(3) unspecific adsorption on themodified surfaces should be minimized;(4) molecules should be attached at
low density to ensure single-molecule
recognition; and (5) for oriented
systems, the best results are usuallyobtained when the molecules areattached in a defined orientation.
To fulfill these requirements, HBHA
was engineered with a His-tag at its
N-terminal end and coupled onto an
AFM tip modified with mixed, selfassembled monolayers (SAMs) of
nitrilotriacetic acid (NTA) andtri(ethylene glycol) (EG) terminatedalkanethiols (see Figure 2). The primary
benefit of employing this site-directedstrategy is that it uniformly exposes theC-terminal binding domains of theadhesins. Biotinylated heparin, used asa model receptor, was attached ongold substrates via a classical sandwich
layer of streptavidin and biotinylated
bovine serum albumin (BBSA).

Force Spectroscopy of theHBHA/heparin Interaction

Since adhesion forces measured
between receptors and ligands usuallydepend on the rate at which the load
is applied5, force curves between theHBHA tip and the heparin surfacewere recorded at various loading rates.
Surprisingly, we found that the meanadhesion force did not depend on theloading rate, indicating that the force
measurements were done close tothermodynamic equilibrium. By contrast,
both the adhesion frequency andadhesion force increased dramaticallywith contact time (see Figures 3b and3c), a finding that suggests theformation of multiple intermolecularbridges between HBHA and its
receptor. Presumably, the prolonged
contact time required to establish astrong HBHA-heparin interaction reflects
the time necessary for conformationalchanges to allow an optimal fitting
between the positive charges of theHBHA heparin-binding domain and thenegative sulphate groups of heparin.

Force-distance curves were recorded
between the HBHA tip and theheparin-coated substrate at a loading
rate of 10,000 pN/s. As shown in
Figure 3a, 32% of a total of 1,000
curves displayed adhesion forces;
the remaining curves exhibited noadhesion. The adhesion force
histogram revealed a bimodal
distribution with average rupture forcesof 50 ± 23 pN and 117 ± 18 pN,
attributed to one and two binding
events between HBHA and heparin.
The ~50 pN binding force may
correspond to the interaction strength
quantum between heparin and amonomeric form of HBHA, while the~117 pN force may be due either tothe simultaneous interaction of two
single HBHA molecules or to thebinding of a HBHA dimer.

b.
a.

c.

Figure 2. Functionalization of AFM tips andsubstrates with HBHA and heparin. HBHA wasengineered with a His-tag for site-directed
immobilization onto a NTA/EG-terminated tip.
This strategy allows the adhesins to be attached
at a low surface density, thereby ensuring
single-molecule binding events. Biotinylatedheparin was bound to gold substrates via
streptavidin and BBSA layers. Adapted withpermission from Nature Methods3.

Figure 3. (a) Representative force-distance curves and adhesion force histogram (n = 1,000)
obtained in PBS between a HBHA tip and a heparin substrate. A significant fraction of thecurves showed adhesion forces, with a mean magnitude of 50 ± 23 pN and 117 ± 18 pN,
which reflected the occurrence of one and two unbinding events between HBHA and heparin.
(b) The adhesion frequency and (c) adhesion force depended strongly on the interaction time(s)(constant loading rate of 10,000 pN/s). Adapted with permission from Nature Methods3.

a.

b.

c.

d.

Figure 4. (a) AFM topographic image recorded in PBS showing two M. bovis BCG cells on a polymer substrate (scale bar:2 µm). (b) Higher magnification image of the cell surface (scale bar: 100 nm). (c) Histogram and (d) spatially resolved map ofadhesion forces recorded with a heparin tip (scale bar: 100 nm). Single HBHA adhesins were detected and found to beorganized in nanodomains (red line). Adapted with permission from Nature Methods3.

Imaging Single Adhesins
on Living Bacteria
Having explored the HBHA-heparininteraction, we then mapped the spatial
distribution of individual HBHA on thesurface of living mycobacteria using
heparin-modified tips. For imaging
native cells, we mechanicallyimmobilized the cells onto porous
polymer membranes, an approach that
does not require chemical fixation or
drying of the specimen3. Figure 4ashows a topographic image of twoM. bovis BCG cells, in buffer solution,immobilized on a membrane. As
shown in Figure 4b, the cell surface
was fairly smooth and homogeneous,in agreement with the expected cellwall architecture. In an attempt to detect

HBHA molecules on the cell surface,multiple force-distance curves wererecorded in the force-volume mode over
300nm x 300nm areas. Importantly, thecurves showed adhesion forces withmean values centered at 53 ± 18 pN
and 110 ± 11 pN (see Figure 4c),which matched remarkably well thoseobtained for the model HBHA-heparininteraction (see Figure 3a). This finding
strongly suggests that the ~50 pN
binding force corresponds to thedetection of single HBHA, a conclusion
further supported by the observation thata mutant M. bovis BCG strain lacking
HBHA did not bind the heparin tip.
To address the question of how theadhesins distribute across the cellsurface, quantitative adhesion maps

were generated from the force-volumedata sets. As shown in Figure 4d,adhesion events (clear pixels) wereobserved in about half of the locations,which again were attributed to thedetection of single HBHA since theseevents were not observed on themutant strain. Interestingly, the HBHA
distribution was not homogeneous,
but apparently concentrated into
nanodomains for which we haveproposed the term adherosomes. These
nanostructures may play an importantbiological function in that they may
promote adhesion to target cells by
inducing the recruitment of receptors
within membrane rafts.