The Bacterial Defensin Resistance Protein MprF Consists of Separable Domains for Lipid Lysinylation and Antimicrobial Peptide Repulsion
et al. (2009) The Bacterial Defensin Resistance Protein MprF Consists of Separable Domains for Lipid
Lysinylation and Antimicrobial Peptide Repulsion. PLoS Pathog 5(11): e1000660. doi:10.1371/journal.ppat.1000660
The Bacterial Defensin Resistance Protein MprF Consists of Separable Domains for Lipid Lysinylation and Antimicrobial Peptide Repulsion
Christoph M. Ernst 0
Petra Staubitz 0
Nagendra N. Mishra 0
Soo-Jin Yang 0
Gabriele Hornig 0
Arnold S. Bayer 0
Dirk Kraus 0
Andreas Peschel 0
Ambrose Cheung, Dartmouth Medical School, United States of America
0 1 Cellular and Molecular Microbiology Division, Interfaculty Institute of Microbiology and Infection Medicine, University of Tu bingen , Tu bingen, Germany , 2 Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-University of California at Los Angeles (UCLA) Medical Center, Torrance, California, United States of America, 3 Medical and Natural Sciences Research Center, University of Tu bingen , Tu bingen, Germany , 4 David Geffen School of Medicine at UCLA , Los Angeles, California , United States of America
Many bacterial pathogens achieve resistance to defensin-like cationic antimicrobial peptides (CAMPs) by the multiple peptide resistance factor (MprF) protein. MprF plays a crucial role in Staphylococcus aureus virulence and it is involved in resistance to the CAMP-like antibiotic daptomycin. MprF is a large membrane protein that modifies the anionic phospholipid phosphatidylglycerol with L-lysine, thereby diminishing the bacterial affinity for CAMPs. Its widespread occurrence recommends MprF as a target for novel antimicrobials, although the mode of action of MprF has remained incompletely understood. We demonstrate that the hydrophilic C-terminal domain and six of the fourteen proposed transmembrane segments of MprF are sufficient for full-level lysyl-phosphatidylglycerol (Lys-PG) production and that several conserved amino acid positions in MprF are indispensable for Lys-PG production. Notably, Lys-PG production did not lead to efficient CAMP resistance and most of the Lys-PG remained in the inner leaflet of the cytoplasmic membrane when the large N-terminal hydrophobic domain of MprF was absent, indicating a crucial role of this protein part. The N-terminal domain alone did not confer CAMP resistance or repulsion of the cationic test protein cytochrome c. However, when the N-terminal domain was coexpressed with the Lys-PG synthase domain either in one protein or as two separate proteins, full-level CAMP resistance was achieved. Moreover, only coexpression of the two domains led to efficient Lys-PG translocation to the outer leaflet of the membrane and to full-level cytochrome c repulsion, indicating that the N-terminal domain facilitates the flipping of Lys-PG. Thus, MprF represents a new class of lipid-biosynthetic enzymes with two separable functional domains that synthesize Lys-PG and facilitate Lys-PG translocation. Our study unravels crucial details on the molecular basis of an important bacterial immune evasion mechanism and it may help to employ MprF as a target for new anti-virulence drugs.
Funding: Our research is supported by grants from the German Research Foundation (SFB685) to HK, from the NIH (AI-39108) to ASB, and from the German
Research Foundation (TR34, FOR449, GRK685, SFB766), the European Union (LSHM-CT-2004-512093), the German Ministry of Education and Research (NGFN2,
SkinStaph), and the IZKF program of the Medical Faculty, University of Tubingen, to AP. 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.
In order to combat increasingly antibiotic-resistant bacteria
such as Staphylococcus aureus, Mycobacterium tuberculosis, Pseudomonas
aeruginosa, and enterococci new antimicrobial strategies based on
compounds with anti-virulence or anti-fitness properties are
increasingly in the focus of research efforts [1,2]. Bacterial
immune evasion mechanisms such as the mprF or
dltABCDencoded pathways are conserved over a wide range of bacterial
species thereby representing attractive targets for broadly active
antimicrobial compounds that would not kill the bacteria directly
but render them susceptible to endogenous host defense molecules
The occurrence of closely related immune evasion factors in
many bacterial pathogens is reflected by the conserved nature of
the most critical antimicrobial host defense molecules. Defensins,
cathelicidins, kinocidins, and related cationic antimicrobial
peptides (CAMPs) are essential components of the antimicrobial
warfare arsenals in humans, vertebrate and invertebrate animals,
and even plants [5,6]. Although peptide structures vary, overall
structural features (cationic, amphipathic properties; often with
c-core motif) and modes of action (damage of microbial
membrane-associated processes) are shared by most of these
peptides . CAMPs appear to take advantage of the fact that
bacterial membranes are formed mostly by anionic phospholipids
. Conversely, the MprF and DltABCD proteins protect many
bacterial pathogens against CAMPs by reducing the negative net
charge of bacterial cell envelopes [3,8]. The dltABCD operon
products neutralize polyanionic teichoic acid polymers by
esterification with D-alanine in many Gram-positive bacteria .
Certain bacterial immune-evasion factors such as the MprF
protein are highly conserved in many bacterial pathogens
and represent attractive targets for new anti-virulence
drugs. MprF, initially discovered in the major human
pathogen Staphylococcus aureus, protects bacteria against
innate human antibiotics such as the defensin peptides.
In addition, MprF has recently been implicated in
resistance to the new defensin-like antibiotic daptomycin.
MprF modifies bacterial membrane lipids with the amino
acid L-lysine, which leads to electrostatic repulsion of the
membrane-damaging peptides. The molecular mechanism
of MprF has remained largely unknown. We demonstrate
that MprF represents a novel bifunctional type of enzyme.
The N- and C-terminal domains of MprF are both required
for mediating antimicrobial peptide resistance but they
can be expressed as two separate proteins without loss of
function indicating that they represent distinct functional
modules. While the C-terminal domain accomplishes lipid
lysinylation the N-terminal membrane-embedded domain
is required to expose the lysine lipid at the outer surface of
the bacterial membrane where it is able to repulse the
antimicrobial peptides. These findings unravel the
molecular basis of an important bacterial immune evasion
mechanism and they may help to employ MprF as a target
for new anti-virulence drugs.
Detailed investigations on this pathway have recently led to the
development of specific DltA inhibitors, which proved to be very
effective anti-virulence drugs for eradication of bacterial infections
Much less is known on the MprF protein, which represents a
particularly interesting antimicrobial drug target because of its
presence in both, Gram-positive and Gram-negative bacteria .
MprF is a large integral membrane protein catalyzing the
modification of the negatively charged lipid phosphatidylglycerol
(PG) with L-lysine thereby neutralizing the membrane surface and
providing CAMP resistance . The resulting
lysyl-phosphatidylglycerol (Lys-PG), described in pioneering biochemical studies
in the 1960es [15,16], is produced by an unusual pathway that
uses PG and Lys-tRNA as substrate molecules . The
LysPG-biosynthetic enzyme has been identified only recently in
Staphylococcus aureus and named multiple peptide resistance factor
(mprF) because mprF mutants lacking Lys-PG are highly susceptible
to CAMPs [12,13]. The loss of Lys-PG in mprF mutants also led to
CAMP susceptibility in Listeria monocytogenes , Bacillus anthracis
, and Rhizobium tropici  thereby demonstrating a general
role of MprF in bacterial immune evasion.
Recently, mprF point mutations or alterations in Lys-PG content
became notorious for spontaneous resistance of S. aureus to
daptomycin [23,24]. This antibiotic has recently been approved
as an antibiotic of last resort for the treatment of
methicillinresistant S. aureus (MRSA), which are responsible for a large
proportion of hospital and, increasingly, community-acquired
bacterial infections . Daptomycin has a negative net charge
but it is believed to have CAMP-like properties and mode of action
upon binding of calcium ions . In addition, MprF has been
implicated in S. aureus susceptibility to the cationic antibiotics
vancomycin, gentamycin, and moenomycin .
mprF expression is upregulated in staphylococci upon contact
with CAMPs by the sensor/regulator system ApsRS [28,29],
which has also been named GraRS [30,31]. Deletion of mprF has
led to profoundly reduced virulence of several bacterial pathogens
in animal models, which underscores the pivotal role of Lys-PG in
bacterial fitness during colonization and infection [12,20,32,33].
Accordingly, it is tempting to elucidate the molecular functions of
MprF as a prerequisite for the development of small inhibitory
molecules that would block Lys-PG biosynthesis and render a large
number of bacterial pathogens highly susceptible to innate host
defenses and cationic antibiotics such as daptomycin,
glycopeptides, or aminoglycosides.
Here we demonstrate that MprF is a bifunctional protein
composed of distinct and separable domains. While the C-terminal
part of MprF is sufficient to synthesize Lys-PG the N-terminal
hydrophobic protein domain is essential for efficient translocation
of Lys-PG from the inner to the outer leaflet of the cytoplasmic
membrane to reduce the bacterial affinity for CAMPs such as
a-defensins, LL-37, daptomycin, or gallidermin.
The first eight N-terminal trans-membrane segments
(TMSs) of MprF are dispensable for Lys-PG synthase
Most MprF-like proteins are composed of large N-terminal
hydrophobic domains followed by hydrophilic C-terminal
domains  (Fig. S1). The hydrophilic portions exhibit much higher
degrees of sequence similarity between different members of the
MprF protein family  suggesting that this domain may play the
most crucial role in Lys-PG biosynthesis. The hydrophobic
domain of S. aureus MprF ranging from amino acid 1 to 509 is
predicted to consist of 14 TMSs (Fig. 1A). In order to study
whether the hydrophobic domain plays a role in Lys-PG
biosynthesis the protein was shortened from the N-terminus in a
step-wise manner by removing two TMSs at a time (Fig. 1B). The
shortened proteins were expressed as N-terminal His-tag fusions
and evaluated for their capacity to mediate Lys-PG production in
E. coli BL21(DE3).
Deletion of the first eight TMSs of MprF from the N-terminus
did not affect the ability of the protein to mediate Lys-PG
production (Fig. 2A). However, further truncations abrogated
LysPG production indicating that at least 6 TMSs are required for
maintaining a functional enzyme and that the N-terminal domain
of MprF may have a separate function. The presence and stability
of the proteins was verified by Western-blotting with a
His-tagspecific antibody. The shorter versions of MprF with no, two, four,
or six predicted TMSs were detectable as singular similarly
pronounced bands indicating that these proteins are largely stable
(Fig. 2B). Longer versions of MprF including the full-length
protein could not be visualized by Coomassie Blue staining or
Western blotting even upon extensive variation of expression,
isolation, and detection methods (data not shown), possibly
because of inaccessibility of the N-terminal His-tag in these
proteins. However, since all proteins ranging from MprF to
MprF(28) yielded similar levels of Lys-PG production the protein
amounts and activities are unlikely to exhibit major differences.
Taken together, our data indicate that the N-terminal eight
TMSs are dispensable for full-level Lys-PG synthesis while any
further shortening completely abrogates the functionality of MprF.
Lys-PG synthase activity depends on several conserved
amino acid residues in the C-terminal part of MprF
Alignment of C-terminal MprF domains from different bacterial
species revealed several conserved sequence motives, which may
represent essential amino acids for substrate binding, enzymatic
reaction, or folding into a stable protein of the Lys-PG synthase
domain (Fig. S2). In order to evaluate the essential nature of such
positions, eight highly conserved amino acid residues were
Figure 1. Structure of MprF and truncated proteins. A) Predicted trans-membrane topology of S. aureus MprF with amino acid positions
predicted to form TMSs indicated. B) Truncated MprF proteins used to study the function of MprF. Length and calculated molecular weight of MprF
variants are shown. Construction of plasmids is described in detail in Table S1.
exchanged with alanine residues by site-directed mutagenesis of
the pET28mprF(28) plasmid. Exchange of K547, K621, E624,
D731, R734, and K806 led to complete abrogation of Lys-PG
production (Fig. 2C). In contrast, replacement of E685 or D546
with alanine resulted in strongly or only slightly reduced Lys-PG
production, respectively. The same results were obtained when the
mutations were introduced into the full-length MprF protein (Fig.
S3A). All the MprF(28)-derived mutant proteins were detectable
in Western Blots as singular protein bands that corresponded to
the MprF(28) protein (Fig. S3B) indicating that even the inactive
Figure 2. TLC and Western Blot analysis of E. coli expressing truncated or mutated variants of MprF. A) Polar lipids from strains
containing expression plasmids without insert (control), with full-length mprF, or truncated mprF genes encoding proteins without the indicated TMS
were separated by TLC and stained with the aminogroup-specific dye ninhydrin. B) The cytosolic fraction of E. coli strains expressing MprF(214) and
the membrane fractions of strains expressing MprF(212), MprF(210), MprF(28) or containing the empty expression plasmid pET28 (control) were
subjected to immunoblot analysis with a His-tag-specific antibody. The TMSs-containing proteins migrated slightly faster than expected, which is
probably due to increased SDS binding capacity and/or incomplete unfolding of TMSs . Molecular weight standard proteins are shown at the
right margin. C) TLC analysis of E. coli strains expressing MprF variants with alanine exchanges. Polar lipids from strains containing the expression
plasmid pET28a without insert (control), with unaltered mprF(28), or with variants encoding proteins with the indicated amino acid exchanges were
separated by TLC and stained with the aminogroup-specific dye ninhydrin. Positions of phosphatidylethanolamine (PE) and lysylphosphatidylglycerol
(Lys-PG) are indicated in A) and C).
proteins were stably produced in E. coli. Taken together, these data
demonstrate essential roles of K547, K621, E624, D731, R734,
and K806 for the enzymatic activity of MprF and less critical but
important roles of D546 and E685.
Expression of mprF (28) in S. aureus DmprF leads to
LysPG production but fails to confer resistance to cationic
In order to investigate if MprF(28) also mediates Lys-PG
production in S. aureus, genes encoding the full-length and the
MprF(28) proteins were cloned in the E. coli/Staphylococcus
shuttle expression vector pRB474 . All the resulting plasmids
led to Lys-PG production in S. aureus SA113 DmprF (Fig. 3A)
thereby reflecting the E. coli results. However, the DmprF mutant
with plasmid-encoded MprF or MprF(28) did not reach the
same level of Lys-PG as the wild-type strain. When the S. aureus
strains were compared for susceptibility to CAMPs such as the
adefensins human neutrophil peptides 13 (HNP 13), the human
cathelicidin LL-37, the bacteriocin gallidermin, or the antibiotic
daptomycin, the mprF mutant was much more susceptible
than the wild-type strain (Fig. 3B), which is in agreement with
previous findings [12,23]. The strain containing the
pRB474mprF(28) plasmid was as susceptible to daptomycin as the mprF
deletion mutant or exhibited only slightly decreased
susceptibilities as in the case of HNP1-3, LL-37, and gallidermin. However,
only the full-length mprF gene led to full resistance to the four
peptides. This result indicates that the N-terminal hydrophobic
domain of MprF is necessary for mediating efficient CAMP
resistance despite the fact that it is dispensable for Lys-PG
The presence of a basic level of Lys-PG seemed to be sufficient
for full-level CAMP resistance provided that the N-terminal
hydrophobic domain of MprF was not absent, while the total
amounts of Lys-PG did not correlate with the levels of CAMP
susceptibility (compare Lys-PG amounts and MIC values for WT
and DmprF containing plasmid pRBmprF). In order to verify this
notion we cloned the minimal Lys-PG synthase domain MprF(28)
in the inducible staphylococcal expression vector pTX15, which
has a higher copy number than pRB474 and permits
xyloseinducible gene expression [35,36]. S. aureus DmprF with the
resulting plasmid pTX15mprF(28) had a 2.53.5-fold increased
Lys-PG content as with the above described pRB474mprF(28)
(Fig. 3C). However, the two strains were inhibited by similarly low
concentrations of daptomycin thereby confirming that Lys-PG
production per se does not necessarily cause CAMP resistance,
irrespective of the produced amounts of Lys-PG.
Both, the hydrophobic domain of MprF and the Lys-PG
synthase domain are required for CAMP resistance but
they do not need to be covalently linked
In order to explore the role of the N-terminal domain of MprF
in CAMP resistance the mprF(2C) gene encoding only the 14
TMSs without the hydrophilic C-terminal domain was expressed
in S. aureus DmprF. Of note, the resulting strain did not show
resistance to any of the tested CAMPs compared to the DmprF
mutant (Fig. 3B) indicating that this protein domain alone cannot
protect the bacteria from CAMPs and depends on the Lys-PG
synthase. In order to evaluate if the two domains need to be fused
or can be separated to achieve CAMP resistance, the mprF(2C)
gene was cloned in pTX15, which is compatible with
pRB474derived plasmids. The resulting plasmid pTX15mprF(2C) or the
empty control plasmid pTX16 were introduced into S. aureus
DmprF bearing pRB474mprF(28). The MIC values of daptomycin
reached much lower levels in the presence of two plasmids
compared to the experiments described above, which is probably
due to increased stress imparted on the two plasmids-containing
bacteria. Notably, when MprF(2C) was co-expressed with
MprF(28) in trans it conferred full CAMP resistance, which
reached the same level as the unchanged MprF protein (Fig. 4B).
Thus, the hydrophobic domain of MprF can only mediate CAMP
resistance if the synthase domain is present but the two proteins
can be separated and do not need to be covalently linked.
The N-terminal hydrophobic domain of MprF is required
for efficient translocation of Lys-PG to the outer leaflet of
the cytoplasmic membrane
While Lys-PG is synthesized at the inner leaflet of the
cytoplasmic membrane where the Lys-tRNA donor substrate is
available, the lipid can only exert its role in CAMP resistance
when present at the outer leaflet of the membrane, where the
antimicrobial peptides are encountered. In order to evaluate the
possibility that the N-terminal hydrophobic domain of MprF
facilitates the translocation and exposure of Lys-PG at the outer
leaflet of the membrane, we first investigated the impact of
MprF(2C) on surface charge neutralization and concomitant
repulsion of cationic peptides . A previously described assay
based on the bacterial binding capacity of the small red-coloured
cationic protein cytochrome c was used for this approach . As
expected, the mprF mutant had a profoundly higher capacity to
bind cytochrome c as the wild-type strain, which reflects the highly
negatively charged membrane surface in the absence of Lys-PG
(Fig. 5A). Likewise, expression of MprF(2C) or of the synthase
Figure 4. Impact on Lys-PG production and resistance to antimicrobial peptides of MprF(28) and MprF(2C) expressed in trans. The
two protein domains were expressed on separate plasmids [pRB474mprF(28) and pTX15mprF(2C)] in S. aureus DmprF. A) Polar lipids were separated
by TLC and stained with the aminogroup-specific dye ninhydrin. B) Minimal inhibitory concentrations of gallidermin and daptomycin. pRB474 and
pTX16 are empty control plasmids. Means and SEM of three independent experiments are shown. ***, P,0.001; ns, not significantly different versus S.
aureus DmprF containing plasmid pRB474mprF and pTX16.
Figure 5. Impact of the hydrophobic N-terminal domain of MprF on the ability of Lys-PG to repulse cationic cytochrome c and to
reach the outer leaflet of the cytoplasmic membrane. A) The capacities of S. aureus wild-type (WT) and DmprF (left panel) or DmprF containing
the indicated plasmids (right panel) to bind cytochrome c were compared. B) Inner and outer-leaflet localization of Lys-PG in DmprF bearing the
indicated plasmids was determined by analyzing the ability of the membrane-impermeable fluorescent dye fluorescamine to react with Lys-PG.
pRB474 and pTX16 are empty control plasmids. Means and SEM of three (A) and four to eight replicas from two (B) independent experiments are
shown. *, P,0.05; **, P,0.01; ns, not significantly different versus S. aureus WT (A, left panel) or DmprF containing plasmids pRB474mprF and pTX16
(A, right panel).
domain MprF(28) in S. aureus DmprF led to substantially reduced
repulsion of cytochrome c compared to the unaltered MprF.
However, when the two protein parts were simultaneously
expressed in trans they led to the same level of cytochrome c
repulsion as expression of the unaltered MprF protein (Fig. 5A).
These results parallel the inability of MprF(28) and MprF(2C) to
confer CAMP resistance individually and they confirm that the
two proteins have complementary functions that can be physically
The ability of the N-terminal hydrophobic domain of MprF to
facilitate the translocation of Lys-PG from the inner to the outer
leaflet of the cytoplasmic membrane was verified by comparing the
capacity of Lys-PG to be modified by the aminogroups-reactive,
membrane-impermeable fluorescent dye fluorescamine in the
absence or presence of MprF(2C). This assay has been developed
to analyze the distribution of amino-phospholipids between inner
or outer leaflets of membranes [38,39] and has been successfully
used to compare Lys-PG distribution in spontaneously
CAMPresistant S. aureus mutants [24,40]. When only the synthase domain
of MprF was expressed in S. aureus DmprF, only a small fraction of
total Lys-PG was found in the outer leaflet (Fig. 5B). However,
when MprF(2C) was coexpressed with the synthase domain, the
amount of Lys-PG in the outer leaflet was strongly increased and
reached a similar level as in the inner leaflet. Thus, the N-terminal
hydrophobic domain of MprF is required for efficient
translocation of Lys-PG.
While the anionic phospholipids PG and cardiolipin are
produced by virtually any bacterial species, zwitterionic or cationic
lipids such as PE or Lys-PG, respectively, are produced only by
certain groups of bacteria . Despite extensive research efforts
the actual roles of the various phospholipids, their biosynthesis,
turnover, and regulation, have remained incompletely understood.
Of note, the same holds true for the identity, specificity, and mode
of action of proposed bacterial translocator proteins required to
flip the lipids, which are generated at the inner cytoplasmic
membrane leaflet, to the outer leaflet. MprF represents the
paradigm of a new class of bifunctional lipid-biosynthetic enzymes
mediating the transfer of amino acids to anionic phospholipids.
While the S. aureus MprF mediates exclusively the biosynthesis of
Lys-PG, the MprF homolog from L. monocytogenes seems to confer
both, Lys-PG and Lys-cardiolipin biosynthesis . MprF
homologs from C. perfringens and P. aeruginosa have been shown to
mediate Ala-PG production [34,42]. Our study represents a basis
for investigating the determinants of substrate specificity of MprF.
Six of the 14 TMSs plus the hydrophilic C-terminal domain
were sufficient to mediate Lys-PG production in E. coli or S. aureus.
The levels of Lys-PG production varied between S. aureus strains
with different plasmid vectors and promoters used to express
MprF or MprF variants but the level of Lys-PG did not correlate
with the level of CAMP resistance indicating that only a basic
amount of Lys-PG is sufficient for repulsing antimicrobial peptides
provided that the lipid is translocated to the outer leaflet of the
membrane. It is amazing that the Lys-PG synthase whose active
center is probably located in the hydrophilic domain of MprF with
its many conserved amino acid positions requires so many TMSs
to function since one or two such segments should be enough to
anchor the hydrophilic C-terminus in the membrane. One might
speculate that six TMSs are required to embrace a PG substrate
molecule and fit it into a position that may allow its lysinylation. It
should be noted that even the MprF homolog with the shortest
integral membrane domain found in Mycobacterium tuberculosis is
predicted to harbor six TMSs (data not shown), which suggests
that the dependence on six TMSs is a general property of
Previous studies on in vitro Lys-PG biosynthesis with artificially
altered aminoacyl tRNAs have demonstrated that the Lys-PG
synthase recognizes features of both, the tRNA and of the bound
amino acid [17,19]. Accordingly, the lysyl group could not be
transferred to PG when it was attached to a cysteinyl tRNA.
However, it did not matter whether the tRNA came from S. aureus
or from another bacterial species such as E. coli . We identified
six conserved amino acids in the C-terminal domain of MprF as
essential for Lys-PG biosynthesis while exchange of two other
amino acid positions led to reduced Lys-PG production. All these
positions are also conserved in MprF homologs with Ala-PG
synthase activity (Fig. S2), which suggests that they are not
involved in specific recognition of the aminoacyl tRNA precursor
and may rather play crucial roles in the enzymatic process or in
non-specific binding of the substrate. Irrespective of the tRNA
structure the substrate-binding domain of MprF may need basic
properties to interact with the polyanionic ribonucleic acid.
Accordingly, four of the six identified essential amino acid position
represent cationic arginine or lysine residues that may participate
in binding of tRNA phosphate groups.
A most intriguing finding of our study was the fact that Lys-PG
production on its own did not lead to CAMP resistance but depended
on the large N-terminal integral membrane domain of MprF. Lys-PG
mediates CAMP resistance by repulsing the cationic peptides from
the outer surface of the membrane, which is only possible upon
translocation of the lipid to the outer leaflet (Fig. 6). Of note, Lys-PG
could only alter the membrane surface charge considerably in the
presence of the N-terminal integral membrane domain indicating
that this part of MprF is required for this lipid to reach the outer
leaflet of the membrane. Moreover, Lys-PG could only be labeled
efficiently by the membrane-impermeable dye fluorescamine in the
presence of the N-terminal hydrophobic domain of MprF, which
confirms the critical role of this protein part in Lys-PG translocation.
Thus, MprF does not only synthesize Lys-PG but also accomplishes
translocation of Lys-PG from the inner to the outer surface of the
membrane. These two functions are allocated in the C-terminal and
N-terminal domains of MprF, respectively, and can be separated into
two functional proteins (Fig. 6).
While lipid translocators have been investigated to some extent
in eukaryotic cells , such proteins have been proposed but
hardly described in bacteria. It is possible that the bacterial
housekeeping translocator(s) are more specific for the standard anionic
phospholipids PG and cardiolipin, while a cationic lipid such as
Lys-PG may require a dedicated translocator. It remains unclear
why a small fraction of Lys-PG was detectable in the outer leaflet
of the cytoplasmic membrane even in the absence of the flippase
domain of MprF. Phospholipids may be able to flip spontaneously
with low efficiency as proposed recently  or one of the
housekeeping flippases may have residual activity for Lys-PG. Lipid
translocators have been classified into energy-dependent (flippases
or floppases) and energy-independent (scramblases) transporters
. MprF does not contain conserved ATP-binding or other
sequence motives indicative of energy consumption. Therefore, it
remains unclear if MprF can accomplish an asymmetric
distribution of Lys-PG. Nevertheless, recent studies suggest that
Lys-PG can be asymmetrically distributed between the inner and
outer leaflets of the membrane in S. aureus depending on the
individual strain background .
The increasing resistance of major bacterial pathogens raises the
specter of untreatable infections as in the pre-antibiotics era.
MRSA are now more and more prevalent in the community and
only a few antibiotics of last resort such as daptomycin have
remained effective against such highly pathogenic S. aureus clones.
As S. aureus can overcome even daptomycin by simple point
mutations in mprF new strategies for antibacterial chemotherapy
are urgently needed. Inhibitors for highly conserved immune
evasion factors such as mprF that would render a wide range of
bacteria susceptible to endogenous human defense mechanisms
and cationic antibiotics such as daptomycin should be increasingly
considered. Our study represents a basis for more detailed
investigations on the structure and mode of action of MprF-like
aminoacylphospholipid synthases and they should enable the
systematic search for inhibitors for this class of enzymes.
Materials and Methods
The plasmids and strains constructed in this study are listed in
Table S1 and primers are listed in Table S2. Construction of
plasmids, growth conditions, alignment and prediction of MprF
structure, Western-blot analysis, lipid extraction, and analysis of
Lys-PG distribution are described in Text S1.
For detection of Lys-PG appropriate amounts of polar lipid
extracts were spotted onto silica 60 F254 HPTLC plates (Merck,
Darmstadt, Germany) using a Linomat 5 sample application unit
(Camag, Berlin, Germany) and developed with chloroform/
methanol/water (65:25:4, by vol.) in an automatic developing
chamber ADC 2 (Camag, Berlin, Germany). Amino groups or
phosphate groups-containing lipids were selectively stained with
ninhydrin spray (0.3 g ninhydrin dissolved in 100 ml 1-butanol and
3 ml 100% acetic acid) or molybdenum blue spray (Sigma).
Integrated lipid spot intensities of molybdenum blue-stained
phospholipids were determined by ImageJ (http://rsbweb.nih.
gov/ij/). MIC values of gallidermin, HNP1-3, and LL-37 were
determined by diluting bacteria from overnight cultures to an
OD600 nm of 0.05 20.1 in fresh MHB medium (gallidermin) or
halfconcentrated MHB (HNP1-3 and LL-37) containing serial dilutions
of antimicrobial peptides as described recently . Gallidermin
was kindly provided by Friedrich G otz. HNP1-3 was isolated from
human neutrophils and purified by reversed-phase
high-performance liquid chromatography (RP-HPLC) as described previously
. LL-37 was synthesized by solid-phase peptide synthesis and
purified by RP-HPLC . Susceptibility to daptomycin was
determined by epsilometer test (E test) in the presence of CaCl2
according to the manufacturers advise (AB Biodisk) .
Differences in bacterial capacity to repulse cationic proteins were
determined by comparing binding of the red-coloured cationic
protein cytochrome c as described recently [37,48].
List of SwissProt accession numbers
Q2G2M2: Staphylococcus aureus MprF; Q5HPI1: Staphylococcus
epidermidis MprF homolog; C0H3X7: Bacillus subtilis MprF
homolog; C0X347: Enterococcus faecalis MprF homolog;
Q8DWT2: Streptococcus agalactiae MprF homolog; Q71YX2: Listeria
monocytogenes MprF homolog; Q88YQ7: Lactobacillus plantarum
MprF homolog; Q8FW76: Brucella suis MprF homolog; Q9I537:
Pseudomonas aeruginosa MprF homolog; Q0SSM7 and Q0STHJ7:
Clostridium perfringens MprF homologs.
Text S1 Supplementary Materials and Methods.
Found at: doi:10.1371/journal.ppat.1000660.s001 (0.07 MB PDF)
Kyte-Doolittle hydrophobicity profile of S. aureus
Found at: doi:10.1371/journal.ppat.1000660.s002 (2.06 MB TIF)
Figure S2 Alignment of the hydrophilic C-terminal parts of
MprF proteins of various bacterial species. Partially and
completely conserved positions are boxed in gray and black,
respectively. Amino acid positions exchanged by site-directed
mutagenesis are indicated. The following proteins were compared
(SwissProt accession numbers are given in brackets): Sa: S. aureus
(Q2G2M2), Se: S. epidermidis (Q5HPI1), Bs: Bacillus subtilis
(C0H3X7), Ef: Enterococcus faecalis (C0X347), Sag: Streptococcus
agalactiae (Q8DWT2), Lm: Listeria monocytogenes (Q71YX2), Lp:
Lactobacillus plantarum (Q88YQ7), Brs: Brucella suis (Q8FW76), PS:
Pseudomonas aeruginosa (Q9I537), Cp1 and Cp2: Clostridium perfringens
(Q0SSM7 and Q0STHJ7, respectively). The C. perfringes Cp1
protein mediates Lys-PG biosynthesis while the C. perfringes Cp2
and the P. aeruginosa protein mediate Ala-PG biosynthesis [15,16].
Found at: doi:10.1371/journal.ppat.1000660.s003 (9.54 MB TIF)
Figure S3 Western Blot analysis of E. coli with pET28mprF(28)
derivatives containing the indicated amino acid exchanges and
TLC analysis of E. coli with mutated full-length mprF genes cloned
in pBAD containing the indicated amino acid exchanges. (A)
Proteins from crude lysates were subjected to immunoblot analysis
with a His-tag-specific antibody. Molecular weight standard
proteins are shown at the right margin. (B) Polar lipids from the
indicated strains were separated by TLC and stained with the
aminogroup-specific dye ninhydrin. Positions of PE and Lys-PG
Found at: doi:10.1371/journal.ppat.1000660.s004 (3.21 MB TIF)
Table S1 Plasmids for expression of truncated or mutated MprF
Found at: doi:10.1371/journal.ppat.1000660.s005 (0.02 MB PDF)
Table S2 Primers used for plasmid construction.
Found at: doi:10.1371/journal.ppat.1000660.s006 (0.02 MB PDF)
Conceived and designed the experiments: CME PS ASB DK AP.
Performed the experiments: CME PS NNM SJY GH HK DK. Analyzed
the data: CME NNM SJY ASB DK AP. Wrote the paper: CME ASB DK
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