Direct observation of Staphylococcus aureus cell wall digestion by lysostaphin.

Abstract

The advent of Staphylococcus aureus strains that are resistant to virtually all antibiotics has increased the need for new antistaphylococcal agents. An example of such a potential therapeutic is lysostaphin, an enzyme that specifically cleaves the S. aureus peptidoglycan, thereby lysing the bacteria. Here we tracked over time the structural and physical dynamics of single S. aureus cells exposed to lysostaphin, using atomic force microscopy. Topographic images of native cells revealed a smooth surface morphology decorated with concentric rings attributed to newly formed peptidoglycan. Time-lapse images collected following addition of lysostaphin revealed major structural changes in the form of cell swelling, splitting of the septum, and creation of nanoscale perforations. Notably, treatment of the cells with lysostaphin was also found to decrease the bacterial spring constant and the cell wall stiffness, demonstrating that structural changes were correlated with major differences in cell wall nanomechanical properties. We interpret these modifications as resulting from the digestion of peptidoglycan by lysostaphin, eventually leading to the formation of osmotically fragile cells. This study provides new insight into the lytic activity of lysostaphin and offers promising prospects for the study of new antistaphylococcal agents.

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JOURNAL OF BACTERIOLOGY, Dec. 2008, p. 7904–7909
0021-9193/08/$08.00?0doi:10.1128/JB.01116-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 24
Direct Observation of Staphylococcus aureus Cell Wall Digestion
by Lysostaphin?
Gre ´gory Francius,1Oscar Domenech,2Marie Paule Mingeot-Leclercq,2and Yves F. Dufre ˆne1*
Unite ´ de Chimie des Interfaces, Universite ´ Catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium,1and
Unite ´ de Pharmacologie Cellulaire et Mole ´culaire, Universite ´ Catholique de Louvain, Avenue E. Mounier 73,
B-1200 Brussels, Belgium2
Received 8 August 2008/Accepted 25 September 2008
The advent of Staphylococcus aureus strains that are resistant to virtually all antibiotics has increased the
need for new antistaphylococcal agents. An example of such a potential therapeutic is lysostaphin, an enzyme
that specifically cleaves the S. aureus peptidoglycan, thereby lysing the bacteria. Here we tracked over time the
structural and physical dynamics of single S. aureus cells exposed to lysostaphin, using atomic force micros-
copy. Topographic images of native cells revealed a smooth surface morphology decorated with concentric rings
attributed to newly formed peptidoglycan. Time-lapse images collected following addition of lysostaphin
revealed major structural changes in the form of cell swelling, splitting of the septum, and creation of
nanoscale perforations. Notably, treatment of the cells with lysostaphin was also found to decrease the
bacterial spring constant and the cell wall stiffness, demonstrating that structural changes were correlated with
major differences in cell wall nanomechanical properties. We interpret these modifications as resulting from
the digestion of peptidoglycan by lysostaphin, eventually leading to the formation of osmotically fragile cells.
This study provides new insight into the lytic activity of lysostaphin and offers promising prospects for the
study of new antistaphylococcal agents.
The gram-positive bacterium Staphylococcus aureus is a ver-
satile pathogen capable of causing a wide range of human
diseases (23, 28). Peptidoglycan, the major cell wall compo-
nents of S. aureus, is known to be the target of important
antibiotics (22, 39). Among these, ?-lactams (e.g., penicillins)
exert their antimicrobial activity by interfering with the en-
zymes (penicillin binding proteins) specifically involved in pep-
tidoglycan synthesis, while glycopeptides (e.g., vancomycin)
bind with high affinity and specificity to peptidoglycan precur-
sors, thereby preventing their incorporation into the bacterial
cell wall (38). Currently, there is a constant need for new
antistaphylococcal drugs owing to the development of antibi-
otic-resistant strains (4, 38, 39). An example of such a novel
drug is lysostaphin, an enzyme secreted by Staphylococcus
simulans which specifically cleaves the peptidoglycan cross-
linking pentaglycine bridges of S. aureus, thereby hydrolyzing
the cell wall and lysing the bacteria (7, 27, 33). Due to its
unique specificity, lysostaphin could have high potential in the
treatment of antibiotic-resistant staphylococcal infections (27).
For instance, lysostaphin, given alone or in combination with
vancomycin, was shown to be more effective in the treatment of
experimental methicillin-resistant S. aureus aortic valve endo-
carditis than vancomycin alone (9). Lysostaphin was also re-
ported to be effective in the treatment of eye infections caused
by S. aureus (12).
Knowledge of the molecular mechanisms underlying pepti-
doglycan digestion by lysostaphin is a key toward its efficient
use in antistaphylococcal therapies. Early structural investiga-
tions using electron microscopy revealed that S. aureus cells
exposed to lysostaphin showed perforations and more exten-
sive damage, including the separation of walls from the plasma
membranes and the disintegration of large sections of the walls
(34). As a consequence, most cells exposed to the enzyme were
rendered osmotically fragile. Among these, cells that retain the
capacity to revert to normal staphylococci were designated
spheroplasts, as opposed to protoplasts, in which the whole cell
wall was digested away. Although powerful, electron micros-
copy techniques are not suited for tracking dynamic, time-
dependent processes on single cells, and they cannot probe the
cell wall mechanical properties.
Recently, atomic force microscopy (AFM) (20, 30) has pro-
vided new opportunities for studying microbial cell walls at the
single-cell and single-molecule levels (14, 15). The technique is
particularly well-suited for visualizing the surfaces of single live
cells while they grow (11, 31, 37), yielding information on cell
wall assembly and dynamics that cannot be obtained with tra-
ditional microscopies. For instance, Touhami et al. (37) were
able to monitor cell growth and division events in S. aureus
using AFM combined with thin-section transmission electron
microscopy. Nanoscale holes were seen around the septal an-
nulus at the onset of division and were attributed to cell wall
structures possessing high autolytic activity. After cell separa-
tion, concentric rings were observed on the surface of the new
cell wall and were suggested to reflect newly formed pepti-
doglycan. AFM can also be used to study the effect of drugs on
microbial cell walls. In one such study, mycobacteria were
observed prior to and after incubation with isoniazid, ethion-
amide, ethambutol, or streptomycin (1). Upon drug treatment,
major structural alterations were observed in the form of lay-
ered structures, striations, and porous morphologies, suggest-
ing that they reflect the inhibition of the synthesis of three
* Corresponding author. Mailing address: Unite ´ de Chimie des In-
terfaces, Universite ´ Catholique de Louvain, Croix du Sud 2/18, B-1348
Louvain-la-Neuve, Belgium. Phone: (32) 10 47 36 00. Fax: (32) 10 47
20 05. E-mail: yves.dufrene@uclouvain.be.
?Published ahead of print on 3 October 2008.
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major cell wall constituents, i.e., mycolic acids, arabinans, and
proteins.
Here, we used AFM to track the structural and physical
dynamics of single S. aureus cells exposed to lysostaphin. AFM
images show that the enzyme causes substantial swelling of the
cells, favors splitting of the septum, and induces the creation of
nanoscale perforations. Consistent with this, nanomechanical
measurements demonstrate that these structural changes cor-
relate with a major decrease of bacterial turgor pressure and
cell wall stiffness, reflecting the formation of osmotically fragile
cells.
MATERIALS AND METHODS
Bacterial strain and growth conditions. Staphylococcus aureus (methicillin-
susceptible S. aureus strain ATCC 25923; American Type Culture Collection
[ATCC], Manassas, VA) was grown in Mueller-Hinter broth (Becton Dickinson,
France) to mid-exponential growth phase (optical density at 600 nm ? 0.6) at
37°C with continuous stirring. Cells were centrifuged (7,000 rpm for 10 min) and
washed twice by resuspension in phosphate-buffered saline (PBS) and centrifu-
gation.
AFM measurements. AFM images and force-distance curves were recorded in
PBS solution (10 mM PBS, 150 mM NaCl, pH 7.4) at room temperature, using
a Nanoscope IV multimode AFM (Veeco Metrology Group, Santa Barbara,
CA). Cells were immobilized by mechanical trapping into porous polycarbonate
membranes (Millipore) with a pore size similar to the bacterial cell size (13, 26).
After a concentrated cell suspension was filtered, the filter was gently rinsed with
PBS, carefully cut (1 cm by 1 cm), and attached to a steel sample puck (Veeco
Metrology Group) using a small piece of double-face adhesive tape, and the
mounted sample was transferred into the AFM liquid cell while avoiding dew-
etting. The spring constants of the cantilevers were measured using the thermal
noise method (Picoforce; Veeco Metrology Group), yielding a mean value of
0.0255 ? 0.001 N/m. For lysostaphin experiments, PBS solutions containing 16
?g/ml lysostaphin from S. simulans (Sigma, Belgium) were injected into the
AFM liquid cell.
Mechanical properties were measured by recording arrays of 32-by-32 force
curves, using a maximum applied force of 1.75 to 2 nN to avoid sample damage.
The bacterial spring constant, kb, was determined from the slope of the linear
portions of the curves for raw deflection (nm) versus piezo displacement (nm)
(5): kb? (kC? s)/(1 ? s), where kCis the spring constant of the cantilever and s
the observed slope. The bacterial Young modulus was obtained by fitting the
curves for force versus indentation using the Hertz model with a conical (tip)-
plane (cell surface) geometry (24): F ? {2Etan?/[?(1 ? ?2)]} ? ?2, where F is the
force, ? is the indentation depth, E is the Young modulus, ? is the Poisson
coefficient, and ? is the semi-top angle of the tip. This simple model is valid for
elastic surfaces and does not take into account tip-surface adhesion. The latter
assumption is reasonable since adhesion forces were negligible in the present
study. The mathematical analysis was performed with an automatic Fortran
C?? algorithm as described elsewhere (16, 17).
RESULTS AND DISCUSSION
High-resolution imaging of S. aureus. Cells immobilized in
porous membranes were imaged in buffer solution. A repre-
sentative low-resolution deflection image of a single cell is
shown in Fig. 1A. Due to the large curvature of the specimen,
height images had fairly poor resolution while deflection im-
ages were much more sensitive to the surface relief. Using
small imaging forces (?100 pN), images of the same area were
obtained repeatedly without detaching the cell or significantly
altering the surface morphology. The cell, located at the center
of the image, was surrounded by artifactual features resulting
from the contact between the AFM tip and the pore edges. The
cell surface was smooth and showed circular or concentric
rings enclosing a central depression. The high-resolution
height image recorded on top of the cell, shown in Fig. 1B,
reveals that the rings were separated by 20 to 50 nm. These
features are similar to those reported earlier by Touhami et al.
(37), who observed concentric rings on the newly formed cell
walls of S. aureus cells but not on older regions of the cell walls.
The ring structures are also reminiscent of the circular patterns
seen by several groups using electron microscopy techniques
(2, 3, 6, 21, 22). We therefore conclude that the rings reflect
structural features of the peptidoglycan strands found on the
surface of the new wall after daughter cells have separated. As
pointed out by Touhami et al. (37), the concentricity of the
rings implies that the peptidoglycan strands are themselves
oriented in a similar fashion and are added or removed in an
orderly manner. Note that a number of cells did not show any
FIG. 1. Imaging of single S. aureus cells. (A) Low-resolution de-
flection image recorded in PBS, showing a single cell trapped in a pore
of the polycarbonate membrane. (B) High-resolution height image
recorded in the square region highlighted in panel A, together with a
vertical cross-section taken along the white line.
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ring structures, presumably because their surfaces consisted of
older cell wall material (Fig. 2, upper panels).
Lysostaphin induces major structural alterations. Pepti-
doglycan is an important component of the S. aureus cell wall,
conferring strength and rigidity to the cell, allowing growth and
division, maintaining cell shape, and protecting against osmotic
lysis (8, 21). In S. aureus and other gram-positive bacteria,
where the external membrane is absent, the multilayered pep-
tidoglycan represents the limit of the cell and therefore the site
for exchanges and interactions with the outside environment
(10). In particular, peptidoglycan is known to be an important
target for antimicrobial compounds (35), including lysostaphin,
which hydrolyzes the pentapeptide cross-linkages (27).
With this in mind, we used in situ AFM imaging to observe
structural changes on single S. aureus cells exposed to lyso-
staphin. Figure 2 shows height and deflection images recorded
for the same cell following incubation with 16 ?g/ml lyso-
staphin for 0, 80, 210, and 260 min. As can be seen in the
vertical cross-sections, a significant increase in cell size was
noted over the course of the experiment, with the cell height
increasing by ?100 nm after 260 min. In agreement with early
FIG. 2. Imaging of single S. aureus cells following incubation with
lysostaphin. A series of height (left) and deflection (right) images
recorded in real time for a single cell prior to and after incubation with
16 ?g/ml lysostaphin in PBS for 80, 210, and 260 min is shown. The
lower panel compares vertical cross-sections taken at 0 min (continu-
ous line) and 260 min (dashed line). White arrows show nanoscale
perforations enlarging with time, while black arrows show splitting of
the septum. Similar data were obtained with different cells from four
independent cultures.
FIG. 3. Imaging of lysostaphin-treated cells at high resolution. A
series of deflection images recorded on top of a single cell after incu-
bation with 16 ?g/ml lysostaphin for 20, 40, 80, 170, 210, and 260 min
is shown. The lower panel shows the temporal evolution of the root
mean square roughness measured on height images.
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viability studies (25, 34), we interpret such cell swelling as
evidence for the formation of lysostaphin-induced osmotically
fragile cells, resulting from peptidoglycan hydrolysis.
Moreover, progressive alteration of the cell surface structure
was clearly observed after lysostaphin addition. After 80 min,
nanoscale perforations were seen, which were about 50 to 100
nm in diameter and 25 to 75 nm in depth (Fig. 2). With time
(e.g., after 260 min), these holes enlarged until they merged
together to form larger perforations. These structures are rem-
iniscent of the small depressions seen at the onset of division
(37) and may reflect so-called murosomes (22), i.e., regions of
the cell wall having high autolytic activity. Interestingly, split-
ting of the septum was observed after 210 min (Fig. 2). We
note that these features, i.e., perforations and splitting of the
septum, were never observed when cells were imaged in PBS in
the absence of lysostaphin, even for prolonged periods of time
(up to 240 min), confirming that they result from the lytic
activity of the enzyme. It is also worth mentioning that proto-
plasts in which the cell wall had been completely digested
could never be observed by AFM, most likely because they
were immediately ruptured by the scanning tip. Besides local-
ized surface modifications, we also found that lysostaphin in-
creased the cell surface roughness (Fig. 3). Power spectral
density analysis of the fast Fourier transform of height images
revealed that the root mean square roughness for native and
treated cells increased with the length scale to reach a plateau
after 200 nm. Notably, the roughness on 500-nm by 500-nm
height images increased from 1.6 ? 0.3 nm for untreated cells
to 5.4 ? 1.2 nm for cells treated for 200 min (Fig. 3). These
results are qualitatively consistent with earlier electron micros-
copy investigations (34) showing progressive disintegration of
the cell wall and separation from the plasma membrane after
exposure to lysostaphin.
Taken together, our AFM images demonstrate that incuba-
tion of S. aureus with lysostaphin causes substantial swelling of
the cell body, favors splitting of the septum, induces nanoscale
perforations, and increases the cell surface roughness. Presum-
ably, these time-dependent cell wall modifications result from
peptidoglycan digestion, eventually leading to the formation of
osmotically fragile spheroplasts.
Lysostaphin alters the cell wall mechanical properties. In
view of the role of peptidoglycan in providing rigidity and
protection against osmotic lysis, we then addressed the perti-
nent question as to whether the observed structural changes
were correlated with differences in cell wall mechanical prop-
erties. To this end, cells incubated with lysostaphin were
probed using nanoindentation measurements (Fig. 4 and 5).
Figure 4A shows typical force-versus-piezo displacement
curves obtained for the polymer support and for a single cell at
increasing incubation times. Consistent with the work of
Gaboriaud et al. (18), the curves recorded on the cell surface
showed two domains, i.e., a nonlinear domain at low loading
forces followed by a linear one at high loading forces. From the
shape of these curves, it can be seen that the cell wall was
FIG. 4. Mechanical properties of native and lysostaphin-treated cells. (A) Representative force-versus-displacement curves recorded on the
polymer support (dashed line, S) and on a single cell prior to (0 min) and after (20, 60, 100, and 300 min) incubation with lysostaphin at 16 ?g/ml.
Open symbols are the raw data, while the solid lines show the theoretical fit (Hooke’s law) used to extract the bacterial spring constant.
(B) Evolution of the bacterial spring constant as a function of incubation time. Each data point represents the mean and standard deviation of 1,024
measurements obtained at different locations in the same cell. (C) Force-indentation curves obtained from the curves shown in panel A. Open
symbols show the data, while the solid lines show the theoretical fits (Hertz model) used to extract the Young modulus values. (D) Evolution of
the Young modulus as a function of incubation time. Each data point represents the mean and standard deviation of 1,024 measurements obtained
at different locations in the same cell. Similar behaviors were observed for six different cells using two different tips.
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already substantially softer than the support and that lyso-
staphin caused a progressive softening of the cell wall.
To provide quantitative information on the cell wall me-
chanical properties, two parameters were extracted from the
force curves, i.e., the bacterial spring constant, which is related
to the inner turgor pressure of the cell (5, 18, 19, 40), and the
Young modulus, which reflects cell surface elasticity (17, 18,
29, 36). Bacterial spring constant values, kb, were determined
from the slopes of the linear portions of the force-versus-piezo
displacement curves (Fig. 4A). The kbvalue of native cells was
found to be 0.013 ? 0.001 N/m (Fig. 4B), which agrees rea-
sonably well with values reported earlier for six Lactobacillus
strains (0.016 to 0.053 N/m) (32). Our slightly lower values
could be due to the higher ionic strength that we used or to
differences in the cell wall organization. Notably, incubation
with lysostaphin caused a progressive decrease of kb,from
0.013 ? 0.001 N/m to 0.006 ? 0.001 N/m after 100 min of
exposure. This change may be attributed to a decrease of the
inner turgor pressure of the cell (5, 18, 19, 40), in agreement
with the observed cell swelling (Fig. 2) and with the notion that
lysostaphin generates osmotically fragile cells.
Next, we estimated Young modulus values by converting the
force curves into force-indentation curves and analyzing them
using the Hertz theory (Fig. 4C). Figure 4D shows that within
100 min, the Young modulus of the cells decreased from 1,764 ?
218 kPa to 189 ? 49 kPa, demonstrating that lysostaphin in-
duces a dramatic reduction of cell wall stiffness. This finding is
fully consistent with the mode of action of the enzyme, which
cleaves the peptidoglycan cross-linking pentaglycine bridges.
Also of note is the comparison between the level of indentation
and cell swelling: for a constant force of 1 nN, the difference in
indentation depth (for native cells versus cells treated for 300
min) was ?100 nm, which is on the order of the height increase
observed in the images (Fig. 2). Finally, arrays of 32-by-32
force curves were recorded across the cell surface to map local
variations of elasticity. As shown in Fig. 5, elasticity maps on
native and treated cells showed rather uniform contrasts, re-
flecting homogeneous distributions of elasticity. The homoge-
neously dark contrast observed after 80 min confirmed that the
entire cell wall was softened upon treatment with the enzyme.
In conclusion, we have observed, for the first time in situ and
on a nanoscale, the digestion of the S. aureus cell wall by
lysostaphin, revealing that the enzyme causes substantial swell-
ing of the cells and major alterations of their surface structure
(septum splitting, nanoscale perforations, and increased
roughness). Using force measurements, we also showed that
these structural changes correlate with major differences in
mechanical properties, i.e., with a decrease of bacterial turgor
pressure and of cell wall stiffness. This study shows that AFM
is a promising tool for exploring the organization and assembly
of peptidoglycan and for investigating its interactions with en-
zymes and drugs.
FIG. 5. Mapping of cell surface elasticity. (A and D) High-resolution deflection images recorded for an S. aureus cell prior to and after
incubation with lysostaphin for 80 min. (B and E) Elasticity maps (z-range ? 3,000 kPa). (C and F) Distribution of elasticity values (n ? 1,024 force
curves) corresponding to the elasticity maps.
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ACKNOWLEDGMENTS
This work was supported by the National Foundation for Scientific
Research (FNRS); the Universite ´ Catholique de Louvain (Fonds Spe ´-
ciaux de Recherche); the Federal Office for Scientific, Technical and
Cultural Affairs (Interuniversity Poles of Attraction Programme); and
the Research Department of the Communaute ´ Franc ¸aise de Belgique
(Concerted Research Action). Y.F.D. is a research associate at the
FNRS.
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