. 2. Principle of surface plasmon resonance. The angle of incident light which
forms maximum excitation is sensitive to changes in the dielectric properties of
thin films forming on the upper side of the gold film. Adapted from [16].
417K. Otto / Research in Microbiology 159 (2008) 415e422
and other cell surface structures that play a key role in medi-ating surface contact.
Bacterial adhesion is controlled by intracellular signaltransduction systems. For instance, the Cpx signaling systemis induced upon cellesurface contact, suggesting a role forthis pathway during the early stage of adhesion. Studyingadhesion with QCM-D supported the hypothesis that theCpx-system is required for an adaptive response of E. coli toadhesion [31]. When components of the Cpx pathway wereabsent, cells not only attached at significantly lower numbersto the surface, but also caused lower DD/Df, indicating thatthe viscoelastic properties of binding were completely differ-ent between wild-type and mutant cells.
In the search for anti-adhesive compounds, the QCM-Dtechnique was used to evaluate the anti-adhesive effect ofN-Acetyl-L-cysteine (NAC) when the compound was boundto a surface [28]. Bacillus strains were grown to differentgrowth states and were allowed to attach to stainless steel orpolystyrene-coated crystals. Frequency changes were affectedby differences in cell surface hydrophobicity. Hydrophiliccells caused positive frequency shifts, a phenomenon thatcould be related to increased viscosity. The viscoelastic ratiofor exponentially grown (hydrophilic) cells was much largerthan that for starved (hydrophobic) cells or spores. The effectof NAC was studied by exposing surfaces to the compound for15 min prior to adhesion of cells. This coating made thesurfaces less hydrophobic, but no general effect of NAC oninitial adhesion could be observed. Viscoelastic propertiesvaried for different strains, growth phases and surfaces.
Most recently, the QCM-D method was used to investigatebiofilm formation of S. mutans [39]. To analyze how flowconditions during initial attachment affect subsequent biofilmformation, bacteria were allowed to attach for 0 h or 2 h understatic conditions before cells were exposed to flow conditionsfor a further 20 h. Viable counts of suspended bacteria afterthe static attachment phase and at the end of the experimentconfirmed that cells attached and proliferated, and increasingfrequency and dissipation shifts could be related to an increasein cell number. Biofilms formed under constant flow conditionshad a greater mass and were more viscoelastic. The massincrease on the surface accelerated over time, reflecting differ-ent phases of biofilm development. To evaluate whether theproperties of the attached cells changed during the experiment,DD/Df versus time was determined. These values increased toa maximum after about 700 min, when a stable structureseemed to be established. However, for cells that were initiallyallowed to attach under static conditions, DD/Df decreased afterthe peak, suggesting that the biofilm formed was not as stable.Thus, steady flow conditions on the surface appeared to beimportant for creating biofilms of greater complexity.
2.2. Surface plasmon resonance (SPR)
SPR is an optical biosensor that detects mass concentrationat a metal sensor chip surface by measuring changes in therefractive index (Fig. 2). Light coming from a medium of higherrefractive index to a medium of lower refractive index is partly
reflected and partly refracted until total internal reflectionoccurs. Above this critical angle of incidence, electromagneticenergy in the form of an evanescent wave appears in themedium with a lower refractive index and decays parallel tothe surface. When light is monochromatic and p-polarizedand the interface between the media is coated with a thin metallayer, the intensity of the reflected light is reduced at a specificincident angle, producing a shadow due to the resonance energytransfer between the evanescent wave and surface plasmons.
To study an interaction by SPR, one of the interaction partnersis immobilized onto the sensor surface and the other is allowed toattach. Bacterial adhesion can be studied by SPR by studying thebinding of purified adhesins to specific receptors immobilized onthe SPR sensor chip. This approach helped to elucidate the adhe-sion of Porphyromonas gingivalis, a periodontal pathogen whosefimbriae are classified into six genotypes [18]. To evaluatewhether strains with type II fimbriae were more virulent thantype I strains, recombinant strains were analyzed with SPR,which demonstrated that type II fimbriae possessed greater adhe-sive abilities than type 1 fimbriae for their receptor.
During recent years, the SPR technique has also gained inpopularity for elucidating molecular interactions involvingwhole bacterial cells. The resolution distance of the instrumentdepends on the decay length of the plasmon wave (about200 nm). Since the sensitivity decreases proportionally withthe distance from the sensor chip surface, the instrumentonly detects the portion of the cell within the detection rangeand a large number of cells are needed to obtain a significantsignal [27,34]. Whole-cell SPR studies on bacterial adhesionmostly address specific binding events. Sensor chips havebeen designed which are presumed to improve sensitivitywhen working with large molecules or whole cells, and recep-tor molecules of different size and complexity have success-fully been immobilized on the sensor surfaces.
To study adhesion of streptococci to salivary components bySPR, whole salivary components were immobilized on thesensor chip surface and the binding of different streptococciwas compared with respect to affinity and separated stages ofassociation and dissociation [19]. This revealed characteristicdifferences between the various strains which may relate to
418 K. Otto / Research in Microbiology 159 (2008) 415e422
their role in early biofilm formation on the tooth surface.Compared to this study, a 10-fold greater change in the reso-nance signal was observed when the interaction of immobilizedsalivary agglutinin with the major adhesin of S. mutans wasmeasured, possibly because a more defined physiologicalligand was used [27]. To rule out the possibility that cell frag-ments or secreted proteins contributed to the measured signal,cells were fixed and washed before use. However, wash super-natants did not give rise to a measurable signal and the changein resonance could be solely attributed to whole-cell binding.
In another SPR study, specific binding of uropathogenic E.coli to globotetraosylceramide (globoside) could be demon-strated [34]. The authors chose a suitable sensor chip to whichglycolipids could be anchored via the lipid portion, whereas thehydrophilic portion of the glycolipids protruded towards thesolution phase. This set-up enabled measurement of the directinteraction between globoside and bacteria. The cell concentra-tion was found to be an important factor in interaction analysis,with a detection limit at about 5� 107 CFU ml�1.
To study the inhibition of binding of uropathogenic E. coli bymultivalent galabiose derivatives under flow conditions [38],the sensor chip was coated with galabiose. To interfere withadhesion, inhibitory galabiose compounds were incubatedwith bacteria prior to the SPR measurement. Comparison ofthe results from the SPR assay with those from conventionalassays showed that, although the actual inhibitory concentra-tions of the compounds slightly differed in the different assays,minimal inhibitory concentration or LC50 values generallydecreased when the valency of the compound was increased.An advantage of SPR over conventional assays was thatsubstantially less ligand and receptor were required (Table 1).
Only rarely was SPR used for bacterial adhesion studieswithout binding a specific ligand to the sensor. In thosestudies, the role of type IV pili in adhesion of Pseudomonasaeruginosa was addressed [17,16]. The authors point outthat quantification of the change in SPR resonance due tonon-specific adhesion by bacteria is difficult. First, adlayersare assumed to be homogeneous in all dimensions, which isnot the case for bacteria. Second, there is no accurate measure-ment of the refractive index of spatially heterogeneouslyattached bacteria. For this reason, results were presented as‘optical thickness’. After introduction of bacteria into theflow chamber, a rapid increase in optical thickness wasobserved, followed by a decline. For non-piliated or hyperpi-liated mutants, the optical thickness was significantly lower,indicating that less adhesion occurred. The different adhesionkinetics of the mutants correlated with a decrease in virulence,but were not reflected in endpoint assays of biofilm formation.
3. Determining the adhesive strength of cellecelland cellesurface interactions
For control of biofilms, the mechanical strength of adhesionis another important characteristic of cellesurface interac-tions. Various methods have been applied to quantify adhesionforces between cell aggregates, single cells and singlemolecules.
3.1. Fluid dynamic gauging
The cohesive strength of biofilms determines their structureand function. It can be quantified by the use of dynamic gaug-ing where a shear force is applied to detach cells [5]. Closeabove the biofilm, a nozzle is located through which a flowis induced. The flow rate depends on the height betweennozzle and sample surface. This height can be used to calcu-late the wall shear stress acting on the biofilm surface, whichin turn can be assumed to be the cohesive strength of thebiofilm. In the case of biofilm detachment, the thickness ofthe biofilm is measured before and after gauging. An advan-tage of this method is that forces acting on the biofilm derivefrom a lateral flow, which is typical for many biofilm systems.This method was used to determine that the cohesive strengthof biofilms from activated sludge ranged between 6 and7.7 N m�2 and it provides a useful tool for comparing thestability of biofilms grown under different conditions [25].
3.2. Microcantilever method
Alternatively, a microcantilever method can be used toestimate the tensile strength of biofilms [33]. A microbialaggregate is held by suction between two micropipettes, oneof which is fixed to a cantilever. The cantilever is attachedto a microscopic stage and can be moved away from thecapturing pipette. The increasing deflection of the cantileverproduces an increasing force until either the sample separatesor suction between the sample and one of the pipettes isbroken. Deflection of the cantilever arm was determined bymeasuring the distance the cantilever moved as the aggregateseparated. In this way, the cohesive strength of Pseudomonasbiofilms on glass disks could be estimated, although a widerange of strengths (395e15,640 N m�2) was measured dueto heterogeneity within the biofilm.
3.3. Micromanipulation
Micromanipulation relies on the same principle as themicrocantilever method but was used for direct measurementof the adhesion force of single bacterial cells [43]. Caulo-bacter crescentus cells were allowed to attach to a thin flexiblepipette. A suction pipette mounted on a micromanipulator wasused to grab the body of an attached cell and pull the cellvertically away from the flexible pipette. The force of adhe-sion was calculated from the amount of bending required tobreak the cellepipette contact. Using this technique, the adhe-sion force of stalked C. crescentus cells was estimated to bemore than 68 N mm�2, which is the strongest adhesionstrength measured for microbial cells.
3.4. Atomic force microscopy (AFM)
AFM consists of a cantilever with a sharp tip at its end, whichserves as a mechanical probe to scan the surface of a sample.When approaching the surface, forces between the tip and thesample cause deflection of the cantilever, which is detected by
Table 1
Comparison of parameters characteristic of biophysical approaches to the study of bacterial adhesion
Method Principle Adhesion parameter
measured
Characteristics Sensitivity, resolution Reference
To measure adhesion kinetics
QCM Acoustic biosensor;
applied alternating
electric field causes a
mechanical oscillation of
quartz crystal
Induced frequency and
dissipation shifts reflect mass
increase and viscoelastic
changes due to adhesion
Non-invasive, real-time
measurement of non-
specific interactions in
gas or liquid phase under
static or flow conditions
Direct contact
required; ng range;
distance resolution
approx. 250 nm
Cooper & Singleton [6]
SPR Optical biosensor;
binding of a film causes
change in dielectric
constant which changes
angle of resonance
Change in refractive index
upon adsorption of cells is
transformed into measurable
signal by light-sensitive
detector
Non-invasive, real-time
measurement of specific
or non-specific
interactions in liquid
phase under flow
conditions; possible to
measure association/
dissociation rates
Direct contact
required; reliable
response signals for
>107 cells; pg mm�2
range; evanescent
decay length of
plasmon wave approx.
200 nm
Hoa et al. [13]
To measure cohesive strength
Fluid dynamic gauging By inducing flow through
a nozzle positioned close
above the sample surface
a shear force is applied
Measuring the thickness of
the biofilm before and after
gauging helps to assume the
cohesive strength of the
sample
Invasive measurement of
non-specific interactions
and aggregation; lateral
flow reflects typical
situation for many biofilm
systems
Cell aggregate level Mohle et al. [25]
To measure adhesive strength
Microcantilever/
micromanipulation
Samples are held by
suction and pulled by
external force; increasing
deflection of cantilever
produces increasing force
upon the sample
Deflection of cantilever arm
is detected by laser light,
quantified and related to the
cohesive or adhesive strength
of the sample
In situ, real-time
measurement of non-
specific interactions
Cell aggregate or
single-cell level
Poppele & Hozalski
[33]
AFM Forces between probe and
sample lead to deflection
of cantilever
Deflection of cantilever arm
by laser corresponds to
adhesive strength of the
sample
Direct measurement of
specific or non-specific
interactions in gas or
liquid phase; bacteria can
be immobilized on a flat
substrate or the tip; probe
can be functionalized
Single cell and cell
surface organelle
level; distance
resolution nm range;
pNenN range
Camesano et al. [4]
OT A focused laser beam is
used to break the force
equilibrium between cell
and surface until
detachment occurs
Difference in indices of
refraction between particle
and its surrounding medium
determines force required for
detachment and correlates
with the adhesive strength of
the sample
Direct force
measurement; either
surface can be cell or
substrate; substrate can be
coated
Single-cell level; pN
range
Camesano et al. [4]
419K. Otto / Research in Microbiology 159 (2008) 415e422
an optical system (Fig. 3). AFM can be used for direct force mea-surements with a sensitivity in the pN to nN range, and in recentyears, this approach has been extensively used to study the phys-ical properties and interaction forces of microbial cell surfaces[11]. For force measurements of bacterial adhesion, cells firstneed to be immobilized. They can be bound on a flat surfaceand scanned by a probe, which is either a clean surface or func-tionalized with specific receptor molecules. Even bacteria canbe attached to the tip, thus comprising a living probe.
Slight differences in cell surface composition of E. colimutants could be determined by the use of AFM [36]. A con-fluent bacterial lawn was established on glass surfaces beforeinteractions with the AFM tip were measured. The adhesionforce was affected by the length of core lipopolysaccharidemolecules on the E. coli cell. Truncated LPS molecules
resulted in increasing electrostatic repulsion to the negativelycharged tip. Similarly, mutants that overproduce colanic acidwere not attracted to the AFM tip.
AFM has been used to assess the interaction of a metal-reducing bacterium with goethite, which simultaneouslyserved as a surface and a terminal electron acceptor [22].Shewanella oneidensis cells were linked to a small beadlocated at the end of the cantilever, thereby creating a biologi-cally active probe. Forces were measured when the mineralcrystal mounted on a scanner was approached and retractedfrom a bacterial cell on the probe. Under anaerobic conditions,S. oneidensis responded to the surface by rapidly developingstronger adhesion energies at the interface.
AFM was also useful for assessing the potential of Candidacells to colonize biomaterial in the presence or absence of
trapped cellstage
external forceforce exerted byoptical trap
amount of displacement
light sourcelight detection
cantilevertip
cell immobilizedon surface
Fig. 3. Principle of the atomic force microscope. A cell is immobilized on a sur-
face and probed by a cantilever equipped with a tip. The laser light deflected
by the bent cantilever is detected by a photodiode. Adapted from [8].
420 K. Otto / Research in Microbiology 159 (2008) 415e422
a biofilm [7]. Candida cells were immobilized on the AFM tipand moved to a silicone surface, which caused strong attractiveinteractions. These forces were three times higher when thesilicone surface was covered with a biofilm of P. aeruginosacells, indicating a higher affinity of Candida for binding tosurfaces on which a biofilm is already growing.
AFM was used to study the mechanical properties of pili[8,24]. In one study, type 1 piliated E. coli were immobi-lized on glass slides and pulled with a mannosylated tip ofan AFM to study elongation and contraction behavior [8].By pulling at a constant force, fimbriae were shown to bestable in length over a range of forces between 25 and60 pN, but they extended or contracted rapidly outside thisrange. Since shear forces enhance fimbriae-mediated attach-ment, the mechanical properties of the fimbrial shaft are thusadapted to optimize attachment under flow. In another study,the mechanical properties of both type 1 pili and P pili wereaddressed by purifying the pili and letting them adsorb ontoa glass surface [24]. Segments of a pilus were then pickedup by adsorption to the AFM tip and stretched. P pili andtype 1 pili proved to be highly extensible, while the forcesrequired to unfold each of these pili were different (35versus 60 pN).
To characterize the interactions between adhesins ofMycobacterium tuberculosis, their adhesion forces weredetermined via AFM [44]. Both the AFM tip and the sup-port were functionalized with recombinant heparin-bindinghemagglutinin (HBHA) proteins. Different binding forceswere measured depending on whether the N-terminal orthe C-terminal domains were exposed. The forces couldbe attributed to specific interactions reflecting the associa-tion of a-helices into coiled-coil structures and to electro-static interactions, respectively. HBHA tips were alsoused to probe the surfaces of living cells. The bindingforces measured resembled those of coiled-coil interactionsvia the N-terminal domains, which may be responsible formycobacterial aggregation.
lightsource
3.5. Optical tweezersFig. 4. Principle of force measuring optical tweezers. A cell trapped by
a strongly focused beam of light is displaced by an external force. The amount
of displacement correlates with the restoring force required to bring the cell
back into the focus of light. Adapted from [9].
Optical tweezers use a strongly focused beam of light totrap small objects ranging from atoms to living cells. Thereare two theoretical models to explain the principle behind
this technique [12]. Particles much larger than the wavelengthof the laser refract some of the light due to the difference inindices of refraction between the particle and its surroundingmedium. This redirects the momentum of the photons in thelaser. As a result of light scattering and gradient forces, theparticle is drawn to the higher flux of photons near the focus.Small objects, on the other hand, develop an electric dipolemoment in response to the light’s electric field, which is drawnup by intensity gradients in the electric field towards the focus.Optical tweezers are also an accurate tool for force measure-ments. If the trapped particle is moved by an external forceand the displacement from the focal region is smaller thanhalf the radius of the particle within the focal region, thenthe amount of displacement is proportional to the restoringforce (Fig. 4). By monitoring the position of a particle in anoptical trap it is possible to calculate the force to which theparticle is exposed [9]. Optical tweezers provide maximumworking forces on the order of about 100 pN and can thereforebe used to make direct measurements of the adhesion forcesbetween micrometer-sized objects, including bacteria.
The binding forces arising from adhesion of Staphylococcuscells to fibronectin were investigated by optically trappingsingle cells and bringing them into contact with small polysty-rene beads coated with fibronectin [40e42]. The minimumforce required to detach the cell from the bead was measuredover a range of fibronectin concentrations [41]. Due to theformation of multiple bonds, the forces measured varied butoccurred as integer multiples of a strain-dependent base valueranging between 18 and 25 pN, which presents the single bondrupture force. Bond formation was shown to be time-depen-dent. To determine the binding capacity of several subregionsof fibronectin binding proteins, mutants expressing recombi-nant proteins with modified subregions were trapped and thedetachment force was determined. While S. aureus was ableto compensate for some degree of mutation without reductionin binding, detachment forces were weakened in mutants withat least two regions in the fibronectin binding protein removed[42].
421K. Otto / Research in Microbiology 159 (2008) 415e422
Optical tweezers were used to show that type IV pili inNeisseria gonorrhoeae promote twitching motility by retrac-tile forces [23]. Isolated cells were pulled towards microcolo-nies over distances of up to 2 pilus lengths. To analyze tfpretraction quantitatively, individual diplococci were immobi-lized on latex beads anchored on a coverslip. Optical tweezerswere used to hold smaller 1 mm beads. When these wereplaced within 1e3 mm of immobilized cells, the beads werepulled towards the immobilized cells through both specificand non-specific pilus-mediated binding interactions thatreached forces up to 80 pN.
In a series of studies, the binding forces of P pili expressedby uropathogenic E. coli to galabiose were investigated usingoptical tweezers. For these experiments, a bacterial cell wastrapped and covalently coupled to a large polystyrene bead.A galabiose-coated small polystyrene bead was then trappedand positioned at a close distance to the bacterium so thatpili were able to contact the beads. The resulting bindingwas due to a single interaction that broke at a force of10e15 pN [9]. When the galabiose-coated bead was posi-tioned in direct contact with the cell, forces measured werein the range of 50e100 pN, which is likely due to multiplebinding sites and other interactions than single receptor-mediated binding. To probe the mechanical properties ofpili, these were stretched and the occurring forces determined[14]. This was done by moving the large bacteria-bound beadfrom the trap and monitoring the displacement of the smallerpili-bound bead. P pili turned out to perform an elastic elonga-tion until the interaction between adjacent PapA subunitsbreaks, measured as an elongation under constant force ofabout 27 pN. A fully unfolded pilus was measured to be aboutseven times longer than its unstretched length. This elongationwas fully reversible [10]. PapA refolded its helical structurespontaneously after elongation. This mechanical behaviormay help bacteria regain close contact with the host cell afterexposure to shear forces.
The same experimental set-up was used to determine themechanical properties of type 1 fimbriae [2]. Compared to Ppili, type 1 pili were shown to be more rigid and to respondfaster to an external force. Their elongation followed similarphases, but refolding of type 1 pili occurred, unlike P pili, attwo different levels.
The dynamic behavior of the elongation of P pili wasaddressed by using so-called dynamic force spectroscopy[1]. Instead of applying steady-state elongation conditions,the binding force between P pili and galabiose was measuredby pulling the pili at higher speeds. An increased elongationspeed caused an increase in the unfolding force, whereas theforce to reconfigurate between the subunits remainedunaffected.
Optical tweezers were also used to extract membranetethers extruding from E. coli cells that may be importantfor initiating attachment to any solid surface [15]. Bacteriabound to a poly-L-lysine coated surface were allowed to attachto polystyrene beads. By pulling a bead, a point-like force wasexerted on the outer membrane, which was measured to beabout 10 pN. The membrane tethers appeared elastic at short
time scales but showed viscous properties during consecutivepulls. An intact LPS core was shown to be critical for tetherformation.
4. Concluding remarks
Currently, antibiotics are used as the main form of therapyagainst bacterial infections. However, with the alarming in-crease in antibiotic resistance among pathogenic and environ-mental strains, new antibacterial strategies must be developed,and anti-adhesion is a promising target. Recent advances in thestudy of bacterial adhesion by biophysical techniques havecontributed unique information and remarkable insights intothe dynamic character of adhesion. By using the different tech-nical approaches which are complementary to each other, andtogether with molecular biological techniques, these newperspectives can be integrated into our increasing understand-ing of the molecular basis of adhesion. This should offera broad platform for the potential development of new anti-adhesive strategies.
Acknowledgements
Work in my laboratory is supported by the Swedish Societyfor Medical Research, the Hedlunds Foundation, the SwedishRoyal Academy of Sciences, the Adlerbertska ScienceFoundation and the Lundgren Science Foundation.
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