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OPEN Deciphering the mode of action

of cell wall-inhibiting antibiotics
using metabolic labeling of growing
Received: 28 November 2016
Accepted: 28 March 2017 peptidoglycan in Streptococcus
pyogenes
Published: xx xx xxxx

Atsushi Sugimoto, Asuka Maeda, Kaori Itto & Hirokazu Arimoto

Because of the scanty pipeline of antibiotics newly obtained from nature, chemical modification of
established drugs is one of the major streams of current antibacterial research. Intuitive and easy-to-
use assays are critical for identifying drug candidates with novel modes of action. In this study, we
demonstrated that metabolic fluorescent staining of growing cell walls is a powerful tool for mode-
of-action analyses of antibiotics using Streptococcus pyogenes. A set of major cell-wall-inhibiting
antibiotics (bacitracin, d-cycloserine, flavomycin, oxacillin, ramoplanin, and vancomycin) was
employed to validate the potential of the assay. The mechanistic differences of these antibiotics were
successfully observed. For instance, d-cycloserine treatment induced fluorescently stained, excessive
peripheral cell wall growth. This may indicate that the switch from the peripheral growth stage to the
succeeding septal growth was disturbed by the treatment. We then applied this assay to analyze a
series of vancomycin derivatives. The assay was sufficiently sensitive to detect the effects of single-
site chemical modification of vancomycin on its modes of action. This metabolic fluorescent labeling
method is easy to perform, especially because it does not require radiolabeled substrates. Thus, it is
suitable for the preliminary evaluation of antibacterial mechanisms during antibacterial research.

Antibiotic resistance is a global challenge, and effective strategies for identifying novel antibacterial compounds
with promising modes of action are in great demand. The minimum inhibitory concentration (MIC) is an essen-
tial criterion for screening large compound libraries or microbial extracts in the pursuit of new antibacterial
agents, but it does not provide insight into the modes of action. Therefore, the incorporation of radiolabeled
precursors into biomacromolecules, e.g., peptidoglycan, nucleotides, and proteins, is often examined to roughly
identify the targets of antibiotics.
Cell wall biosynthesis is a major target of antibiotics, including beta-lactam and glycopeptide antibiotics. In
vitro reconstitution of peptidoglycan biosynthesis using semi- or completely purified enzymes and radiolabeled
precursors has been utilized to analyze the action of cell wall-inhibiting antibiotics at the molecular level. A major
drawback of this in vitro assay is a limited supply of radiolabeled precursors, specifically those for late-stage pep-
tidoglycan synthesis. Chemical synthesis is currently the most practical means of supplying these key precursors,
but it often requires years of laborious synthetic efforts to provide several milligrams of precursors. In this context,
we recently reported the first chemical synthesis of depsi-lipid intermediates of vancomycin-resistant strains1.
Another concern regarding the reconstituted assay using a purified enzyme is that the system may not reflect the
true situation of cell wall biosynthesis as orchestrated by the dynamic interplay among multiple enzymes.
We envisaged that whole cell-based assays could compensate for the drawbacks of the in vitro enzyme-based
assay. To monitor the actions of cell wall-inhibiting antibiotics, efficient labeling methods for newly forming cell
walls are needed. Recently, Nelson et al. used endogenous sortase to label living Staphylococcus aureus2. Sortase
recognizes the specific C-terminal sequence of cell wall-binding proteins and connects them with the cell wall

Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan. Atsushi Sugimoto and Asuka Maeda
contributed equally to this work. Correspondence and requests for materials should be addressed to H.A. (email:
[email protected])

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Figure 1. Distribution of cell sizes and phenotypes of log-phase Streptococcus pyogenes in the absence of
antibiotics. (A) Schematic presentations of peripheral and septal cell wall synthesis in Streptococcus. (B) Typical
labeling patterns in metabolic labeling of Streptococcus pyogenes cell wall synthesis using a fluorescent sortase
A substrate. Phenotype A: cells without active cell wall synthesis. Phenotype B: cells undergoing peripheral
growth. Phenotype C: cells undergoing septal growth. Each phenotype reflects the bacterial cell cycle. (C)
Definition of axial and equatorial length in this study. (D) Typical fluorescent images and the average cell-size
distribution of phenotypes A–C (n = 3). Images: PG (growing peptidoglycan stained using the sortase method),
Membrane (Nile red staining), DNA (DAPI), and Overlay (overlay of PG and Membrane images); scale bar:
2 μm. Blue bars in histograms represent the cell size distribution of all cells. Orange bars in histograms represent
the size distribution cells with the indicated phenotypes. Statistical analysis regarding the histograms is available
in Supplementary Table S1.

precursor lipid II3. By employing an artificial sortase-substrate with a fluorescent chromophore, microscopic
observation of newly synthesized S. aureus cell walls was successfully demonstrated2. However, the application
of this strategy for living cells has been limited to S. aureus4 because sortases have different substrate specificities
among bacteria.
In this study, we demonstrated that metabolic labeling can be a powerful and quick means to obtain insight
into the modes of action of new antibacterial compounds. This imaging-based assay with group A streptococcus
(GAS, Streptococcus pyogenes) was able to distinguish the actions of six major cell wall-inhibiting antibiotics
together with a series of structurally similar vancomycin derivatives.

Results
Live monitoring of S. pyogenes cell wall biosynthesis using sortase-mediated metabolic labe-
ling. GAS causes a wide variety of diseases in humans. The first important step toward a new imaging-based
assay was the optimization of labeling conditions because no study had examined labeling of the GAS cell wall
by the sortase method before. Sortase A was known to accept peptides with a C-terminal LPXTG sequence in
both GAS and S. aureus, and form an intermediate that is subsequently linked to peptidoglycan. We found that
sufficient labeling of a mid-log phase GAS proceeded within 1 h with the fluorescent substrate FL-KLPETG-NH2
while using the sortase method, whereas it required 12 h with S. aureus originally used by Nelson et al.2. Regarding
live cell monitoring of cell wall biosynthesis, the observed large difference in labeling efficiency is noteworthy
because labeling should be as fast as possible. Thus, we decided to use the GAS JRS4 strain and sortase-mediated
labeling for our study (Fig. 1).
Streptococcal cell wall synthesis consists of cylindrical peripheral synthesis and septal synthesis (Fig. 1A). A
study using Streptococcus pneumoniae illustrated that the serine-threonine kinase StkP controls the switch from
peripheral synthesis to septal synthesis5. Splitting of the septum (cell separation) is then mediated by the action of
autolysin. We labeled the GAS cell wall using the sortase method in the absence of antibiotics, and the observed
labeling patterns of GAS were classified into three phenotypes (Fig. 1A–C). Phenotype A cells are newly divided

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cells without specific fluorescent labeling. Phenotype B cells are in the peripheral growth stage. A characteris-
tic two-elongated-dot image or an open ring corresponds to peripheral growth near the bacterial division site.
Phenotype C cells are in the septal growth stage, and the dividing septum is fluorescently stained. The distribution
(%) of phenotypes A, B, and C among cells was 37 ± 2, 47 ± 2, and 16 ± 1, respectively, in log-phase GAS. Data
represent the mean ± sem (n = 3). Subsequently, we constructed a histogram of each phenotype population as a
function of bacterial cell length, as defined in Fig. 1C (orange, Fig. 1D). The subpopulation of cells with a specific
phenotype is overlaid on the total cell size distribution (blue). The histograms suggested that GAS elongates
mostly along the axial direction in the progression from phenotype A to phenotype C, and growth along the
equatorial direction is smaller. The histograms also illustrate that cells grow from phenotype A, through pheno-
type B, to phenotype C (followed by cell separation), confirming that peripheral growth precedes septal growth in
GAS. We speculated that the changes of this histogram would provide information on antibiotic modes of action.

Histogram analyses of cell size and phenotypes in the presence of cell wall-inhibiting anti-
biotics. We then performed similar histogram analyses in the presence of cell wall-inhibiting antibiotics
namely bacitracin, flavomycin, d-cycloserine, oxacillin, and ramoplanin. Because we employed these drugs at
their subbacteriostatic concentrations, metabolic-fluorescent labeling could proceed slowly in living cells (see
the Materials and methods section for determination of subbacteriostatic concentration for each antibiotic).
Although all of these antibiotics are known to inhibit peptidoglycan synthesis, the observed abnormalities in
bacterial size and shape varied among the antibiotic treatments. These results may be due to the differences in the
stages of cell wall biosynthesis inhibited by the compounds.

Bacitracin and ramoplanin halted cell wall growth and reduced the size of GAS cells. Bacitracin
suppresses the formation of late-stage peptidoglycan intermediates (lipid intermediates) by inhibiting lipid phos-
phorylase. These lipid intermediates are used in both peripheral and septal growth. In the bacitracin-treated
bacteria, the distribution (%) of each phenotype among the cells was 31 ± 5, 52 ± 5, and 12 ± 0 for phenotypes A,
B, and C respectively (n = 3). A slight accumulation of phenotype-B cells (peripheral growth stage) was observed,
but the overall distribution was similar to that of non-treated cells. A rapid decline of essential substrates by bac-
itracin treatment might halt each stage of cell wall synthesis. Bacitracin was also found to reduce both axial and
equatorial length distributions (Fig. 2A) compared with the findings in non-treated cells (Fig. 1).
Ramoplanin is a lipopeptide antibiotic that inhibits the intracellular glycosyltransferase MurG which converts
lipid I to lipid II via the addition of an N-acetylglucosamine. As the outcome of this effect, ramoplanin can also
suppress the formation of essential lipid intermediates for cell wall synthesis. In the ramoplanin-treated bacteria
(Fig. 2B), the average distribution (%) of each phenotype among cells was 27 ± 3 (phenotype A), 50 ± 4 (pheno-
type B), and 23 ± 5 (phenotype C) (n = 3). The distribution was similar to that of bacitracin, and this may reflect
the modes of action of these antibiotics.
Although the specific actions of ramoplanin on S. pyogenes are not fully known, a previous study reported
using purified E. coli MurG and E. coli PBP1b, the major transglycosylase in the organism, and the transglyco-
sylation step was found to be preferentially blocked compared with the MurG step6. Thus, we were interested in
whether this second action of ramoplanin (inhibition of cell wall polymerization) resulted in a different phe-
notype distribution from that of bacitracin. However, the results suggested that ramoplanin acts mainly as an
inhibitor of lipid intermediate formation in S. pyogenes at subbacteriostatic concentration.

Flavomycin inhibited cell separation. Flavomycin is a glycolipid antibiotic that suppresses peptidoglycan
polymerization by inhibiting transglycosylase. In the flavomycin-treated bacteria (Fig. 3), the average propor-
tions (%) of the phenotypes among the cells was 4 ± 1 (phenotype A), 20 ± 6 (phenotype B), 22 ± 2 (phenotype
C), and 54 ± 5 (phenotype D) (n = 3). The predominant phenotype (D), which was not observed under normal
conditions, had multiple septa in an elongated cell (Fig. 3A). The elongated cell with several septa could also be
regarded as a cell cluster with defective cell separation, but we treated this phenotype D as a single cell that had
not completed a normal cell division cycle in this study. The cell separation step is a necessary step for normal
growth. Labeling of these multiple septa suggested that septal growth proceeded in the presence of flavomycin at
subbacteriostatic concentration. The accumulation of phenotype D thus would imply that flavomycin selectively
stopped the cell separation step of S. pyogenes and that the subsequent peripheral and septal cell wall synthesis
could proceed regardless of the failure of the preceding cell separation step. This process may eventually provide
an additional septum in an elongated cell. The low population of phenotype A also supported this interpreta-
tion because it implied the failure of normal cell division in most growing cells. As flavomycin is considered an
inhibitor of the transglycosylation step of peptidoglycan polymerization mediated by penicillin-binding proteins
(PBPs), these results (inhibition of cell separation rather than cell wall synthesis) at subbacteriostatic concentra-
tion were unexpected. In fact, the inhibitory effect of flavomycin on cell separation was reported recently for S.
aureus7, but to our knowledge, it has not been well described for S. pyogenes. Strepcococci use multiple enzymes
(PBP1A, PBP1B, and PBP2A) for peptidoglycan polymerization8. An interesting future study would involve iden-
tification of specific PBPs responsible for the defect of cell separation.

GAS exhibited enlarged cell size and phenotype B was accumulated after d-cycloserine or oxa-
cillin treatment. d-Cycloserine is a cyclic analog of d-alanine that blocks the early cytoplasmic phase of
peptidoglycan synthesis by inhibiting d-alanyl-d-alanyl ligase (Ddl). Thus, d-cycloserine treatment produces
abnormal tripeptidic cell wall precursors (vs normal pentapeptidic precursors)9. By contrast, oxacillin blocks the
late stage of peptidoglycan biosynthesis by inhibiting the action of PBPs that cross-link the immature peptidogly-
can. It is intriguing to observe that these mechanistically different inhibitors induced apparently similar enlarged
cell morphology.

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Figure 2. Average distribution of cell sizes and phenotypes of bacitracin (4 μg/mL)-treated Streptococcus
pyogenes (A) and ramoplanin (2 μg/mL)-treated S. pyogenes (B) (n = 3). Images: PG (growing peptidoglycan
stained using the sortase method), Membrane (Nile red staining), DNA (DAPI), and Overlay (overlay of PG
and Membrane images); scale bar: 2 μm. Blue bars in histograms represent the cell size distribution of all cells.
Orange bars in histograms represent the size distribution cells with the indicated phenotypes. Statistical analysis
regarding the histograms is available in Supplementary Table S2.

In the d-cycloserine-treated bacteria (Fig. 4A), the average proportions (%) of the phenotypes among cells
were 25 ± 6 (phenotype A), 68 ± 7 (phenotype B), 2 ± 1 (phenotype C), and 5 ± 1 (phenotype D) in three inde-
pendent experiments. The accumulation of cells staying at the peripheral growth stage (phenotype B) was there-
fore characteristic. Spherical enlarged cells, in which both axial and equatorial lengths increased, were observed
for most phenotype-B cells (Fig. 4A). Conversely, cells at the septal growth stage (phenotype C) were scarce.
The abnormal tripeptidic precursors might inhibit switching from the peripheral (phenotype B) to the septal
growth stage (phenotype C). As previously mentioned, the streptococci serine-threonine kinase StkP regulates
the switch from peripheral synthesis to septal synthesis5. The phenotype-D population, which was predominant
in the flavomycin-treated cells, was small (5%) under these conditions.
In the oxacillin-treated bacteria (Fig. 4B), the average proportions (%) of each phenotype among the cells were
19 ± 4 (phenotype A), 60 ± 4 (phenotype B), 12 ± 5 (phenotype C), and 9 ± 2 (phenotype D) (n = 3). The accu-
mulation of phenotype-B cells and enlargement of cells were also observed in the presence of oxacillin. Although
the cell size distributions of the d-cycloserine- and oxacillin-treated cells (blue, Fig. 4A and B) were similar, we
further investigated the differences of mechanisms of the two antibiotics other than the abnormal enlarged mor-
phology in the following section.

Imaging of peripheral cell wall growth revealed the differences of d-cycloserine and oxacillin
concerning the mechanism of cell enlargement. The observed enlargement of cells implied that cell
wall synthesis could proceed, at least partly, in the presence of these antibiotics at their subbacteriostatic con-
centrations. We therefore studied the relationship between peripheral growth length and axial length for cells
via dual-color staining. Nile red was used to stain the bacterial cell membrane, and the fluorescent d-amino acid
(FDAA) labeling10–12 was employed for old cell wall (preexisting prior to antibiotic treatment). In the presence of
antibiotic, peripheral growth length was measured as a gap between the old (stained) regions of the preexisting
cell wall (Fig. 5A and B).
As depicted in Fig. 4, both d-cycloserine- and oxacillin-treated cells had similar cell size distribu-
tions. However, a dot blot analysis (Fig. 5C) demonstrated that the peripheral growth/axial length ratio of

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Figure 3. Average distribution of cell sizes and phenotypes of flavomycin (also known as moenomycin) (1 μg/
mL)-treated Streptococcus pyogenes (n = 3). (A) Phenotype D represents cells with multiple septal growths
(left: schematic presentation of phenotype D in flavomycin-treated S. pyogenes; right: microscopic image of a
phenotype-D cell stained using the sortase method). (B) Typical fluorescent images and cell size distributions
of phenotypes A–D. Images: PG (growing peptidoglycan stained using the sortase method), Membrane (Nile
red staining), DNA (DAPI), and Overlay (overlay of PG and Membrane images); scale bar: 2 μm. Blue bars
in histograms represent the cell size distribution of all cells. Orange bars in histograms represent the size
distribution of cells with the indicated phenotypes. Statistical analysis regarding the histograms is available in
Supplementary Table S3.

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Figure 4. Average distribution of cell sizes and phenotypes of d -cycloserine (100 μg/mL)-treated Streptococcus
pyogenes (A) and oxacillin (4 μg/mL)-treated S. pyogenes (B) (n = 3). Images: PG (growing peptidoglycan
stained using the sortase method), Membrane (Nile red staining), DNA (DAPI), and Overlay (overlay of PG
and Membrane images); scale bar: 2 μm. Blue bars in histograms represent the cell size distribution of all cells.
Orange bars in histograms represent the size distribution cells with the indicated phenotypes. Statistical analysis
regarding the histograms is available in Supplementary Table S4.

d-cycloserine-treated cells was considerably larger than that of oxacillin-treated cells. This result indicated that
enlargement of d-cycloserine-treated cells was the result of continued excessive peripheral growth, which may
have been due to suppression of the switch from peripheral to septal growth. We then examined whether the
region of the excessive peripheral growth lacked the d-Ala- d-Ala peptide using Van-FL staining (Fig. 5D).
Van-FL, a fluorescent vancomycin derivative, has been used for detecting the d-Ala- d-Ala substructure because
of its specific binding to this peptide. This indicates that this region was created from the abnormal tripeptidic
precursors. Because the d-Ala- d-Ala sequence is essential for the cross-linking (transpeptidation) of immature
peptidoglycan, the region may lack sufficient strength as a cell wall. Accordingly, site-specific bulge formation was
often observed in the d-cycloserine-treated S. pyogenes (Fig. 5E).
In contrast to the results of d-cycloserine treatment, the peripheral growth length could not explain the
increased size of oxacillin-treated cells as assessed by dot blot analysis. Cell enlargement induced by oxacillin
should be mediated by the growth of the remaining preexisting region (presumably the outer wall region). In S.
aureus, oxacillin was found to delocalize PBP2, a major S. aureus enzyme responsible for peptidoglycan polymer-
ization, over the entire surface of the cell13. Similar PBP delocalization may also be the cause of un-orchestrated
cell wall synthesis in the outer wall region of S. pyogenes.

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Figure 5. Excessive peripheral growth is the major cause of cell enlargement in d-cycloserine-treated cells,
whereas peripheral growth has little contribution to cell size in oxacillin-treated cells. (A) Streptococcus pyogenes
was pre-labeled with FDAA (1 h) and then allowed to grow without FDAA in the presence of d-cycloserine/
oxacillin (3 h). scale bar: 2 μm. (B) Schematic presentation of peripheral and axial lengths in these experiments.
The length of peripheral growth was estimated as the gap between old (stained) cell wall regions. (C) A dot blot
analysis of the peripheral-growth/axial-length relationships of each cell suggested that peripheral growth barely
contributed to cell enlargement in oxacillin-treated cells. Cells were treated with antibiotics for 3 h. Non-treated
cells were analyzed 1 h after the FDAA pre-labeling because of their rapid growth. (D) Van-FL was used to stain
cell walls with d-Ala-d-Ala terminus (30 min prior to analysis). The peripheral wall is expected to exist at a gap
between old peptidoglycan regions. Van-FL staining did not localize at the newly synthesized peripheral cell
wall (white arrow heads) in the presence of d-cycloserine. (E) Cell lysis in the presence of d-cycloserine at an
excessive peripheral growth region; scale bar: 2 μm.

Application of cell wall labeling for mechanistic analyses of vancomycin and its semi-synthetic
derivatives CBPV and ΔNCBPV. Vancomycin is the drug of choice for treating infections caused by
multi-resistant gram-positive pathogens, including MRSA. CBPV is a synthetic derivative of vancomycin with a
chlorobiphenylmethyl modification at the sugar moiety of vancomycin (Fig. 6A)14. Oritavancin, which also pos-
sesses a chlorobiphenylmethyl substituent was recently approved by US Food and Drug Administration (FDA) as
antibacterial agent. These lipoglycopeptides display excellent activity against vancomycin-resistant strains, and
the mechanism of the enhanced activity has been the subject of other studies15, 16. We applied our imaging-based
analysis to obtain further insights into the mechanisms of these vancomycin derivatives.
We first examined the effect of unmodified vancomycin on GAS morphology and cell cycle progression.
Vancomycin exerts its antimicrobial activity via a specific interaction with the d-Ala-d-Ala terminus of cell wall
intermediates on the bacterial surface. This mechanism is unique among the antibiotics used in this study, as
only vancomycin targets the enzymatic substrate opposed to the enzyme itself. We found that vancomycin (4 μg/
mL) inhibited the growth of GAS, but the cell size distribution was not affected along the axial and equatorial
directions (Fig. 6B). The cell size distribution was similar to that in the absence of antibiotics at the log phase
(Fig. 1). Moreover, the distribution of phenotypes A–C, which reflected the cell cycle stage of each cell, was also
least affected. The proportions (%) were 34 ± 4 (phenotype A), 49 ± 4 (phenotype B), and 17 ± 1 (phenotype C)
(n = 3). The results were similar to those for bacitracin and ramoplanin but distinct from those with other cell
wall-inhibiting antibiotics used in this study (e.g., d-cycloserine, flavomycin, and oxacillin). This can be ration-
alized because all of these three antibiotics reduce the levels of the precursors available for cell wall synthesis.
Bacitracin and ramoplanin are established inhibitors of cell wall precursor biosynthesis, whereas vancomycin

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Figure 6. Average distributions of cell sizes and phenotypes of vancomycin (4 μg/mL)-treated Streptococcus
pyogenes (n = 3). (A) Chemical structures of vancomycin, chlorobiphenyl vancomycin (CBPV), and des-N-
methylleucyl CBPV (ΔNCBPV). (B) Typical fluorescent images and cell size distribution of phenotypes A–C
in the presence of vancomycin. Images: PG (growing peptidoglycan was stained using the sortase method),
Membrane (Nile red staining), DNA (DAPI), and Overlay (overlay of PG and Membrane images); scale bar:
2 μm. Blue bars in histograms represent the cell size distribution of all cells. Orange bars in histograms represent
the size distribution of cells with the indicated phenotypes. Statistical analysis regarding the histograms is
available in Supplementary Table S5.

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Figure 7. Average distributions of cell sizes and phenotypes of chlorobiphenyl vancomycin (0.25 μg/mL)-
treated Streptococcus pyogenes (A,B) and des-N-methylleucyl chlorobiphenyl vancomycin (ΔNCBPV)
(3 μg/mL)-treated S. pyogenes (C) (n = 3). (A and C) Typical fluorescent images and cell size distribution of
phenotypes A–D′. Images: PG (growing peptidoglycan stained using the sortase method), Membrane (Nile
red staining), DNA (DAPI), and Overlay (overlay of PG and Membrane images); scale bar: 2 μm. Blue bars
in histograms represent the cell size distribution of all cells. Orange bars in histograms represent the size
distribution cells with the indicated phenotypes. Statistical analysis regarding the histograms is available in
Supplementary Table S6. (B) Schematic drawing and labeled PG image of phenotype D′.

strongly binds to the precursors to sterically suppress their use for enzymatic reactions. Regardless of these differ-
ences, the overall outcome is the depletion of cell wall precursors.
Next, we investigated the effects of CBPV on S. pyogenes. The cells exhibited an axially elongated morphology
(phenotype D′) that was not observed in vancomycin-treated cells. This implies distinct molecular modes of inhi-
bition of CBPV from vancomycin (Fig. 7A). CBPV-treated cells were distributed into phenotypes A (20 ± 1%), B
(58 ± 6%), C (9 ± 2%), and D′ (13 ± 4%) in three independent experiments. Phenotype D′ had multiple division
sites in axially elongated cells similar to that of the related phenotype D (see the data for flavomycin-treated

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Figure 8. Inhibitory effects of antibiotics on S. pyogenes cell division.

cells, Fig. 3). Phenotype D cells had multiple completed septa in cells. Phenotype D′ cells were different from
phenotype-D cells, because they had multiple peripheral growth sites but rarely exhibited completed septa
(Fig. 7B).
Only 4% of the CBPV-treated cells with multiple division sites (n = 55) possessed one or more completed
septa, versus 98% (n = 114) of flavomycin-treated cells. This marked difference probably indicates the difference
of antibacterial mechanisms for these two antibiotics, namely that flavomycin inhibited the cell separation step
of the cell cycle, whereas CBPV inhibited the septal growth stage. This idea may also be supported by the fact
that an extremely small population of cells remained at septal synthesis (phenotype C) during CBPV treatment.
The cause of this potential selectivity among peripheral and septal synthesis is unclear at this stage. Collectively,
vancomycin and CBPV have distinct in the modes of cell wall inhibition.
Subsequently, we focus on the derivative ΔNCBPV (Fig. 6A), which suppresses S. pyogenes growth with simi-
lar potency (3 μg/mL) as vancomycin (4 μg/mL). ΔNCBPV is a derivative of CBPV that lacks an N-methyl-leucyl
residue at its terminus. Many studies established that d-Ala-d-Ala-binding by vancomycin involves this
N-methyl-leucyl residue and that des-N-methyl-leucyl-vancomycin totally loses its antibacterial activity
against gram-positive strains. However, ΔNCBPV has reduced but strong antibacterial potency. Consequently,
CBPV is likely to be a dual-mechanism inhibitor of cell wall synthesis based on its vancomycin-derived
d-Ala-d-Ala-binding ability (requiring N-methylleucine) and the effects of the lipophilic modification at its sugar
moiety. ΔNCBPV appears to be a useful compound to analyze the roles of the lipophilic modification on antibac-
terial activity (Fig. 7C).
We found the morphologies of ΔNCBPV-treated GAS cells were distinct from those of vancomycin-treated
and CBPV-treated cells, although the morphologies of ΔNCBPV-treated GAS cells were more similar to those
of CBPV-treated cells. The proportions (%) of each phenotype were 14 ± 1 (phenotype A), 45 ± 12 (phenotype
B), 17 ± 8 (phenotype C), and 24 ± 3 (phenotype D′) (n = 3). The accumulation of phenotype D′ indicated that
ΔNCBPV also efficiently suppressed septal synthesis but allowed peripheral synthesis. The ratio of cells with at
least one completed septum among all phenotype-D cells was 52% in the ΔNCBPV-treated cells (n = 105), versus
98% (n = 114) in flavomycin-treated S. pyogenes.
A previous kinetic study using purified enzyme and radiolabeled substrate revealed that CBPV and ΔNCBPV
inhibit the same transglycosylation step of peptidoglycan biosynthesis via a direct interaction with PBPs16.
Flavomycin is also known to bind to the same transglycosylase domain of purified high-molecular-weight PBPs.
Our whole-cell-based assay suggested the possibility that flavomycin may behave as a cell separation inhibitor
rather than a transglycosylase inhibitor for living bacteria.
Most previous synthetic studies of vancomycin derivatives speculated that the resulting derivatives should
exhibit enhanced antibacterial actions with similar mechanisms as vancomycin17. This is in part due to the lack
of a concise, easy-to-perform assay. The results in this section clearly indicate that even a single-site modification
could dramatically change the modes of action of glycopeptides, in line with previous biochemical studies16, 18.
However, our assay may be a more concise, readily available alternative means by which to analyze such changes
in actions.

Discussion
In this study, we observed peripheral and septal cell wall syntheses in ovococcal S. pyogenes using
“sortase-mediated” metabolic labeling for the first time. Fluorescent labeling of peripheral growth was observed
in close proximity to the mid-cell division site in accordance with previous results obtained with S. pneumo-
niae19. The fluorescent labeling of peripheral cell wall synthesis is a significant advantage of our assay because this
synthesis cannot be observed by optical microscopy without labeling. This novel imaging-based assay was then
applied to analyze the actions of major antibiotics (Fig. 8). Because sortase exists in most gram-positive bacteria,
our technique may also be applied in bacteria other than S. pyogenes.
Metabolic labeling techniques are not limited to the sortase-based method, and are attracting significant atten-
tion in microbiology and its related fields. FDAA is also incorporated into growing bacterial cell wall10–12. This
FDAA-based method may also be applied for imaging-based mode-of-action analyses of antibiotics in the near
future. However, the incorporation of FDAA may be interfered by antibiotics, because it is mediated by PBPs20
that are the targets of antibiotics, including beta-lactams. Sortase is less likely to be interfered by these antibiotics,
and thus, we decide to use sortase in this study.

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By visualizing the growing peptidoglycan, we first illustrated that the enlarged morphology of
d-cycloserine-treated cells is ascribed to the excessive peripheral synthesis. We also demonstrated that flavo-
mycin or CBPV treatment resulted in abnormally elongated cells with multiple division sites, which may be
related to a recent report of S. pneumoniae21. The S. pneumoniae study demonstrated that PBPs migrate to the
next division site and that they initiate peripheral synthesis before the completion of septal synthesis. Thus, it
may be possible to assume that neither of these drugs suppresses the migration of PBPs or peripheral synthesis
resulting in abnormally elongated cells possessing multiple division sites. Other examined antibiotics (bacitracin,
ramoplanin, oxacillin, and vancomycin) displayed unique inhibitory patterns that were different from those of
the aforementioned drugs.
The above intriguing observations with CBPV, a lipoglycopeptide, may support the importance of our tech-
nique in current antibacterial research. Lipoglycopeptides have recently been attracting exceptional attention
because of recent approval of oritavancin, telavancin, and dalbavancin from the FDA as antibacterial agents22.
Oritavancin and CBPV share identical lipophilic modifications in their structures. Several lipoglycopeptides are
being investigated in non-/pre-clinical studies18, 23–25. Our assay would be useful in the medicinal chemistry of
lipoglycopeptide antibiotics.
In conclusion, our imaging-based integrated protocols can offer invaluable rapid feedback to the medicinal
chemists regarding newly synthesized or isolated antibacterial compounds and thus can accelerate the selection of
drug leads based on both potent activity and antibacterial modes of action. These protocols may also be applicable
to other pathogens.

Materials and Methods

Bacterial strain. S. pyogenes strain JRS4 (M6+ F1+) was grown in Todd-Hewitt broth supplemented with
0.3% yeast extract (THY) at 37 °C.

Chemicals. Bacitracin, d-cycloserine, oxacillin, vancomycin, DAPI and Nile red stain were purchased
from Wako Chemical Industries, Ltd. (Tokyo, Japan). Flavomycin and ramoplanin were obtained from
Sigma-Aldrich Co. (St. Louis, MO). Van-FL stain is a product of Molecular ProbesTM. Chlorobiphenyl vancomy-
cin (CBPV), des-N-methylleucyl CBPV (ΔNCBPV), the fluorescently labeled sortase substrate FL-KLPETG-NH2
(FL = Nε-fluorescein-lysine)2, and FDAAs12 (FL-d-Lys for Fig. 5A and E; tetramethylrhodamine- d-Lys for
Fig. 5D) were synthesized according to the literature procedures.

Determination of subbacteriostatic concentrations. S. pyogenes were grown in THY to an OD600 of

0.4 (exponential phase) at 37 °C and then added a solution of antibiotic of varying concentration. The culture
mixture was further incubated for 4 h at 37 °C. Subbacteriostatic concentration in this study is the minimum dose
of antibiotic that makes growth curve reach a plateau at OD600 of <1.0 after the 4-h incubation. In the absence of
antibiotics, GAS cells grew up to OD600 of approximately 1.6. Subbacteriostatic concentrations were determined
for each antibiotic, and mode-of-action analyses were performed at these concentrations (Figs 2, 3, 4, 5, 6 and 7).

Live cell labeling with sortase A substrate in the presence of antibiotics. Bacterial cells grown in
THY to an OD600 of 0.35–0.4 (exponential phase) at 37 °C were exposed to antibiotics and incubated for 1 h, and
100-μL aliquots of culture mixture were taken and added to a solution of sortase substrate (FL-KLPETG-NH2,
final concentration = 1 mM). After incubation for 2.5 h at 37 °C in the dark, Nile Red (10 μg/mL) and DAPI (1 μg/
mL) were added. The mixture was allowed to stand for 30 min and then centrifuged (12,000 rpm, 2 min, 4 °C).
The supernatant was removed, and the pellet was washed thrice with PBS buffer to remove unreacted sortase sub-
strate. Cells were re-suspended in PBS buffer, mounted on a glass slide, and examined by fluorescent microscopy.
Images were analyzed manually using ImageJ software (ver. 1.50). Sets of axial- and equatorial-length were also
recorded for each cell. At least 200 cells were analyzed in each analysis. All experiments were performed at least
thrice, and representative results are shown.

Imaging of peripheral growths in d-cycloserine/oxacillin-treated cells. Bacterial cells grown in

THY to an OD600 of 0.2 (exponential phase) at 37 °C, and 100-μL aliquots of culture mixture were taken (Fig. 5).
FDAA solution (final concentration = 1 mM; FL-d-Lys for Fig. 5A and E, tetramethylrhodamine- d-Lys for
Fig. 5D) was added, and the mixture was incubated for 1 h at 37 °C in the dark. The mixture was centrifuged
(12,000 rpm, 2 min, 4 °C), the supernatant was removed, and the pellet was washed with PBS buffer thrice to
remove unreacted FDAA. Cells were re-suspended in 100-μL THY and incubated at 37 °C in the dark with antibi-
otics for 2.5 h. A reagent (10 μg/mL Nile Red for Fig. 5A and E; 1 μg/mL Van-FL for Fig. 5D) was then added to the
mixture, which was allowed to stand for 30 min before centrifugation (12,000 rpm, 2 min, 4 °C). The supernatant
was removed, and the pellet was washed thrice with PBS buffer. Cells were re-suspended in PBS buffer, mounted
on a glass slide, and examined by fluorescent microscopy.
The datasets generated during and/or analysed during the current study are available from the corresponding
author on reasonable request.

References
1. Nakamura, J. et al. Staphylococcus aureus Penicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant
Strains for Cell Wall Synthesis. Chem. Eur. J. 19, 12104–12112 (2013).
2. Nelson, J. W. et al. A biosynthetic strategy for re-engineering the Staphylococcus aureus cell wall with non-native small molecules.
ACS Chem. Biol. 5, 1147–1155 (2010).
3. Mazmanian, S. K., Liu, G., Ton-That, H. & Schneewind, O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins
to the cell wall. Science 285, 760–763 (1999).
4. Hansenová Maňásková, S. et al. Synthetic LPETG-containing peptide incorporation in the Staphylococcus aureus cell-wall in a
sortase A- and growth phase-dependent manner. PLoS One 9, e89260 (2014).

Scientific Reports | 7: 1129 | DOI:10.1038/s41598-017-01267-5 11

www.nature.com/scientificreports/

5. Fleurie, A. et al. Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation
and division. PLoS Genet. 10, e1004275 (2014).
6. Fang, X. et al. The mechanism of action of ramoplanin and enduracidin. Mol. Biosyst. 2, 69–76 (2006).
7. Yuan, Y. et al. Structural Analysis of the Contacts Anchoring Moenomycin to Peptidoglycan Glycosyltransferases and Implications
for Antibiotic Design. ACS Chem. Biol. 3, 429–436 (2014).
8. Massidda, O., Nováková, L. & Vollmer, W. From models to pathogens: how much have we learned about Streptococcus pneumoniae
cell division? Environ. Microbiol. 15, 3133–3157 (2013).
9. Sieradzki, K. & Tomasz, A. Suppression of beta-lactam antibiotic resistance in a methicillin-resistant Staphylococcus aureus through
synergic action of early cell wall inhibitors and some other antibiotics. J. Antimicrob. Chemother. 39, 47–51 (1997).
10. Lam, H. et al. D-Amino acids govern stationary phase cell wall remodeling in bacteria. Science 325, 1552–1555 (2009).
11. Cava, F., de Pedro, M. A., Lam, H., Davis, B. M. & Waldor, M. K. Distinct pathways for modification of the bacterial cell wall by non-
canonical D-amino acids. EMBO J 30, 3442–3453 (2011).
12. Kuru, E. et al. In Situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angew. Chem. Int.
Ed. Engl. 51, 12519–12523 (2012).
13. Pinho, M. G. & Errington, J. Recruitment of penicillin-binding protein PBP2 to the division site of Staphylococcus aureus is
dependent on its transpeptidation substrates. Mol. Microbiol 55, 799–807 (2005).
14. Eggert, U. S. et al. Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin. Science 294,
361–364 (2001).
15. Butler, M. S., Hansford, K. A., Blaskovich, M. A., Halai, R. & Cooper, M. A. Glycopeptide antibiotics: back to the future. J Antibiot
(Tokyo) 67, 631–644 (2014). Qiao, Y. et al. Detection of lipid-linked peptidoglycan precursors by exploiting an unexpected
transpeptidase reaction. J. Am. Chem. Soc. 136,14678–14681 (2014).
16. Ge, M. et al. Vancomycin derivatives that inhibit peptidoglycan biosynthesis without binding D-Ala-D-Ala. Science 284, 507–511
(1999).
17. Williams, D. H. & Bardsley, B. The vancomycin group of antibiotics and the fight against resistant bacteria. Angew. Chem. Int. Ed.
Engl. 38, 1173–1193 (1999).
18. Miura, K. et al. Mode of action of Van-M-02, a novel glycopeptide inhibitor of peptidoglycan synthesis, in vancomycin-resistant
bacteria. Antimicrob. Agents Chemother. 54, 960–962 (2010).
19. Pinho, M. G., Kjos, M. & Veening, J. W. How to get (a)round: mechanisms controlling growth and division of coccoid bacteria. Nat.
Rev. Microbiol. 11, 601–614 (2013).
20. Qiao, Y. et al. Detection of lipid-linked peptidoglycan precursors by exploiting an unexpected transpeptidase reaction. J. Am. Chem.
Soc. 136, 14678–14681 (2014).
21. Tsui, H. C. et al. Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division
of Streptococcus pneumoniae D39. Mol. Microbiol. 94, 21–40 (2014).
22. Zeng, D. et al. Approved glycopeptide antibacterial drugs: mechanism of action and resistance. Cold Spring Harb. Perspect. Med 6,
a026989 (2016).
23. James, R. C., Pierce, J. G., Okano, A., Xie, J. & Boger, D. L. Redesign of glycopeptide antibiotics: back to the future. ACS Chem. Biol.
7, 797–804 (2012).
24. Yoganathan, S. & Miller, S. J. Structure diversification of vancomycin through peptide-catalyzed, site-selective lipidation: a catalysis-
based approach to combat glycopeptide-resistant pathogens. J. Med. Chem. 58, 2397–2377 (2015).
25. Yarlagadda, V. et al. Glycopeptide antibiotic to overcome the intrinsic resistance of Gram-negative bacteria. ACS Infect. Dis 2,
132–139 (2016).

Acknowledgements
This work was funded in part by JSPS KAKENHI Grant Number JP21310136.

Author Contributions
H.A. and A.S. designed the study. A.S. and A.M. performed experiments and analyzed data. K.I. analyzed data.
H.A. and A.M. wrote the manuscript.

Additional Information
Supplementary information accompanies this paper at doi:10.1038/s41598-017-01267-5
Competing Interests: The authors declare that they have no competing interests.
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