Increased Cell Wall Teichoic Acid Production and D-alanylation Are Common Phenotypes among Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus (MRSA) Clinical Isolates
et al. (2013) Increased Cell Wall Teichoic Acid Production and D-
alanylation Are Common Phenotypes among Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus (MRSA) Clinical Isolates.
PLoS ONE 8(6): e67398. doi:10.1371/journal.pone.0067398
Increased Cell Wall Teichoic Acid Production and D-alanylation Are Common Phenotypes among Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus (MRSA) Clinical Isolates
Ute Bertsche 0
Soo-Jin Yang 0
Daniel Kuehner 0
Stefanie Wanner 0
Nagendra N. 0
Tobias Roth 0
Mulugeta Nega 0
Alexander Schneider 0
Christoph Mayer 0
Arnold S. Bayer 0
Christopher Weidenmaier 0
Willem van Schaik, University Medical Center Utrecht, The Netherlands
0 1 Interfakultares Institut fur Mikrobiologie und Infektionsmedizin , Microbial Genetics , University of Tubingen , Tubingen, Germany , 2 Division of Infectious Diseases, LA Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, United States of America, 3 David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America , 4 Cecolabs UG, Tubingen, Germany , 5 Interfakultares Institut fur Mikrobiologie und Infektionsmedizin, University of Tubingen , Tubingen, Germany , 6 Interfakultares Institut fur Mikrobiologie und Infektionsmedizin, Microbiology/Biotechnology, University of Tubingen , Tubingen , Germany
Multiple mechanisms have been correlated with daptomycin-resistance (DAP-R) in Staphylococcus aureus. However, one common phenotype observed in many DAP-R S. Aureus strains is a thickened cell wall (CW). The first evidence for an impact of CW-linked glycopolymers on this phenotype was recently demonstrated in a single, well-characterized DAP-R methicillin-susceptible S. aureus (MSSA) strain. In this isolate the thickened CW phenotype was linked to an increased production and Dalanylation of wall teichoic acids (WTA). In the current report, we extended these observations to methicillin-resistant daptomycin-sensitive/daptomyin-resistant (DAP-S/DAP-R) strain-pairs. These pairs included methicillin-resistant S. aureus (MRSA) isolates with and without single nucleotide polymorphisms (SNPs) in mprF (a genetic locus linked to DAP-R phenotype). We found increased CW dry mass in all DAP-R vs DAP-S isolates. This correlated with an increased expression of the WTA biosynthesis gene tagA, as well as an increased amount of WTA in the DAP-R vs DAP-S isolates. In addition, all DAP-R isolates showed a higher proportion of WTA D-alanylation vs their corresponding DAP-S isolate. We also detected an increased positive surface charge amongst the DAP-R strains (presumably related to the enhanced D-alanylation). In comparing the detailed CW composition of all isolate pairs, substantive differences were only detected in one DAP-S/DAP-R pair. The thickened CW phenotype, together with an increased surface charge most likely contributes to either: i) a chargedependent repulsion of calcium complexed-DAP; and/or ii) steric-limited access of DAP to the bacterial cell envelope target. Taken together well-defined perturbations of CW structural and functional metrics contribute to the DAP-R phenotype and are common phenotypes in DAP-R S. Aureus isolates, both MSSA and MRSA.
Funding: UB was supported by German Research Foundation Grant SFB766. CW was supported by German Research Foundation
Grants TR-SFB34 and SFB766. This study was supported in part by a grant from the National Institutes of Health (NIAID)
RO1AI-039108-15 (to ASB), a Beginning Grant-in-Aid from the American Heart Association (Western States Affiliate) 12BGIA11780035 (to
SJY) and a Research Seed Grant from the Los Angeles Biomedical Research Institute (to S-JY). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Daniel Kuehner is employed by the University of Tuebingen as a technical assistant in the department of
Microbial Genetics. Simultaneously he recently founded the company Cecolabs Unternehmergesellschaft (http://www.cecolabs.de) as a
spin-off of the department of Microbial Genetics. The company is specialized in analyses of bacterial peptidoglycan structures.
Regarding the results presented here there is no financial or non-financial competing interest. As a technician, DK isolated, digested,
and analyzed cell wall of the test strains. Experiments were designed and interpreted by Ute Bertsche. UB and CM are members of the
advisory board of Cecolabs UG. This does not alter the authors' adherence to all the PLOS ONE policies and sharing data and materials.
The rising number of multi-antibiotic-resistant strains
has seriously limited the treatment options in severe S.
aureus infections (e.g. MRSA; VISA) [1,2]. In this regard,
daptomycin (DAP) has become one of the most
important therapeutic agents [3,4]. The recent
emergence of DAP-resistant (DAP-R) strains,
associated with clinical treatment failures , has
spiked an interest in determining the molecular bases
of DAP-R. Interestingly, DAP-R can be linked to several
distinct, and perhaps, unrelated mechanisms, and is
often multifactorial. In a number of DAP-R isolates,
expression of genes that are involved in maintenance
of the bacterial surface positive charge (e.g., dltA-D;
mprF) is perturbed, usually translating into
gain-infunction phenotypes . The phenotypic readout
of such gains-in-function has been enhanced positive
envelope surface charge, presumably creating a
charge-repulsive milieu, mitigating calcium-DAP: cell
membrane (CM) interactions . In addition,
DAPinduced changes in CM permeabilization , as well
as alterations in CM biophysical order (resulting in
extremes of CM fluidity or rigidity) have also been
observed in relation to the DAP-R phenotype .
Although not a universal association , the most
frequently described genetic mutations observed in
DAP-R S. aureus strains are single point mutations
(SNPs) in various regions of the mprF open reading
frame, with or without additional point mutations in the
yyc operon [11,1416]. MprF is responsible for the
lysinylation of phosphatidylglycerol (PG)  and flips
the positively-charged product, lysyl-PG (L-PG) to the
outer CM leaflet . The yyc operon encodes for the
YycFG (WalKR) two-component regulatory system,
which is believed to regulate fatty acid biosynthesis
 and also to modulate general CW homeostasis to a
variety of stressors .
Of interest, in many, but not all, DAP-R S. aureus
strains, a thickened CW phenotype has been
documented by electron microscopy [8,9,13]. In this
regard, our labs have recently provided the first
evidence that this thickened CW phenotype is linked to
an increased expression of wall teichoic acid (WTA)
biosynthesis genes (tag), in a single, well-characterized
methicillin-susceptible S. aureus (MSSA) DAP-R isolate
. WTA biosynthesis is a complicated process (Figure
S1), starting with synthesis of a disaccharide linkage
unit, which requires the enzymes TagO and TagA
[22,23]. These enzymes transfer GlcNAc-1-phosphate
and ManNAc, respectively, from UDP-activated
precursor molecules to undecaprenyl-phosphate (C55
P). The repeating units are then incorporated by
several priming and polymerizing enzymes, and after
biosynthesis is completed, the repeating units are
modified with D-alanine . The dltABCD operon
encodes the required enzymes, and is therefore
responsible for the modulation of the net charge of the
teichoic acid polymers . The enhanced expression
of the tagA gene in the single DAP-R MSSA strain noted
above correlated with elevated WTA production; this
DAP-R strain also demonstrated increased dltA
expression, which was associated with augmentation in
the proportionality of WTA D-alanylation. On the other
hand no significant changes in CW peptidoglycan
crosslinkage or in the O-acetylation profiles (as had been
previously reported for other DAP-R strains ) were
found in this DAP-R MSSA strain.
In the current report, we expand upon the
preliminary report above  by: i) investigating WTA
production and D-alanylation profiles in a cadre of
DAPS/DAP-R MRSA strain-pairs; ii) studying DAP-R strains,
both with and without mprF SNPs; and iii) utilizing
advanced HPLC techniques to adjudicate comparative
CW muropeptide compositional analyses of the DAP-S/
Material and Methods
The four DAP-S/DAP-R MRSA study pairs used in this
investigation were clinical bloodstream isolates from
the Cubist Pharmaceuticals isolate collection (courtesy
of Dr. Aileen Rubio; Lexington, MA). This strain-set was
prioritized for the current study because it has been
previously well-characterized in terms of: i) strain-pair
isogenicity ; ii) antimicrobial peptide
crossresistances ; iii) CM metrics ; and iv)
demonstration of a thickened CW phenotype among
the DAP-R isolates . As previously documented, the
DAP-S and DAP-R isolates within a strain-pair were
isogenic on the basis of PFGE analysis, agr typing, spa
typing, inferred clonal complex typing and SCCmec
typing . The DAP-R isolates of the CB5021-CB5020
(resistant) and CB5062-CB5063 (resistant) pairs
contain no mprF or yyc operon SNPs, whereas the
DAPR strain of the CB1663/CB1664 strain-pair carries single
point mutations in both mprF and yycG that lead to
amino acid exchanges L826F in MprF and R86H in
YycG, respectively . The genotyping and SNP data
have been previously reported . The strain-pair,
CB5088/CB5089 exhibits no CW thickening in the
DAPR strain, and was included as a control. Strain CB5089
contains a point mutation that leads to the amino acid
exchange S295L in MprF.The daptomycin MICs and SNP
characteristics are listed in Table 1. These data have
been previously reported 
Wall teichoic acid (WTA) isolation and
We isolated CW and WTA specifically as described in
detail before [24,25]. In brief, bacteria were cultivated
overnight in B-Medium (1% peptone, 0.5% yeast
extract, 0.1% glucose, 0.5% NaCl and 0.1% K2HPO4)
containing 0.25% (wt/vol) glucose, washed twice in
sodium acetate buffer (20 mM, pH 4.7) and disrupted in
the same buffer with glass beads for 1h on ice in a cell
disruptor (Euler). We determined the total amount of
protein-free CW contained within our strain-sets by
weighing the CW preparation after drying. The CW dry
weight determinations were derived from 5
independent isolations. To allow better strain to strain
comparability cell wall dry weight was expressed as mg
cell wall dry weight per g cell wall wet weight. In
parallel, WTA was released from purified CWs by
treatment with 5% trichloroacetic acid in sodium
acetate buffer for 4 h at 60C. CWs were removed by
centrifugation. WTA was quantified by determining its
inorganic phosphate (Pi) content as described . The
isolation was performed in triplicate for each strain,
and assayed in triplicate for their respective Pi content.
Quantification of D-alanine content
D-alanylation of the WTA polymers was assayed and
quantified as described before [21,26]. In brief,
Dalanine esters were hydrolyzed by a mild alkaline
hydrolysis carried out at 37C for 1 h in 0.1 M NaOH.
The supernatant was neutralized, dried under vacuum,
and used for precolumn derivatization with Marfeys
reagent (1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide;
Sigma). Amino acid derivates (detection at 340 nm)
were then separated as described before  and
analyzed with the ChemStation software. Data were
expressed as percent of WTA ( SD) that was
Dalanylated. A minimum of three independent runs was
Quantification of dlt and tagA expression
We examined the relationship between WTA
production and D-alanylation profiles with gene
expression related to these two phenotypes (tagA and
dltA, respectively) [21,27,28]. For RNA sample
preparation, fresh overnight cultures of S. aureus
strains were used to inoculate TSB to an optical density
at 600 nm of 0.1. Cells were harvested during both
exponential and stationary growth phases. Total RNA
was isolated from the cell pellets by using the RNeasy
kit (Qiagen, Valencia, CA) and the FASTPREP FP120
instrument (BIO 101, Vista, CA), according to the
manufacturers recommended protocols.
Primers to amplify dltA were dlt-F-1 and dlt-R [8,21].
Primers for tagA were tagA-F and tagA-R . All
RTPCR experiments were performed in triplicate, with the
gyrB gene expression used as a control and baseline
for fold-changes in expression of tagA and dltA.
Surface charge assays
We determined the relative surface charge with a
cytochrome c binding assay as described previously
. BHI broth overnight cultures were washed with 20
mM MOPS buffer (pH 7.0) and then resuspended in the
same buffer at OD578 = 1.0. Cells were incubated with
0.5 mg/ml cytochrome c for 10 min, and the amount of
cytochrome c remaining in the supernatant was
determined spectrophotometrically at OD530 nm. The
more unbound cytochrome c was detected in the
supernatant, the more relative positive charge on the
bacterial surface. Data were expressed as mean ( SD)
amount of unbound cytochrome c. At least three
independent runs were performed on separate days.
Muropeptide analysis by HPLC
All strains were grown in Mueller-Hinton broth to an
OD578 = 0.7 or for 24 hrs. If indicated, 0.7 g/l glycine or
alanine was added. The CW of the study strains was
isolated, then digested with a muramidase, and
analyzed via HPLC essentially as described before 
(Cecolabs; Tuebingen, Germany). The analyses were
done on an Agilent 1200 system with a Prontosil
C18RP column (Bischoff Chromatography, Leonberg,
Table 1. Bacterial strains.
Amino acid analysis
The CW of the strain-pair, CB1663 and CB1664, was
isolated after 24 h in triplicate (see above). The
lyophilized CW was hydrolyzed by adding 600 l 6N HCl
to 45 mg substrate and incubated by 110C for at least
18 h. The released amino acids were then neutralized
for 72 h in NaOH atmosphere under vacuum conditions.
OPA derivatization was performed in the
injectionneedle of the HPLC as pre-column derivatization.
OrthoPhthaldialdehyde (OPA) was purchased from GRACE,
Davison (Lokeren, Belgium). The stock solution of 10
mg/ml was diluted 1:10 in 1 M Borate-buffer (61.8 g
borate in 1 liter of HPLC-grade-water). 6 l OPA and 1.5
l substrate were mixed for 90 sec in the injection
needle and then separated via HPLC with an Agilent
1200 series HPLC-system using a Grom-SIL OPA-3
(5m) 4.0 x 150 mm column. The gradient was run in
24 min from 100% buffer A (25 mM Sodium-phosphate
buffer with pH =7.2) to 100% buffer B (50% 25 mM
sodium-phosphate buffer, pH =7.2, 35% methanol, and
15% acetonitrile) in a stepwise manner. The column
temperature was 25C and the flow rate was 1.1 ml/
min. The detector was set on fluorescence with 330 nm
excitation and 450 nm emission. The data was
analyzed with the ChemStation software.
Total CW and WTA content
We detected significant differences in the amount of
total CW produced between the DAP-S vs DAP-R
isolates within each strain-pair (Table 2). For example,
the ratio of mg CW dry weight/g of CW wet weight in
the DAP-S strain CB5062 was 7.5 ( 5.7) vs. DAP-R
strain CB5063 at 18.2 ( 9.9) (p < 0.05).
In addition, there were significant differences in the
amount of WTA found in the CWs of each strain-pair in
which the DAP-R strain exhibited the thickened CW
phenotype, with DAP-R strains producing significantly
more WTA than their respective DAP-S strains. In
contrast, in the strain-pair CB5088/5089 in which the
CWs were of equivalent thickness, neither CW dry
weight nor WTA amount was significantly different
In addition to the significant increases in overall WTA
content in the DAP-R strains above, there were also
substantial differences in the proportion of WTA that
was D-alanylated when comparing the DAP-R vs DAP-S
isolates. The percentage of D-alanine contained within
Cell wall (CW) dry mass and WTA amount
CW mass [mg dry Amount of WTA
Dweight/g wet Amount of WTA alanylation [% nmol
weight] [nmol Pi/mg CW] D-alanine/nmol Pi]
CB1663 12.6 4.2 93.4 24.0 45.2 4.8
CB1664 21.8. 6.6* 144.6 22.0* 82.1 16.7*
CB5021 12.9 5.9 87.7 37.2 32.9 5.8
CB5020 25.3 10.3* 175.3 42.7* 53.7 9.3*
CB5062 7.5 5.7 67.4 18.3 32.8 8.8
CB5063 18.2 9.9* 169.5 70.5* 57.7 18.8*
CB5088 10.7 2.6 99.1 50.1 41.4 15.4
CB5089 9.2 3.0ns 106.5 54.1ns 38.8 14.7 ns
Dry mass of CW was quantified as [mg dry weight/ g wet weight] n 5 The
amount of WTA was determined by a colorimetric assay and expressed
as [nmol Pi/mg cell wall] n 4 (except 5062/5063). The rate of
Dalanylation of WTA repeating units was determined by HPLC n 3
Statistical analysis was performed by Students t-test (except
CB5062/5063 Welch corrected t-test). Significance: p-value <0.05 vs.
Mass spectrometry (MS) analysis
HPLC peaks-of-interest from the muropeptide
analysis were collected and analyzed by LC-MS. The
liquid chromatography system used was a Dionex
Ultimate 300 RS coupled to a BrukermicrOTOF II set on
positive ion mode. CW components were separated on
a Phenomenex Gemini 150 x 4.6 mm C18 110 5M
column (Phenomenex, Aschaffenburg, Germany). The
45 min program was run with a flow rate of 0.2 ml/min
and 0.1% formic acid with 0.05% ammonium formate
as buffer A and 100% acetonitrile as buffer B. After a 5
min washing step with 100% buffer A, a 30 min linear
gradient to 40% buffer B followed. A 5 min gradient
delay and 5 min of re-equilibration completed the
method. The injection volume of the single peaks was
Table 3. Relative surface charge of DAP-S/DAP-R
the WTA (nmol D-alanine/nmol Pi) in all DAP-R strains
was significantly higher than that observed in their
respective DAP-S parental strains. In the control strain
pair, CB5088/CB5089, without differences in CW
thickness, no differences in D-alanylation of WTA were
detected (Table 2).
Gene expression analysis
As shown in Figure 1, during exponential growth
phase, in all three of the strain-pairs, dltA expression
was significantly greater in the DAP-R isolate as
compared to the respective DAP-S parental strain. A
similar outcome was observed for tagA, with
expression of this gene being significantly higher in two
of the DAP-R isolates as compared to their respective
DAP-S parental strains. This pattern of differential
expression between the DAP-S/DAP-R strain pairs was
even more notable during stationary phase of growth.
For dltA, all three DAP-R strains exhibited substantially
higher expression than their respective DAP-S parental
strains. Moreover, for tagA, all three DAP-R strains
exhibited increased expression as compared to their
DAP-S parental strains, reaching statistical significance
in two of the three comparisons. It should be pointed
out that the overall level of expression of both tagA and
dltA was substantially higher during exponential as
compared to stationary growth phases. In the control
strain-pair (CB5088/CB5089) there were no differences
in tagA or dltA expression levels noted (Figure S2).
We tested all 3 DAP-S/DAP-R study pairs that
exhibited differences in their D-alanine contents. In all
three strain-pairs, the DAP-R isolate exhibited
significantly more relative positive surface charge vs.
its respective parental DAP-S parental strain (Table 3).
Muropeptide analysis and calculation of
In order to determine whether there were structural
differences in the CW of the DAP-S vs. respective DAP-R
strains, the peptidoglycan was isolated, digested into
muropeptides and analyzed by HPLC (Figure S3). We
determined the distribution of monomeric, dimeric,
trimeric, and oligomeric muropeptides, and calculated
the amount of cross-linkage for each strain (Table 4).
For the CB5021/CB5020, CB5062/CB5063, and CB5088/
CB5089 strain-pairs, no reduction in cross-linkage was
detected, and therefore, no increase in the monomeric,
dimeric, and trimeric muropeptides. In contrast, strain
pair CB1663/CB1664 showed a significant reduction in
cross-linkage (73.8 2.4 vs 65.5 1.5, p = 0.0011),
and a concomitantly significant increase in monomers
(8.8 1.7 vs 15.7 1.0, p = 0.0005), dimers (14.9
2.1 vs. 18.7 1.4, p = 0.0314), and trimers (9.8 1.3
vs. 11.9 0.9, p = 0.0025). In addition, in DAP-R
isolate CB1664, a notable increase of monomeric
muropeptide species was seen (only present in very
small amounts in CB1663) (Figure S3, structures are
depicted in Figure S4). In contrast, several
muropeptide peaks were slightly reduced in DAP-R
strain CB1664 vs. DAP-S strain CB1663.
A recent publication indicated that structural changes
in the peptidoglycan of S. aureus can depend on the
available nutrients . We, therefore, analyzed the
muropeptide pattern of strain set CB1663/CB1664 at
different growth time-points, and tested whether the
addition of glycine or alanine to the medium had any
effect (Figure S5). Only this single strain-pair was
analyzed because of its obvious differences in the
muropeptide composition between the DAP-S and
DAPR isolates and the significant reduction in cross-linkage
with a concomitant doubling in monomeric
muropeptides. These differences were not seen in the
other three strain pairs. The peaks of strain CB1663
and CB1664 without the addition of extra amino acids
were collected and analyzed by mass spectrometry
(MS) and the percentage of each muropeptide was
calculated (Table S1). An overview on muropeptide
structures is given in Figure S4. At OD578=0.7, there
was an increase of 4.5 fold in peak 3 (Penta-(Gln)) and
3.9 fold in peak 5 (Penta(Gln) Gly) of DAP-R strain
CB1664 vs DAP-S strain CB1663, while peak 11 (the
cyclic dimer) was diminished by 50%. While the
addition of alanine had no obvious effect on the
muropeptide patterns of either CB1663 or CB1664, we
saw an 1.9 fold increase in peak 4 (Tetra(Gln) Gly6 to
Tetra(Gln) Gly9) of the DAP-S strain CB1663 when
glycine was added. After 24 hrs of growth, the
muropeptide pattern of the DAP-R strain, CB1664,
exhibited a very strong increase in two monomeric
muropeptides (peaks 3 (9.8 fold), and 5 (4.8 fold) and
four new monomeric peaks (peak 1 (Tetra(Gln) AlaGly),
peak 2 (Tetra(Gln), peak 7 (Penta(Gln) Ala) and peak 8
(acetylated Penta(Gln) Ala)) appeared. Again, there
Figure 1. Expression profiles of dltA and tagA. Expression in exponential (A) and stationary growth phase
(B). Values from exponential and stationary growth phase RNA samples were normalized vs. housekeeping gene,
gyrB, expression levels; data from the DAP-S strains were set to 1 to allow comparison of data from different
samples with their respective DAP-R isolates. *P < 0.05 and **P < 0.001.
was a decrease in the cyclic dimeric peak (peak 11) by
66%. Peaks 3 and 7 were almost completely lost when
glycine was added to the growth medium of strain
CB1664. However, alanine had no effect on the
muropeptide patterns of either strain. For both strains,
the relative percentage of each muropeptide also
differed between OD578=0.7 and the 24 hr time-point,
but to a lesser extent than the differences between the
DAP-S and the DAP-R strain when compared at the
same harvesting point (Table S1).
Table 4. Distribution of muropeptides and amount
CB1663 8.8 1.7
Since the MS data suggested an increase of
muropeptides that contained an alanine within the
interpeptide bridge, we analyzed the amino acid
composition of the whole cell wall of strains CB1663
and CB1664 after 24 hrs of incubation. In strain
CB1664, the amounts of glycine, alanine, and lysine
were ~twice as high as in strain CB1663 (Table S2).
In S. aureus, there is growing evidence for the
involvement of CW in the development of the DAP-R
phenotype . Several studies have shown that
DAPR S. aureus isolates derived from both in vitro passage
selection, as well as from patients treated with failing
regimens of DAP, exhibited significantly thicker CWs as
compared to their respective DAP-S parental strain
[13,31,32]. This thickened CW phenotype is very
reminiscent of that described for VISA isolates ;
many (but not all) of these DAP-R strains with
thickened CWs were, in fact, isolated from patients
previously treated with vancomycin [31,32]. These data
argue for common molecular mechanisms between the
thickened CW phenotype induced by vancomycin and
DAP. We recently provided the first evidence for a link
between the thickened CW phenotype and an
increased production and D-alanylation of WTA.
However this study only included a single,
wellcharacterized DAP-R MSSA strain. Since staphylococcal
isolates can differ substantially in their phenotypes due
to their genetic variability, we extended our
observations in the recent study to now include DAP-R
Regulation of CW biosynthesis is very complex
process, and the physiological stress imposed by
antibiotic treatment can lead to massive changes in
pathways responsible for CW biosynthesis. For
example, a gene belonging to the CW stress stimulon,
cwrA (cell wallresponsive antibiotics; SA2343), was
found to be both highly upregulated in several clinical
VISA strains  and also upregulated upon DAP
challenge . However, the complete regulatory
mechanisms underlying the VISA and DAP-R
phenotypes remain largely elusive, and are most likely
multifactorial. For example, Yang et al.  confirmed
amongst non-VISA, that DAP-R S. aureus strains often,
but not universally, display thickened CWs (~50%
frequency). In contrast, Boyle-Vavra et al. found neither
a thick CW phenotype in one DAP-R isolate, nor
sequence or transcriptional profiling differences
between this DAP-S/DAP-R clinical strain-pair in terms
of genes involved in CW metabolism . Therefore,
we also included a DAP-S/DAP-R strain-pair that did not
show differences in CW thickness as relevant controls.
Furthermore, Muthaiyan et al.  investigated the
transcriptional activation profile of in vitro DAP-exposed
S. aureus cells. They observed that, in addition to
inducing genes consistent with CM depolarization, a
number of genes involved in the CW stress stimulon
were also impacted by in vitro DAP exposures.
Interestingly, when the transcriptomic inductioprofiles
of DAP vs vancomycin vs oxacillin were compared, a
large consensus cadre of genes involved in CW
synthesis were induced by all three agents (including
vraSR, murAB, pbpB, tcaA and the various tag genes).
Thus, DAP can clearly induce the CW stress stimulon in
a manner similar to classical CW-active agents. Fischer
et al.  recently confirmed some of these
observations in comparing the transcriptomic and
proteomic profiles of a DAP-S/DAP-R MSSA strain-pair.
These investigators found a number of genes involved
in CW metabolism were up-regulated in the DAP-R
isolate, including the WTA biosynthesis enzymes tagA
and tagG, among others.
In the DAP-S/DAP-R strain-pairs in which the DAP-R
isolate demonstrated a thickened CW phenotype, the
DAP-R strains all showed notable increases in terms of
CW dry mass. In turn, this phenotype was likely
explicable, at least in part, by the increased amount of
CW-attached WTA found in these same DAP-R strains
as compared to their respective parental DAP-S
isolates. In addition, all DAP-R isolates exhibited a
higher percentage of WTA D-alanylation when
compared to their DAP-S parental isolates. The control
strain-pair CB5088/CB5089 (without CW thickness
differences) showed neither differences in WTA amount
nor in WTA D-alanylation. This thickened CW
phenotype, together with the documented increased
positive surface envelope charge amongst the DAP-R
strains (presumably related to the enhanced
Dalanylation) most likely contributes to either: i) a
charge-dependent repulsion milieu, limiting
calciumcomplexed DAPs interaction with the bacterial surface;
and/or ii) steric-limited access of DAP due to a
physically denser CW. It should be pointed out that the
above CW perturbations were demonstrated in all three
DAP-S/DAP-R strain pairs, irrespective of the presence
or absence of SNPs within mprF and yycG. This
suggests that the contribution of perturbations in these
gene loci are independent of, and additive to, those
involved in the modified CW parameters noted above.
This is consistent with the CM (not CW) specificity of
these latter two genes which is also underlined by the
fact that the altered MprF in the strain CB5089 does
not lead to any changes in cell wall composition. MprF
is responsible for the lysinylation of CM
phosphotidylglycerol, which generates the
positivelycharged CM phospholipid, L-PG [17,18]. In addition to
this synthetic function, MprF is also involved in the
inner-to-outer CM flipping of L-PG . On the other
hand, the yyc operon is involved in the CM stress
stimulon and fatty acid metabolism .
We have previously compared relevant gene
expression profiles in DAP-R vs respective DAP-S strain
pairs. For example, for the mprF gene, one of two
expression profiles distinguish the DAP-R vs DAP-S
pairs: i) increased expression during exponential
growth (point of expected maximal expression of this
gene); and/or ii) unexpected retention of expression
during stationary phase of growth [8,10]. In the current
study, we saw similar outcome patterns for both tagA
and dltA expression, i.e., i) increased dltA expression at
both exponential and stationary phases of growth for
two of the three DAP-R isolates vs their respective
DAPS parental strains; and ii) unexpected enhancement of
dltA expression during stationary growth for the
remaining DAP-R isolate. A very similar pattern of
increased expression profiles was noted for tagA, i.e. a
substantially increased level of expression at both
exponential and/or stationary growth phases. These
data speak to a notable deregulation of these two
operons which are critically responsible for the target
CW phenotypes investigated in this study amongst
DAP-R isolates: WTA production and D-alanylation of
WTA. The genetic network perturbations responsible for
this deregulation are under active investigation in our
When we investigated the peptidoglycan composition
to rule out additional CW perturbations in the strain
sets, we could not detect any major changes in the CW
composition of these strain-pairs, with the exception of
strain-pair CB1663/CB1664. For CB1664 we saw a
significant reduction in cross-linkage, and a
concomitant increase in the monomeric, dimeric, and
trimeric muropeptide content. The increase in some
monomeric muropeptides seen at OD578=0.7 shows,
that the remodeling of the peptidoglycan of the DAP-R
strain CB1664 has already started in exponential
phase, becoming more extensive later during
stationary growth phase. Similar to the report of Zhou
and Cegelski for an MSSA strain , we observed in
our DAP-R study strain, CB1664, an increase in the
monomeric muropeptide Penta(Gln) (peak 3) and in
muropeptides with an alanine, instead of glycine, in the
interpeptide bridge (peaks 1, 7, and 8). We, therefore,
suggest that the DAP-R strain modifies its CW by the
incorporation of alanine, which leads to a reduced
cross-linking of peptidoglycan. These changes are not
present in the other strain pairs. Interestingly, we saw
a notable decrease in the cyclic dimeric muropeptide
(Tetra(Gln) Gly5-Tetra(Gln) Gly5), previously noted to be
increased in a -lactam-resistant strain . While the
increase in certain monomers can be reversed by the
addition of glycine to the growth medium, the decrease
of the cyclic peak cannot, indicating that these two
events have different causes. As the increased
monomeric peaks only appeared in stationary growth
phase (i.e. glycine limited conditions ), one could
speculate that the DAP-R strain CB1664 buffers stem
peptide-containing muropeptides (peak 3: Penta(Gln),
peak 5: Penta(Gln) Gly and peak 7: Penta(Gln) Ala)) and
alanine until glycine becomes available again.
We tested the effect of additional glycine on the MIC
against DAP, but saw no differences compared to
normal medium (data not shown). This indicates that
the remodeling of the peptidoglycan has no influence
on DAP-R in the strain-pair CB1663/CB1664.
When we analyzed the amino acid composition of the
peptidoglycan of strain pair CB1663/CB1664 after 24 h
growth, we noted an increased alanine and glycine
content for the DAP-R strains. This finding fits with the
proposed monomeric muropeptide structures, which
were increased. We did not observe them in another
set of DAP-R clinical isolates .
Taken together, we provide new evidence here for
the fact that an increase in CW thickness, as a
consequence of an increased WTA content, and
increased WTA D-alanylation is a relatively common
phenotype amongst DAP-R S. aureus strains (including
both MSSA  and MRSA). These phenotypic
alterations are consistent with both observed changes
in the positive surface charge characteristics and
transcriptional enhancement of expression profiles of
genes involved in the above CW phenotypes. Lastly, it
appears clear that, in addition to a plethora of CM
adaptations, well-defined perturbations of CW
structural and functional metrics contribute to the
DAPR phenotype in S. aureus.
Figure S1. Cell wall biosynthesis. Peptidoglycan
biosynthesis starts in the cytoplasm with the step-wise
assembly of the precursor UDP-MurNAc-pentapeptide.
This precursor is then added to
undecaprenolphosphate at the cytoplasmic membrane, resulting in
Lipid I. The addition of GlcNAc from UDP-GlcNAc forms
Lipid II. In staphylocci, five glycine-residues from tRNAs
are added before Lipid II is finally flipped over the
cytoplasmic membrane by a yet unknown enzyme.
Outside the cell, Lipid II is incorporated into the existing
cell wall by the transpeptidase and transglycosylase
reactions of penicillin-binding proteins (PBPs). WTA
biosynthesis occurs directly at the cytoplasmic
membrane, starting with the addition of GlcNAc-P from
UDP-GlcNAc to undecaprenol-phosphate (bracket).
After the addition of ManNAc the anchor structure is
finished by adding 3 glycerol-P molecules. Then up to
40 ribitol-P molecules are polymerized step-wise until
the WTA molecule is completed and finally transported
across the CM by TagGH. The mature polymer is linked
to the C6 atom of MurNAc in the peptidoglycan by a yet
unidentified enzyme and then modified with GlcNAc
and D-alanine (circled) (A). The organisation of WTA
biosynthesis genes (B).
Figure S2. Expression profiles of dltA and tagA
for strains CB5088/CB5098. Values from
exponential (A) and stationary (B) growth phase RNA
samples were normalized vs. housekeeping gene, gyrB,
expression levels; data from the DAP-S strains were set
to 1 to allow comparison of data from different samples
with their respective DAP-R isolates.
Figure S3. Muropeptide pattern by HPLC
analysis. The CW was isolated at OD578=0.7. The
peptidoglycan was digested by the muraminidase
mutanolysin and analyzed by HPLC. The overall
muropeptide pattern of all strains was typical for
Staphylococcus aureus. However, DAP-R strain CB1664
showed an increase in certain monomeric
muropeptides vs. its respective DAP-S isolate (CB1663),
which was not seen in the other three strain pairs.
Figure S4. Muropeptide structures. . (A)
Muropeptides are the subunits of the bacterial CW. The
glycan part consists of N-acetylglucosamine (G) linked
by a -1,4 glycosidic bond to N-acetylmuramic acid (M).
A polymer of these disaccharides forms the glycan
backbone of the CW. Attached to M is the stem peptide
(L-Ala D-Gln L-Lys D-Ala D-Ala). Added to the
amino group of L-Lys is the interpeptide bridge, which
mainly consists of five Gly residues. The first Gly is
sometimes seen to be replaced by Ala  and the
second one by Ser . Some muropeptides also
contain Gly residues attached to the D-Ala on position
four. They persist from former cross-links between two
adjacent peptides from two different glycan strands.
The peptide parts of the CW are indirectly cross-linked
by the interpeptide bridge, forming a bond between the
D-Ala on position four of the donor peptide and the fifth
Gly of the interpeptide bridge of the adjacent stem
peptide. Thereby, the terminal D-Ala of the donor
peptide is cleaved off. Part (B) gives two examples of
dimeric muropeptides. The upper part shows a classical
Penta-Tetra dimer coming from two cross-linked glycan
chains. Cross-linking in S. aureus can result in bigger
muropeptides (e.g. trimers, tetramers,) The bottom
part shows the unique cyclic dimer with a double
crosslink between two stem peptides .
Figure S5. Muropeptide analyses at different
time points with the addition of glycine or
alanine to the medium. We analyzed the
muropeptide pattern of strain set CB1663/CB1664 at
different time points, and tested whether the addition
of glycine or alanine (~8 times the normal amount) to
the medium had any effect. The peaks of strain CB1663
and CB1664 after 24h without the addition of extra
amino acids were collected and analyzed by mass
spectrometry (MS). The peaks at OD578=0.7 were
labeled according to the retention time at 24 hrs.
Table S1. Muropeptide composition.
Table S2. Relative amounts of amino acids.
We thank Annika Vass and Larissa Kull for technical
assistance with WTA D-alanylation determination.
Conceived and designed the experiments: UB S-JY CM
ASB CW. Performed the experiments: UB S-JY SW NM
TR MN AS DK TG CW. Analyzed the data: UB ASB CW.
Contributed reagents/materials/analysis tools: UB S-JY
ASB CM CW. Wrote the manuscript: UB ASB CW.
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