Daptomycin Resistance in Enterococci Is Associated with Distinct Alterations of Cell Membrane Phospholipid Content
et al. (2012) Daptomycin Resistance in Enterococci Is Associated with Distinct Alterations of
Cell Membrane Phospholipid Content. PLoS ONE 7(8): e43958. doi:10.1371/journal.pone.0043958
Daptomycin Resistance in Enterococci Is Associated with Distinct Alterations of Cell Membrane Phospholipid Content
Nagendra N. Mishra
Arnold S. Bayer
Truc T. Tran
Cesar A. Arias
Tarek Msadek, Institut Pasteur, France
Background: The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains. Methodology: Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs. Principal Findings: Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i ) phosphatidic acid; and ii ) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM. Conclusion: Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.
Funding: CAA is supported by National Institutes of Health (NIH) grants R00 AI72961 and R01 AI093749 to CAA from the National Institute of Allergy and
Infectious Diseases (NIAID). ASB and YS are supported by NIH grants R01 AI39108 and R01 AI080714 also from NIAID. This work was also supported in part by a
John S. Dunn Foundation Collaborative Research Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Enterococci are leading causes of nosocomial infections in the
US , causing a variety of life-threatening syndromes such as
bacteremic infections (including endocarditis), urosepsis and
meningitis, among others. Enterococcal disease occurs frequently
in patients that are seriously ill and/or with important degrees of
immunosuppression. Two species are responsible for the vast
majority of enterococcal infections, E. faecalis and E. faecium. The
treatment of such infections is often impacted by the increased
prevalence of multidrug resistance in these isolates. Indeed,
ampicillin and vancomycin resistance is now present in more
than 80% of E. faecium isolates , making these compounds
almost obsolete for the treatment of this pathogen. Moreover, E.
faecium is one of the no ESKAPE pathogens (E. faecium,
Staphylococus aureus, Klebsiella pneumoniae, Acinetobacter baumanii,
Pseudomonas aeruginosa, Enterobacter spp.), highlighted by the
Infectious Diseases Society of America as nosocomial organisms
requiring new therapeutic approaches, both because of their
commonality and challenging clinical presentations, but
importantly also due to multiple antibiotic resistances . Although most
E. faecalis isolates remain susceptible to ampicillin, the increased
prevalence of resistance to aminoglycosides and vancomycin
within this species also limits the therapeutic alternatives, as well
as the ability to achieve bactericidal killing of this organism [1,3].
Daptomycin (DAP) is a lipopeptide antibiotic approved by the
Food and Drug Administration (FDA) in 2003 for the treatment of
skin and soft tissue infections caused by susceptible Gram-positive
organisms and, subsequently in 2006, for S. aureus bacteremia and
right-sided endocarditis . DAP has potent in vitro bactericidal
activity against vancomycin-resistant enterococci (VRE), although
it does not have an FDA approval for VRE infections.
Nonetheless, many clinicians often use this compound for the
treatment of severe enterococcal infections, particularly those
caused by E. faecium. Two retrospective studies have shown that
outcomes of patients with serious enterococcal infections treated
with DAP were similar to those treated with linezolid (an
FDAapproved antibiotic for VRE infections) [5,6]. However, there are
several recent reports of patients failing DAP mono-therapy in
association with emergence of DAP resistance during therapy
The mechanism of action of DAP involves a calcium-dependent
interaction with the bacterial cell membrane (CM) [10,11]. The
insertion of the drug into the CM causes disruption in its
homeostasis that is associated with a leakage of potassium ions
from the cytoplasm of the bacterial cell . These alterations of
the CM lead to bacterial cell death by mechanisms that are not
fully elucidated but are likely to involve alterations in cell division
homeostasis . Emergence of DAP non-susceptibility has been
described in both S. aureus and enterococci. Several genes have
been associated with this phenomenon and include: i ) genes
encoding two or three-component regulatory systems involved in
cell envelope homeostasis: yvqF-vraSR in S. aureus, and liaFSR in
enterococci and Bacillus subtilis [12,13]; ii ) genes coding for
enzymes involved in CM phospholipid metabolism: mprF (multiple
peptide resistance factor) [12,14,15], cls (cardiolipin synthase) ,
pgsA (phosphatidylglycerol synthase)  in S. aureus; gdpD
(glycerophosphodiesterphosphodiesterase) , cls [13,16] in
enterococci, and pgsA  in B. subtillis; and iii ) genes encoding
for b subunits of RNA polymerase in S. aureus (rpoB/rpoC) [14,18].
In the present study, we delineate the following CM
characteristics of both two enterococcal clinical strain-pairs
(DAP-susceptible and DAP-resistant), each isolated from a patient during failed
DAP therapy: i ) phospholipid repertoire, ii ) fatty acid
composition, iii ) fluidity, and iv ) DAP-induced
CM phospholipids (PLs) of E. faecalis vs. those of E.
Each strain-pair had an identical PFGE profile (data not
shown). The DAP minimal inhibitory concentrations (MICs) for
the E. faecalis pair were 1 mg/ml and 12 mg/ml for S613 and
R712, respectively. For E. faecium S447 and R446, the DAP MICs
were 2 mg/ml and 16 mg/ml, respectively. Table 1 shows the CM
PL content of the clinical strain-pairs of E. faecalis and E. faecium;
Figure 1 shows these PL repertoires as identified on
twodimensional thin layer chromatography (2D-TLC) plates with
each major spot subsequently confirmed by liquid
chromatography/ electrospray ionization-mass spectrometry/mass
spectrometry (LC/ESI-MS/MS) analysis. The CM PLs of the E. faecium pair
were less complex than those of E. faecalis. For example,
lysylphosphatidylglycerol (L-PG) was the only amino-containing
(positively-charged) PL detected in E. faecium (Table 1), whereas
E. faecalis CMs contained L-PG plus two additional
aminocontaining PLs of unknown identity (Table 1, Figure 1).
Moreover, the PLs of E. faecium were composed mainly of
phosphatidylglycerol (PG), cardiolipin (CL) and a
glycerophosphoglycolipid identified by LC/ESI-MS/MS as
glycerolphosphodiglycodiacylglycerol (GP-DGDAG), whereas phosphatidic acid
(PA) and an unidentified non-amino-containing PL were also
detected in E. faecalis CMs (Table 1, Figure 1).
Interestingly, in both enterococcal pairs, a significant decrease
in PG content was observed in the DAP-resistant variants
(Table 1), with the reduction in PG being quite pronounced in
E. faecium R446 (,50% vs the parental S447 strain). This decrease
in PG content in both DAP-resistant strains (E. faecalis R712 and
E. faecium R446) was accompanied by a significant increase in the
glycerophosphoglycolipid GP-DGDAG content as compared to
their parental strains. Also, in E. faecalis R712, there was a
concomitant increase in an unidentified negatively-charged PL
species (Table 1). We were unable to detect a significant
difference in the CL content of the DAP-resistant derivatives of
either E. faecalis or E. faecium (R712 and R446, respectively) when
compared with their DAP-susceptible parental isolates (Table 1)
although, a trend towards increased CL content was observed in
E. faecium R446 vs S447 (p = 0.067). Another important finding
was that, in contrast to DAP resistance in S. aureus , the
amount of amino-containing PLs in the inner vs. outer CM leaflet
(asymmetry) did not change significantly between the
DAPsusceptible and DAP-resistant clinical strain-pairs of either E.
faecalis or E. faecium (Tables 1).
CM fluidity in enterococcal strain pairs
Table 2 shows that the DAP-resistant derivatives of both E.
faecalis and E. faecium (R712 and R446, respectively) had a
significantly higher polarization index value as compared with
their respective parental DAP-susceptible isolates (S613 and S447,
respectively), indicating less fluid (more rigid) membranes.
Fatty acid compositional analysis
Table 3 shows the CM fatty acid composition of the
enterococcal clinical strain-pairs. We found no statistically
significant difference in the patterns of saturated fatty acids
(SFAs), unsaturated fatty acids (UFAs), and cyclic fatty acid (CFA)
profiles between the E. faecalis S613 (DAP-susceptible) and its
DAP-resistant derivative (R712). In contrast, a decrease in the
proportion of total UFAs and an increase in CFAs were observed
in the DAP-resistant E. faecium R446 as compared to its
DAPsusceptible parental strain, S447 (P = 0.0121 and 0.044,
respectively). This decrease in total UFAs appeared to be driven by a
substantial reduction in the major UFA species, C18:1v7c
Ultrastructural cell envelope alterations and cell wall
We previously observed several unique ultrastructural cell
envelope alterations in association with DAP resistance in E.
faecalis . Hence, we similarly characterized the cell envelope of
our clinical strain-pair of E. faecium using transmission electron
PLs, phospholipid; ACP-1, amino-containing phospholipid 1; ACP-2, amino-containing phospholipid 2; LPG, lysyl-phosphatidylglycerol; PG, phosphatidylglycerol; CL,
cardiolipin; GP-DGDAG, glycerolphospho-diglycodiacylglycerol; PA, phosphatidic acid; Un, unidentified phospholipid.
*P,0.05 in relation to S613; **P,0.01 in relation to S447. Statistically significant values are underlined.
microscopy. Similar to the above prior E. faecalis analyses, we
confirmed a significant increase in the thickness of the cell wall in
the DAP-resistant E. faecium R446 as compared to its parental
DAP-susceptible strain, S447 (35.167.1 nm vs. 25.663.8 nm,
respectively, P,0.001). Moreover, we also found impressive
disruptions in the architecture of the cell envelope of the
DAPresistant E. faecium strain, the most striking of which included the
appearance of cell envelope protrusions (Figure 2), similar to
those we previously described in DAP-resistant E. faecalis .
Relative cell surface positive charge and DAP-mediated
CM permeabilization-depolarization profiles
Similar to data generated previously in our E. faecalis clinical
strain-pair , emergence of resistance to DAP in E. faecium was
associated with an increase in the relative net surface positive
charge, reflected by a significant decrease in the percentage of
surface-bound cytochrome c (,7% in E. faecium R446 vs ,17% in
S447; P = 0.0016).
Figure 3 quantifies the ability of DAP to depolarize the CM of
the E. faecium clinical-strain pair. There was a statistically
significant reduction in the ability of DAP to depolarize the CM
in DAP-resistant E. faecium R446 as compared to its
DAPsusceptible parental strain, S447. This difference was observed
immediately after exposure to DAP (time 0), especially at
concentrations .8 mg/ml. The reduction in DAP-mediated CM
depolarization observed in R446 was maintained at 5, 15 and
30 min after DAP exposure, and was most prominent at DAP
concentrations of 16 and 32 mg/ml. Of note, these differences in
depolarization profiles between the DAP-susceptible and
DAPresistant strains were less evident at the highest DAP
concentrations tested (32 and 64 mg/ml) in a time-dependent manner.
% of total PL composition and asymmetry
% of total PL composition and asymmetry
*p,0.005 in relation to the susceptible isolate.
Similarly, we observed a substantial decrease in the ability of
DAP to permeabilize the CM of the DAP-resistant E. faecium R446
vs. the DAP-susceptible parental isolate (S447) (Figure 4). There
was a reduction in DAP-dependent CM permeabilization of E.
faecium R446 vs S447, particularly at DAP concentrations of 8 and
16 mg/ml after 5 minutes of exposure to daptomycin (Figure 4).
DAP is one of the few antibiotics that exhibit in vitro bactericidal
activity against enterococci, including those that are
vancomycinand ampicillin-resistant . This antibiotic is now commonly
used off-label for the treatment of serious enterococcal infections
due to the lack of other therapeutic alternatives. A challenging
issue for the use of DAP against enterococci is the emergence of
resistance during therapy. Indeed, several cases of patients infected
with E. faecalis and E. faecium who developed resistance during the
course of DAP treatment have now been documented [7,8,9].
Furthermore, enterococci are less susceptible to DAP in vitro than
staphylococci, and this relatively reduced ability of DAP to kill
enterococci may contribute to the development of DAP-resistance
in vitro, and subsequent failure during DAP therapy of serious
The mechanism of action of DAP involves interactions with the
bacterial CM in a calcium-dependent manner . We have
previously shown that the development of DAP-resistance in a
clinical-strain pair of E. faecalis was associated with important
ultrastructural changes in the cell envelope, including an increase
in cell-wall thickness, as well as notable perturbations in cell
surface charge and a reduced ability of DAP to depolarize and
permeabilize the CM . In the current work, we extended the
above observations to a clinical strain-pair of E. faecium and, most
importantly, focused our studies on a comparative analysis of the
CM PL profile of both clinical-strain pairs. A number of
interesting themes emerged from our investigations. Firstly, as we
observed previously in E. faecalis, development of DAP resistance
in E. faecium was associated with marked structural and functional
changes in the cell envelope, and in the ability of DAP to alter the
CM integrity. Thus, development of DAP resistance in E. faecium
was accompanied by structural perturbations within the cell
envelope (e.g., peri-septal CM protrusions) and in the cell wall
(thickening). Also, biophysically, the CMs of both DAP-resistant
enterococci became less fluid. Our findings confirm that structural
alterations of the cell envelope and changes in the biophysical
properties of the CM are strongly associated with the mechanism
of DAP resistance, as previously described in S. aureus [13,19] and
B. subtillis .
Secondly, detailed analysis of CM PL content in both clinical
strain-pairs revealed important differences between E. faecalis and
E. faecium. Indeed, whereas in E. faecium, the only
aminocontaining PL detected was L-PG (similar to S. aureus) , E.
faecalis CMs contained at least three amino-PLs; one was identified
as L-PG (,15% of total PLs), with two other amino-PLs of
unknown identity. To our knowledge, a study that
comprehensively characterized the amino-containing PLs of E. faecalis was
published over 40 years ago , and provided compelling
Nature of Fatty Acid
Saturated fatty acids (SFA)
Unsaturated fatty acids (UFA)
Cyclic fatty acid (CFA)
% of fatty acid composition
*p,0.05 in relation with S447. Statistically significant values are underlined.
% of fatty acid composition
evidence that lysyl-, alanyl- and arginyl-containing PLs were
present in their CM extracts. A recent study also noted the
presence of alanyl- and arginyl-containing PLs in their membrane
lipid extract . Although our experimental growth conditions
for PL extraction were different from those used in the former
study (BHI vs medium containing 1% tryptone, 0.5% yeast
extract, 0.5% dipotassium phosphate and 1% glucose,
respectively), it is tempting to speculate that the other amino-containing PLs
identified in our E. faecalis strain-pair are likely to represent
alanylPG and arginyl-PG. Of interest, we did not find any significant
difference in the amino-containing PL content or L-PG flipping
between the DAP-resistant E. faecalis R712 compared to its
parental DAP-susceptible S613. In contrast, in our E. faecium
strain-pair, a trend towards increased content of L-PG in the outer
CM of the DAP-resistant isolate was observed, although this did
not quite reach statistical significance. Our findings suggest that
differences in content and distribution (flipping) of
aminocontaining PLs are not a major determinant of DAP resistance in
enterococci. This is in contrast to data from S. aureus where
increased L-PG translocation to the outer CM leaflet (likely
mediated by gain-in-function mutations in the CM lipid-modifier
protein, MprF) appears to play a pivotal role in DAP-R in selected
Thirdly, a striking finding in our studies was that the in vivo
development of DAP resistance in enterococci was associated with
a significant decrease in PG content. Such striking reductions in
CM PG have been previously associated with resistance to DAP
and other cationic antimicrobial peptides in Gram-positive
bacteria [14,25]. For example, using an in vitro passage strategy
for developing DAP-resistance, Hachmann et al. 
demonstrated that development of DAP resistance was associated with a
marked decrease in CM PG content in a laboratory strain of B.
subtilis, although changes in other CM PLs were not described.
The reduction in PG was correlated with the presence of a single
nucleotide polymorphism (A64V) in pgsA, a gene encoding a key
PG synthase. Of interest, mutations in pgsA have also been recently
associated with DAP-resistance in S. aureus . Similarly,
characterization of E. faecium mutants resistant to the class II
bacteriocin, mundticin KS (a 43 amino acid peptide produced by
E. mundtii and active against E. faecium), was associated with
significant reductions in PG (and also CL) . PG is an abundant
anionic PL of bacterial CMs in both Gram-positive and
Gramnegative bacteria . In E. coli, the balance of zwitterionic and
acidic (negatively-charged) PLs is the result of the formation of PG,
which is tightly coupled with the regulation of the synthesis of
other PLs . Maintaining the PL balance is crucial for the
homeostasis of several bacterial processes including CM protein
topology  and cell division , among others. Interestingly,
we previously showed that a codon deletion in a gene encoding a
putative glycerophosphodiesterphosphodiesterase (GdpD, an
enzyme that hydrolyzes PLs) was necessary (but not sufficient) to
confer DAP resistance in E. faecalis R712 . Therefore, one
could hypothesize that the decrease in PG associated with
development of DAP resistance in enterococci could be due to
the rapid recycling of PG; of note, the glycerol phosphate moiety
of PG undergoes rapid turnover in bacteria [26,29]. It is, thus,
tempting to speculate that changes in the PG pools are
important in the development of DAP resistance by providing
the necessary substrates for synthesis of other PLs or
glycerolcontaining molecules (e.g., lipoteichoic acids) [26,30] in order to
avoid killing by cationic antimicrobial molecules. Of interest, we
did not observe any major changes in CL content associated with
development of DAP-resistance in E. faecalis or E. faecium despite
the fact that mutations in the CL synthase gene (cls) have been
Figure 3. CM depolarization by DiSC3(5) after daptomycin exposure of daptomycin-susceptible (S447) and daptomycin-resistant
(R446) Enterococcus faecium. Data represent the mean (6SD). * p,0.05; ** p,0.001.
previously linked to DAP resistance in E. faecalis , E. faecium
 and S. aureus . Nonetheless, as PG is a substrate for Cls, it
is plausible that changes in Cls activity or specificity may also
contribute to the shift in PG pools.
Fourthly, a significant increase in a negatively charged
glycerophosphoglycolipid ultimately identified as GP-DGDAG by LC/
ESI-MS/MS was found in both DAP-resistant enterococci
studied. It has been previously shown that E. faecalis contains
several glycolipids including
glycerophosphoryl-diglucosyl-diglyceride , phosphatidyldiglucosyldiglyceride [32,33] and
Interestingly, some of the above glycolipids appear to be the precursors of
enterococcal lipoteichoic acids (LTA), which are important
constituents of the cell surface of Gram-positive organisms .
Indeed, Ganfield & Pieringer showed that PG is the donor of
snglycerol 1-phosphate moieties to
phosphatidyl-kojibiosyldiacylglycerol in the in vitro synthesis of enterococcal membrane LTA .
Furthermore, alanylation of wall teichoic acids (TA) has been
shown to play a role in the defense against antimicrobial peptides
in S. aureus  and overexpression of the dlt operon, which
encodes 4 genes involved in the alanylation of wall TAs in S. aureus,
has been linked to DAP resistance . Thus, our findings suggest
that the emergence of DAP-resistance in enterococci is associated
with increase production of membrane glycerophosphoglycolipids
which may serve as precursors of LTA using PG as the donor of
glycerol-phosphate moiety, which, upon alanylation, might
increase relative positive surface charge and contribute to DAP
Fifthly, changes in the fatty acid composition of bacterial CMs
are essential in the optimization of CM function for growth and
response to modifications in environmental conditions (including
the presence of antimicrobial molecules) . Fatty acid
composition is also a major determinant of CM order (fluidity-rigidity)
that affects the homeostasis of the bacterial cell. In the current
study, the development of DAP resistance in both enterococcal
isolates was associated with less fluid (more rigid) membranes. Of
interest, this is in contrast to previous reports in clinically-derived
DAP-resistant S. aureus strains, whose CMs were significantly more
fluid than their respective DAP-susceptible parental strains .
Conversely, a modest decrease in CM fluidity was observed in a
DAP-resistant S. aureus strain selected by serial in vitro passages in
DAP . These apparent paradoxes in CM order responses
indicate that such CM changes may well be either strain-specific
and/or dependent on diverse DAP exposure conditions .
Additionally, factors that influence the development of DAP
resistance in vivo are highly likely to be different from those in vitro,
including a cadre of host factors such as serum components, host
immune cells and innate host defense peptides . Interestingly,
the decrease in CM fluidity observed in our DAP-resistant E.
faecalis strain could not be linked to changes in the proportion of
Figure 4. CM permeabilization by LIVE/DEAD BacLight after daptomycin exposure of daptomycin-susceptible (S447) and
daptomycin-resistant (R446) E. faecium. Data represent mean (6SD); * p,0.05, ** p,0.001.
saturated vs unsaturated (or cyclic) fatty acids, and, thus, other
factors may be important. In contrast, a significant decrease in
unsaturated fatty acids was observed in the DAP-resistant E.
faecium isolate (mostly due to a decrease in the C18:1v7c species),
representing a potential explanation for the observed reduction in
CM fluidity in this strain. It should be noted that there was a
significant increase in cyclopropane fatty acids observed in this
same DAP-resistant E. faecium isolate as compared to its
DAPsusceptible parent. Cyclopropane fatty acids have been shown to
stabilize CMs to a variety of environmental challenges .
In summary, we present evidence that development of DAP
resistance in clinical enterococcal strains is associated with a
significant shift in PL profiles (i.e., decreases in PG and increases in
GP-DGDAG). A common feature of DAP resistance in both E.
faecalis and E. faecium include marked ultrastructural changes in the
cell envelope, as well as increases in the net surface positive charge
and increased rigidity of the CMs. Our findings suggest that
important biochemical and biophysical modifications in CM lipid
metabolism occur in response to DAP exposures in enterococci.
Materials and Methods
Bacterial isolates and susceptibility testing
The bacterial isolates used in this study are clinical-strain pairs
of DAP-susceptible and DAP-resistant E. faecalis (S613 and R712,
respectively) and E. faecium (S447 and R446 respectively); the
DAP-resistant derivatives emerged during DAP therapy, and have
been described previously [7,8]. Briefly, the E. faecalis pair was
recovered from the bloodstream of a patient presenting with
recurrent bacteremia in 2005 . The E. faecium pair was obtained
in 2006 from the urine and blood of a hospitalized patient with
acute leukemia . Both clinical strain-pairs exhibited high-level
resistance to vancomycin (MIC.128 mg/ml) and the presence of
the vanA gene was confirmed using a PCR assay as described
before . Each strain-pair was utilized for all assays related to
phospholipid (PL) analysis, while characterization of the cell
envelope was performed only in the E. faecium (cell envelope
characteristics of the E. faecalis pair have been previously reported
by us ). DAP MICs were performed by Etest on
MuellerHinton agar following the recommendation of the Clinical
Laboratory Standards Institute. In order to confirm the genetic
relatedness of each strain pair, pulsed field gel electrophoresis was
performed in all isolates following the protocol previously
CM PL composition and asymmetry
PLs were extracted from enterococcal isolates using a
methodology described before [43,44]. Briefly, extraction of PLs was
performed on enterococcal isolates grown in brain heart infusion
(BHI) broth for 18 h (late stationary phase). The major
enterococcal PLs were separated using two-dimensional thin-layer
chromatography (2D-TLC) using Silica 60 F254 HPTLC plates
(Merck). The protocol used first-dimension chloroform-methanol
25% ammonium hydroxide (65:25:6, by volume) in the vertical
orientation, and a second-dimension
chloroform:water:methanol:glacial acetic acid:acetone (45:4:8:9:16, by volume) in the
horizontal orientation for the separation of the PLs and for
additional quantitation by phosphate estimation . The
identification of individual TLC PL spots was made in comparison
to control 2D-TLC plates of known PL standards (Figure 1). All
PL standards were purchased from Avanti Polar Lipids (Alabaster,
AL). Quantitative analysis of individual PLs isolated from TLC
plates was performed by digesting with 70% perchloric acid
(0.3 mL) at 180uC for 3 h. The PLs were quantified
spectrophotometrically at A660 as described before . The results are
reported as the mean (6 SD) of at least three independent
experiments performed on separate days.
Since the relative outer-to-inner CM asymmetry of PLs can
influence the overall surface charge in Gram-positive bacteria
, the CM distribution of amino-containing (positively-charged)
PLs in the outer vs. inner CM bilayers was determined. For this
purpose, we used quantitative fluorescamine analysis, since this
fluorophore specifically labels only surface-exposed amino-PLs in
the outer CM leaflet. Fluorescamine assays for labeling,
quantitative estimation of PLs, and chemical analysis of inner vs. outer
CM leaflet PLs have been previously described [19,44]. These
latter studies were performed in parallel with the PL analyses
above. All PL spots on the TLC plate were identified and
confirmed by exposure to iodine vapors and spraying with CuSO4
(100 mg/ml) containing 8% phosphoric acid (v/v) and heated at
180uC . I-LPG (positively charged), and other
aminocontaining PLs were visualized by ninhydrin staining.
Liquid chromatography/electrospray ionization-mass
spectrometry/mass spectrometry (LC/ESI-MS/MS) analysis
Major lipids separated on the 2D-TLC plate were scrapped off,
extracted with chloroform:methanol (1:1) and analyzed by LC/
MS. The major spots observed by 2D-TLC were confirmed by
normal phase LC/ESI-MS/MS analysis. Normal phase LC was
performed on an Agilent 1200 Quaternary LC system equipped
with an Ascentis Silica HPLC column, 5 mm, 25 cm62.1 mm
(Sigma-Aldrich, St. Louis, MO). Mobile phase A consisted of
chloroform/methanol/aqueous ammonium hydroxide (800:195:5,
v/v); mobile phase B consisted of chloroform/methanol/water/
aqueous ammonium hydroxide (600:340:50:5, v/v); mobile phase
C consisted of chloroform/methanol/water/aqueous ammonium
hydroxide (450:450:95:5, v/v). The elution program consisted of
the following: 100% mobile phase A was held isocratically for
2 min and then linearly increased to 100% mobile phase B over
14 min and held at 100% B for 11 min. The LC gradient was then
changed to 100% mobile phase C over 3 min and held at 100% C
for 3 min, and finally returned to 100% A over 0.5 min and held
at 100% A for 5 min. The total LC flow rate was 300 ml/min. To
achieve optimum ESI efficiency, a post-column splitter was used to
divert ,10% of the LC effluent into the mass spectrometer, a
QSTAR XL quadrupole time-of-flight tandem mass spectrometer
(Applied Biosystem, Foster City, CA). Instrumental settings for
negative ion electrospray (ESI) and MS/MS analysis of lipid
species were as follows: IS = 24500 V; CUR = 20 psi; GSI = 20
psi; DP = 255 V; and FP = 2150 V. The MS/MS analysis used
nitrogen as the collision gas. Data analysis was performed using
Analyst QS software (Applied Biosystem, Foster City, CA).
CM fatty acid composition
Gas-liquid chromatography was used to study the fatty acid
composition of total lipids extracted from enterococcal CMs, and
analyzed after conversion to their methyl-ester form (using fatty
acid standards) as previously described (courtesy of Microbial ID
Inc.,Newark, DE) .
CM fluidity was assessed using the fluorescent probe
1,6diphenyl-1,3,5-hexatriene (DPH). The protocol followed
previously-published techniques for DPH incorporation into target CMs,
measurement of fluorescence polarization and calculation of the
degree of fluorescence polarization (polarization index) .
Excitation and emission wavelengths of DPH are 360 nm and
426 nm, respectively, and were measured using a Biotek Model
SFM 25 spectrofluorimeter. The results were interpreted
according to the polarization index, since an inverse correlation exists
between polarization index values and fluidity (i.e., a lower index
equates to a greater extent of CM fluidity ). The experiments
were conducted at least 3 times for each isolate on separate days.
Ultrastructural analysis of the cell envelope of the E.
faecium clinical strain-pair
Comparative visualization of the cell envelope of E. faecium S447
and R446 was performed using transmission electron microscopy
following standard methodology . Cell wall thickness was
evaluated in both isolates by performing 75 separate observations
of each isolate (minimum of 50 cells) at 200,000 X magnification
in cells from different fields. Thickness of the cell walls of each
isolate was measured from the outer border of the CM to the outer
edge of the cell wall. The means of cell wall thickness (6 SD) were
determined for each isolate.
CM surface charge and DAP-induced CM
permeabilization and depolarization
A cytochrome c assay  was performed to measure the overall
relative cell surface positive charge of the clinical E. faecium
strainpair following the methodology used before for the E. faecalis
strain-pair . The amount of cytochrome c (a highly
positivelycharged molecule) remaining in the supernatant after 15 min
exposure to each enterococcal strain was determined at A530.
Cytochrome c interacts with the CM in a charge-dependent
manner . Thus, the greater the amount of residual supernatant
cytochrome c, the greater the relative surface positive charge.
DAP-induced CM permeabilization was measured with the
LIVE/DEAD BacLight kit which is based on the nucleic
acidspecific viability dyes, propidium iodide and SYTO9, as described
before [48,49]. The reaction is based on the observation that
viable bacterial cells with an intact plasma membrane are stained
by the CM-permeant green fluorescent dye SYTO9. If the
membrane is compromised and membrane permeabilization
occurs, SYTO9 fluorescence is quenched by entry of propidium
iodide into the cytoplasm [13,49]. SYTO9 fluorescence was
measured following excitation at 488 nm and emission at 510 nm.
The CM potential-sensitive 3,3-dipentyoxacarbocyanine
[DiSC3(5)] assay  was used to measure DAP-induced changes
in CM potential in E. faecium S447 and R446 as previously
described . Fluorescence was measured with an excitation
wavelength of 622 nm and an emission wavelength of 670 nm.
Loss of red fluorescence indicated CM depolarization.
CM permeabilization and potential were both measured in the
presence of increasing concentrations of DAP supplemented with
50 mg/L of calcium chloride in the buffer. A positive control of
100% ethanol and a negative control of buffer alone were included
for these two assays. Percent fluorescence change was calculated,
setting the ethanol control as 100% fluorescence change and
buffer control as 0% fluorescence change. Pilot studies confirmed
that there was no spontaneous CM permeabilization or
depolarization observed over the study time-periods of these investigations
(data not shown).
Differences in cell wall thickness, PL composition and
polarization index profiles were compared using a Students t-test. A P
value ,0.05 was consider significant.
We are grateful to Silvia Munoz-Price, John P. Quinn and James Jorgensen
for providing the enterococcal strains used in these studies. We thank
Danielle M. McGrath, I-Hsiu Huang, and Hung Ton-That for excellent
Conceived and designed the experiments: CAA ASB. Performed the
experiments: NNM TTT ZG. Analyzed the data: NNM ASB TTT YS EM
WD ZG CAA. Contributed reagents/materials/analysis tools: NNM ASB
TTT ZG CAA. Wrote the paper: NNM ASB TTT YS EM WD ZG CAA.
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