B

15Piezo displacement

10520100 pN

20 nm

Frequency (%)

2025 85%
Force100 pN

25ForceFrequency (%)

A20 nm15Piezo displacement

105000100

2000300

100200300

Adhesion force (pN)

D

100

300
250

Frequency (%)

Adhesion force (pN)

C200
150
100
50

80
60
40
20000

1000

10000

100000

Loading rate (pN/s)

0500

1000

1500

2000

Interaction time (ms)Figure 2. Single-molecule force spectroscopy of the vancomycin/D-Ala-D-Ala interaction on synthetic support. Gold tips are functionalized with bis(vancomycin)
cysteamide while gold supports terminated with OEG and OEG propionic acid moieties are covalently reacted with D-Ala-D-Ala peptides. (A) Representative
force curve and adhesion histogram (n = 978) obtained in PBS buffer between a vancomycin tip and a D-Ala-D-Ala support, with a high reproducibility. (B) Control
experiment achieved with free D-Ala-D-Ala peptides showing a dramatic decrease of adhesion frequency. (C) Plot of the adhesion force as a function of thelogarithm of the loading rate during the retraction, while keeping constant the interaction time and the approach speed (100 measurements for each data point). (D)Plot of the adhesion frequency as a function of the interaction time while keeping constant the approach and the retraction speed (100 measurements for each datapoint)13.

were performed using Veeco’s
triangular-shaped silicon nitride
cantilevers. For force measurements,
the maximum applied force was kept at
250 pN to minimize indentation. Affinity
images were obtained by recording
16 x 16 force distance curve array,and calculating the adhesion force foreach over a certain area. The spring
constants of the cantilevers were foundto be ~0.011 N/m13.
DYNAMIC FORCE SPECTROSCOPY USINGVANCOMYCIN TIPS ON SYNTHETICSUPPORT

Force distance curve measurements
confirm single and specific adhesion
forces between vancomycin and
D-Ala-D-Ala on synthetic support. The
dynamic spectroscopy data show aslow binding process. The equilibrium
dissociation constant of this event
was found to be 0.4 mM, i.e. higher
than in previous studies17. This can beExperiment

Validation

Results

Recording force distancecurves Figure 2A vs. control
experiment Figure 2B

Single moleculeadhesion forces

98 ± 33 pN(n = 978)

Recording force curves withvarying loading rates
Figure 2C

Length scale of energy
barrierxB ~ 0.36 nm

Kinetic off-rate constantof dissociation at zeroforce

Koff = 2x10–3 s–1

Interaction time needed
for half-maximal
probability of bindingt0.5 = 0.25 s

Association rate constant
Kon = t0.5–1NAVeff

Kon = 5 M–1 s–1

Equilibrium constant
KD = Koff/Kon

KD = 0.4 mM

Varying interaction time
while keeping the loading
rate constant Figure 2D

The chemical strategy is described in Figure 1 with a summary of the experimental data measured andextrapolated accordingly13, 14-17

PAGE 2 DRUG INTERACTIONS INVESTIGATED USING ATOMIC FORCE MICROSCOPY WITH FUNCTIONALIZED TIPS

B

C88%

100 pNA

100 nm

DE

2.0

20 nm

1.5
1.0

Piezo displacement

0.5
00F

100

200

98%
2.5

Force

Frequency (%)

3.0

2.0
1.5

300

100 pN

500 nm

2.5Force

Frequency (%)

3.0

20 nm

Piezo displacement

1.0
0.50

500 nm

100 nm0100

200300Adhesion force (pN)
Figure 3. Imaging and probing of D-Ala-D-Ala sites on living bacteria. (A) AFM image showing a single wt Lactococcus latics in versalia cell trapped into a porous
polymer membrane especially adapted for non-invasive and in situ imaging during a division process. The septum area as well as ring-like structures are thought
to be rich in newly synthesized peptidoglycan (white box). (B, C) Adhesion cartography and adhesion force histogram (n = 1536) with representative force curves
recorded between vancomycin tips and the living bacterium in the septum area. (D, E, F) Same experiments carried out on a mutant bacterium exhibiting
D-Ala-D-Lac endings instead of D-Ala-D-Ala, resulting in a spectacular reduction of the unbinding events, both on the adhesion mapping and on the histogram13.

for vancomycin22. The specificity ofthis interaction was proven by theuse of a mutant bacterium producing
peptidoglycan precursors ending by
D-Ala-D-Lac instead of D-Ala-D-Ala.

explained by steric hindrance effects
around vancomycin and D-Ala-DAla peptides, which may alter therecognition process.
MAPPING UNBINDING EVENTS ON
LIVING BACTERIACONCLUSIONAs shown in Figure 313, vancomycin tips
were used to map the distribution of
single D-Ala-D-Ala peptides on living
Lactococcus lactis19-22. Force curves
were recorded showing unbinding
events in about 12% of the cases.
As opposed to previous experiments
on a planar support, the adhesion force
histogram (n = 1536) revealed a bimodal
distribution of adhesion forces withmaxima at 83 ± 22 pN and 150 ± 16 pN(Figure 3C). The first peak is thought
to reflect single interactions since its
value is close to the one found on theplanar support (~98 pN). The otherpeak may reflect cooperative binding,which is a well-known phenomenon

Single-molecule AFM force
measurements with antibiotic-modified
tips enable the measurement ofantibiotic unbinding forces and mappingof their corresponding ligands on live
bacteria. Relevant parameters canbe extracted from force spectroscopy
measurements, providing information
about the specificity and dynamics ofthe drug-target interaction, which canbe of great help for the development
and improvement of new drugs.

de Recherche), the Federal Officefor Scientific, Technical and Cultural
Affairs (Interuniversity Poles ofAttraction Programme), and theAustrian Science Foundation (projectP-15295). Work in the laboratory ofJ. Errington was funded by a grant fromthe BBSRC. We thank L. Piraux for theuse of the thermal evaporator, E. Ferain
for the use of the scanning electron
microscope. Y.F. Dufrêne and P. Hols
are Research Associates of the FNRS.
Published with permission of theAmerican Chemical Society.

ACKNOWLEDGEMENTS

This work was supported by theNational Foundation for Scientific
Research (FNRS), the Université
catholique de Louvain (Fonds Spéciaux

DRUG INTERACTIONS INVESTIGATED USING ATOMIC FORCE MICROSCOPY WITH FUNCTIONALIZED TIPS PAGE 3