Mechanism for sortase localization and the role of sortase localization in efficient pilus assembly in Enterococcus faecalis.
ABSTRACT Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.
- [Show abstract] [Hide abstract]
ABSTRACT: Signal peptide-driven secretion of precursor proteins directs polypeptides across the plasma membrane of bacteria. Two pathways, Sec- and SRP-dependent, converge at the SecYEG translocon to thread unfolded precursor proteins across the membrane, whereas folded preproteins are routed via the Tat secretion pathway. Gram-positive bacteria lack an outer membrane and are surrounded by a rigid layer of peptidoglycan. Interactions with their environment are mediated by proteins that are retained in the cell wall, often through covalent attachment to the peptidoglycan. In this review, we describe the mechanisms for both Sec-dependent secretion and sortase-dependent assembly of proteins in the envelope of Gram-positive bacteria. This article is part of a Special Issue entitled: Protein Trafficking & Secretion.Biochimica et Biophysica Acta 11/2013; · 4.66 Impact Factor
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ABSTRACT: ABSTRACT The ExPortal of Streptococcus pyogenes is a focal microdomain of the cytoplasmic membrane that clusters the translocons of the general secretory pathway with accessory factors to facilitate the maturation of secreted polypeptides. While it is known that the ExPortal is enriched in anionic lipids, the mechanisms that organize the ExPortal are poorly understood. In the present study, we examined the role of the cell wall in organizing and maintaining the ExPortal. Removal of the cell wall resulted in a loss of ExPortal focal integrity accompanied by the circumferential redistribution of ExPortal lipid and protein components. A similar loss occurred upon treatment with gallidermin, a nonpermeabilizing lantibiotic that targets the lipid II precursor of peptidoglycan synthesis, and this treatment disrupted the secretion of several ExPortal substrates. Furthermore, several enzymes involved in the membrane-associated steps of lipid II synthesis, including MraY and MurN, were found to localize to a single discrete focus in the membrane that was coincident with the focal location of the secretory translocons and the anionic lipid microdomain. These data suggest that the ExPortal is associated with the site of peptidoglycan precursor synthesis and that peptidoglycan biogenesis influences ExPortal organization. These data add to an emerging literature indicating that cell wall biogenesis, cell division, and protein secretion are spatially coorganized processes. IMPORTANCE Since Gram-positive bacteria lack a periplasmic space, they lack a protected compartment to spatially coordinate interaction between newly secreted proteins and the factors required to process them. This represents a significant problem for pathogens that depend on the secretion of toxins and cell wall-associated adhesins to cause disease. Streptococci solve this dilemma by restricting secretion and processing factors to a defined region of the membrane. However, the mechanisms that promote restriction are not understood. In this study, we show that restriction of these factors in the pathogen Streptococcus pyogenes is intimately linked with the presence of the cell wall and its synthesis. Furthermore, several cell wall synthesis proteins are also restricted to the site of protein secretion. This study contributes to our understanding of how the Gram-positive cell is organized to coordinate protein secretion and biogenesis with cell wall synthesis and to the ongoing development of antibiotics that target these processes.mBio 01/2013; 4(5). · 6.88 Impact Factor
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ABSTRACT: Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human β-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.Proceedings of the National Academy of Sciences 11/2013; · 9.81 Impact Factor
JOURNAL OF BACTERIOLOGY, May 2009, p. 3237–3247
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 10
Mechanism for Sortase Localization and the Role of Sortase
Localization in Efficient Pilus Assembly in
Kimberly A. Kline,1‡ Andrew L. Kau,1‡§ Swaine L. Chen,1Adeline Lim,1Jerome S. Pinkner,1
Jason Rosch,1¶ Sreedhar R. Nallapareddy,2Barbara E. Murray,2Birgitta Henriques-Normark,3
Wandy Beatty,1Michael G. Caparon,1* and Scott J. Hultgren1*
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 631101; Division of
Infectious Diseases, Department of Internal Medicine, University of Texas Medical School at Houston, Houston,
Texas 770302; and Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden3
Received 31 December 2008/Accepted 6 March 2009
Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the
translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in
gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought
to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We
discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the entero-
coccus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted,
implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing
SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found
to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in
SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that
this positively charged domain can act as a localization retention signal for the focal compartmentalization of
Understanding the transport and processing of proteins in
their journey from the cytosol to the extracellular milieu has
driven significant advances in elucidating the molecular inter-
actions between an organism and its environment. These in-
teractions are particularly important at the host-pathogen in-
terface, where bacterial adhesins, toxins, and other virulence
factors interact with host tissues (31). In gram-negative organ-
isms, transit from the cytosol to the extracellular environment
occurs by several mechanisms that either bypass the periplasm
or use it as an organelle to process and fold proteins destined
for secretion (46). Gram-positive organisms lack a membrane-
bound periplasm but nevertheless secrete many virulence fac-
tors that require posttranslational modification (21). It has
been proposed that the space between the cell membrane and
cell wall provides a protected environment for folding and
processing of secreted proteins in gram-positive bacteria (23,
24, 36, 52). Once translocated across the membrane, many
virulence factors, such as the Streptococcus pyogenes SpeB pro-
tease, are secreted into the extracellular milieu (4), while ad-
hesins are retained at the bacterial surface, where they mediate
attachment to host tissues. A large subset of adhesins charac-
terized as virulence factors in gram-positive organisms, such as
S. pyogenes M protein and Staphylococcus aureus protein A, are
covalently linked to the cell wall by the presence of a cell wall
sorting (CWS) signal (1, 8, 41). The CWS signal is comprised
of a C-terminal LPXTG motif, a transmembrane domain, and
a positively charged tail (41). Proteins containing this CWS
signal are recognized by a sortase enzyme, which cleaves the
CWS motif between the threonine-glycine bond. Subsequent
transpeptidation links the protein to a lipid II intermediate
prior to its incorporation into the cell wall (26, 47). The pro-
tein-lipid II complex is processed by penicillin binding pro-
teins, which results in the incorporation of the CWS protein
into the mature cell wall (48). Despite the great deal known
about the biochemical and mechanistic aspects of cell wall
synthesis and sorting, details of the spatio-temporal coordina-
tion of cell wall synthesis, sorting, and secretion are unclear.
Nevertheless, a close association linking these separate pro-
cesses appears to be critical, because CWS proteins become
properly exposed on the surface of the bacteria only after their
sortase-mediated incorporation into the cell wall (25).
Enterococcus faecalis commonly causes urinary tract infec-
tions, endocarditis, intra-abdominal infections, and bactere-
mia, and it relies on CWS proteins, including Esp, aggregation
substance (AS), and pili, to cause disease (18, 27, 39, 42).
While these studies demonstrate the importance of cell wall
proteins in E. faecalis pathogenesis, the basic mechanisms by
* Corresponding authors. Mailing address: Department of Molecu-
lar Microbiology, Washington University School of Medicine, 660 S.
Euclid Ave., Campus Box 8230, Saint Louis, MO 63110-1093. Phone:
(314) 362-6772. Fax: (314) 362-1998. E-mail for Michael G. Caparon:
email@example.com. E-mail for Scott J. Hultgren: hultgren
† Supplemental material for this article is available at http://jb.asm
‡ K.A.K. and A.L.K. contributed equally to this work.
§ Present address: Department of Allergy and Immunology, Wash-
ington University School of Medicine, St. Louis, MO 63110.
¶ Present address: Department of Infectious Diseases, St. Jude Chil-
dren’s Research Hospital, Memphis, TN 38105.
?Published ahead of print on 13 March 2009.
which these proteins are localized to the cell surface or se-
creted remains unclear. We show here that secretion, protein
trafficking, and cell wall processing are colocalized at single
foci in E. faecalis through the presence of a positively charged
retention domain within the localized protein itself, indicating
that these processes are compartmentalized into an organelle.
MATERIALS AND METHODS
Bacterial strains and culture. Strains used in this study are listed in Table 1.
Escherichia coli MC1061 (50) was grown in Luria-Bertani broth or agar at 37°C
and used to propagate plasmids. E. faecalis strains were inoculated 1:1,000 and
grown statically in brain heart infusion (BHI) broth or agar at 37°C for 15 to 18 h
for all assays unless otherwise noted. Antibiotics were added at the following
concentrations for E. coli: chloramphenicol, 20 mg/liter; kanamycin, 50 mg/liter;
and erythromycin (Erm), 750 mg/liter. For E. faecalis strains, the antibiotics were
added as follows: chloramphenicol, 20 mg/liter; erythromycin, 25 mg/liter; fusidic
acid, 25 mg/liter, kanamycin, 500 mg/liter; rifampin (rifampicin), 25 mg/liter;
streptomycin, 500 mg/liter; tetracycline, 15 mg/liter.
Genetic manipulations. Genes targeted for mutation were identified based on
the annotated complete genome of E. faecalis V583 (32); all references to
genomic loci are based on this annotation (GenBank accession number
AE016830). In-frame deletions of srtA (EF3056) and srtC (EF1094) were created
according to previously described methods (38). SrtB, a third sortase present in
a subset of strains (18, 32), was not investigated. To construct in-frame deletions
of srtA (EF3056) and srtC (EF1094), regions approximately 800 bp upstream and
downstream of the genes were amplified from OG1RF using primer pairs
EF3056e-f3/EF3056 sew-r or EF1094e-f3/EF1094 sew-r for upstream regions and
EF3056e-r3/EF3056 sew-f or EF1094e-r3/EF1094 sew-r for downstream regions
(Table 2). These products were sewn together and amplified using EF3056e-f3/
EF3056e-r3 or EF1094e-f3/EF1094e-r3. These PCR products were then cloned
into the pCR2.1 vector (Invitrogen) according to the manufacturer’s protocol
and sequenced. Correct clones were subsequently subcloned into pJRS233, a
temperature sensitive gram-positive plasmid, using the flanking XbaI sites to
generate deletion constructs pJRS233-?EF3056 and pJRS233-?EF1094 (33).
Deletion constructs were then transformed into OG1SS/pCF10 or OG1X (12) by
electroporation and the transformants selected at 30°C on Erm. Chromosomal
integrants were selected by growth at 42°C in the presence of Erm. Selection for
excision of the integrated plasmid by homologous recombination was accom-
plished by growing the bacteria at 30°C in the absence of Erm. Loss of the
EF3056 or EF1094 loci in Erm-sensitive bacteria was demonstrated by PCR
using primer pair EF3056e-f1/EF3056e-r1 or EF1094e-f1/EF1094e-r1.
Complementation constructs of sortase, sortase mutants, and hemagglutinin
(HA)-tagged sortases were made in pAL1, a derivative of pABG5 that has the
Cmrdeterminant inactivated by NcoI and StuI digestion, blunting, and religation
(16). Wild-type srtA expressed from the rofA promoter present in the plasmid was
cloned into pAL1 by amplifying the srtA coding sequence from OG1RF with
EF3056i-f2/EF3056i-r3, digesting with EcoRI and PstI, and ligating into pAL1. A
gene encoding a C-terminal HA-tagged SrtA expressed under the control of its
native promoter was constructed by amplifying this region from OG1RF (10)
using the primer pair EF3056e-f5/EF3056i-r7. This construct was then cloned
into pAL1 using the BamHI and PstI restriction sites, removing the rofA pro-
moter. Tail mutants of SrtC containing a C-terminal HA2tag were generated in
one step using primers EF1094i-r5, EF1094 tail delete-r, EF1094 (?/?) tail, or
EF1094 (?) tail and EF1094i-f2 and cloned into pAL1 using EcoRI and PstI
restriction sites. All constructs were confirmed by sequencing and transformed
into E. faecalis. The expression and stability of complementation constructs was
verified by anti-HA immunoblotting of whole-cell E. faecalis lysates.
Clumping response protocol. The bacterial clumping protocol was adapted
from previous studies (11) with the following modifications. Overnight starter
cultures were diluted to an optical density (OD) at 600 nm of ?0.06 in 5 ml BHI
supplemented with 0.25 ml sterile supernatant from OG1X. These cultures were
then grown with shaking for 2 to 2.5 h at 37°C before visualization and quanti-
tative OD measurement. These bacteria were also used for localization of AS.
Electron microscopy. Immunolocalization was performed as described previ-
ously for S. pyogenes focal protein localization with the following modifications
(35, 36). Bacteria were fixed in 4% paraformaldehyde–0.5% glutaraldehyde in
100 mM PIPES [piperazine-N,N?-bis(2-ethanesulfonic acid)]–0.5 mM MgCl2
(pH 7.2) for 1 h at 4°C. Samples were then embedded in 10% gelatin and
infiltrated overnight with 2.3 M sucrose–20% polyvinyl pyrrolidone in PIPES-
MgCl2at 4°C. Samples were trimmed, frozen in liquid nitrogen, and sectioned
with a Leica Ultracut UCT cryo-ultramicrotome (Leica Microsystems Inc., Ban-
nockburn, IL). Seventy-nanometer sections were blocked with 0.01 M gly-
cine–5% fetal bovine serum–5% normal goat serum for 30 min and subsequently
incubated with polyclonal anti-HA (Sigma), anti-SecA, anti-Asc10, or affinity-
purified anti-EbpA or anti-EbpC primary antibody overnight at 4°C. Sections
were then washed in blocking buffer and probed with 18-nm colloidal gold-
conjugated anti-rabbit immunoglobulin G (Jackson ImmunoResearch Labora-
tories, Inc., West Grove PA) for 1 h at room temperature. Sections were washed
in PIPES buffer followed by an extensive water rinse and stained with 1% uranyl
acetate–1.6% methylcellulose. Samples were viewed with a JEOL 1200EX trans-
mission electron microscope (JEOL USA Inc., Peabody, MA). Parallel controls
with the primary antibody omitted were consistently negative at the concentra-
tion of colloidal gold-conjugated secondary antibodies used in these studies.
Negative-stain immunogold electron microscopy experiments for labeling of pili
were carried out as described previously (27) with the following modifications.
Bacterial strains were grown statically overnight in BHI, diluted 1:1,000 in tryptic soy
broth containing 0.25% glucose (TSBG), and again grown overnight (?16 h) stati-
cally at 37°C. All mutant strains exhibited growth curves in TSBG that were similar
to those of their wild-type controls (data not shown). The bacteria were then pel-
leted, washed in phosphate-buffered saline (PBS), and resuspended in PBS contain-
ing 5% calf serum. The cells were adsorbed to grids and incubated with affinity-
purified rabbit anti-EbpA or anti-EbpC for 1 h. The grids were then washed with
gold particles for 30 min. The grids were again washed with PBS, fixed with 1%
glutaraldehyde for 20 min, and stained with 0.1% uranyl acetate for 30 s. After three
subsequent washes with PBS, the grids were examined with a JEOL 1200 transmis-
preimmune serum were consistently negative.
Quantification of SecA and sortase foci. Quantitative analysis of the frequency
and region of localization was done from electron micrographs of ?600 repre-
TABLE 1. Strains and plasmids used in this study
Species and strain or plasmid
OG1SS/pCF10 (Str, Tet)
OG1RF (Rif, Fus)
OG1X ?SrtA (Str)
OG1X ?SrtC (Str)
OG1X ?SrtA ?SrtC (Str)
prgB srtA srtC?
prgB srtA srtC
E. coli MC106150
pABG5 (Kan, Cm)
Derivative of pABG5
srtA promoter (Kan)
aStr, streptomycin; Tet, tetracycline; Kan, kanamycin; Erm, erythromycin; Rif,
rifampin; Fus, fusidic acid. See Materials and Methods for antibiotic concentra-
3238 KLINE ET AL.J. BACTERIOL.
sentative bacteria per strain. A focus of localization was defined as a focus
containing ?3 gold particles clustered together. The frequency of focal forma-
tion was determined as the total number of bacterial cells containing foci divided
by the total number of cells counted. Focal localization was determined by
dividing each focus-containing bacterium into three regions from youngest visi-
ble septum to pole. Cells in which septa were not visible were not included in this
analysis. Fisher’s exact test of significance was performed to compare the asso-
ciation between the numbers of foci in each region. Quantification of SrtC focal
reduction in mutant strains was assessed by comparing the number of foci to the
total number of bacterial cells containing ?3 gold particle anywhere on the cell.
Significance was measured by Fisher’s exact test.
Theoretical mathematical predictions for SrtC foci were based on the condi-
tions that the diameter of an enterococcal cell is ?500 nm, that the bacteria are
sectioned into 70-nm sections (see above), and that the microdomain where SrtC
localizes is small relative to the section size and cell size. If the SrtC microdomain
exists as only a single spot, it should be present in one-seventh (14%) of the
sections analyzed and multiple spots should never be observed. If the SrtC
microdomain exists as two spots, the number of foci predicted to be observed
depends on how far apart the foci are. The maximum predicted number would
be for diametrically opposed foci; the minimum predicted number would be
observed for foci that are very close together, where the numbers will eventually
converge to the case of just one spot. If there are two spots diametrically
opposed, then the probability of seeing any spot in a given section is p(spot 1) ?
p(spot 2) ? p(spot 1 and spot 2) ? 28.56%. p(spot 1 and spot 2) is at most 1/7
if they are very close together. p(spot 1 and spot 2) decreases to p(equatorial
slice) ? p(spots rotated into slice) ? 1/7 ? arcsin(70/250)/180° ? 1.2%. As the
two spots get closer together, the probability of seeing two spots increases to
14.28% and the probability of seeing a single spot decreases to 14.28%. It is likely
that if a proportion of cells have two SrtC foci during replication (see Discus-
sion), they will be somewhere between adjacent and diametrically opposed. If
there is a mixture of cells with one spot and two spots, then these probabilities
scale linearly with the proportion of cells with one spot and two spots.
Pilus localization and quantification. For studies of pilus expression and
localization, bacteria were grown in TSBG to enhance pilus production and
immunoblotting was performed as described previously (18, 27, 39, 42), with the
following modifications. Ten milliliters of bacteria (equivalent units of OD at 600
nm) were pelleted, and the supernatants were filtered through 0.2-?m filters and
subsequently concentrated 100? by trichloroacetic acid precipitation. The pellet
from 1 ml of each culture was resuspended in 1 ml of 50 mM Tris-HCl (pH 6.8)
containing 125 units of mutanolysin (Sigma), incubated 2 h at 37°C with gentle
rotation, and centrifuged for 15 min at 14,000 rpm. The supernatant containing
cell wall constituents was collected.
Quantification of pilus expression on whole cells was carried on out negatively
stained, immunolabeled bacteria. At least 100 bacteria per strain per experiment
were scored for pilus expression, as determined by the presence of gold particles
on the cell surface. Quantification of pilus subunit focal localization was assessed
by electron microscopy on immunolabeled thin sections as described for SecA
and sortases above.
Immunofluorescence microscopy was performed as described previously (14)
with the following modifications: Bacteria were grown to stationary phase in
TSBG, washed once in PBS, applied to glass slides, allowed to air dry, fixed in 3%
paraformaldehyde for 10 minutes, washed again, and incubated for 1 hour with
a 1:10,000 dilution of rabbit anti-EbpA or anti-EbpC in PBS–1% bovine serum
albumin. Slides were washed, incubated with Cy3-labeled anti-rabbit secondary
antibody, washed, and stained with DAPI (4?,6?-diamidino-2-phenylindole) (1
mg/ml) and wheat germ agglutinin for 10 min. All imaging was performed at the
Karolinska Institutet Core Visualization Facility at Microbiology, Tumor and
Cell Biology on a Leica (Wetzlar, Germany) fluorescence microscope equipped
with Hamamatsu digital cameras operated by HiPic software (Hamamatsu).
Images were prepared and processed in Adobe Photoshop. Pilus subunit local-
ization was assessed for ?200 bacteria per strain per experiment, where a single
fluorescent spot on a cell was scored as a single focus and multiple spots or
circumferential staining around the cell surface was scored as nonfocal.
Sortase A localizes in single foci in E. faecalis. To investigate
the overlap of secretion with cell wall assembly, we first con-
structed in-frame deletions of both sortases A and C (SrtA and
SrtC), which are present in all examined strains of E. faecalis
(27), creating strains ?SrtA and ?SrtC. To verify the loss of
SrtA activity in E. faecalis, we monitored the phenotypic ex-
TABLE 2. Primers used in this study
Primer name Sequence (5? 3 3?)
EF1094 tail delete-r ..........AAAACTGCAGCTAAGCATAATCTGGAACATCATATGGATAAGCATAATCTGGAACATCATATGGATAG
VOL. 191, 2009 ENTEROCOCCAL SORTASE AND SUBSTRATE LOCALIZATION3239
pression of the SrtA CWS-containing substrate, AS (15). AS
expression leads to a marked, SrtA-dependent clumping of
bacteria (11, 19). E. faecalis ?SrtA, but not ?SrtC, lost the
ability to aggregate compared to the wild type (Fig. 1A). In
complementation analyses, plasmids expressing either wild-
type srtA (?SrtA ? SrtA) or srtA constructed to express SrtA
fused with a dual influenza virus HA epitope tag (SrtA-HA)
restored AS-mediated aggregation in ?SrtA to levels identical
to wild type (Fig. 1A and data not shown, respectively). Intro-
duction of the empty vector did not restore the aggregation
phenotype to ?SrtA. Immunoblot analysis of fractionated
?SrtA cells verified that AS was no longer efficiently incorpo-
rated into the cell wall fraction (data not shown) (13).
Immunogold electron microscopy of E. faecalis grown to
early stationary phase and probed for SrtA-HA revealed that
SrtA appeared clustered in foci in 71% (39/55) of labeled cells
(defined as the presence of three or more gold particles on a
single bacterium). SrtA foci in stationary-phase bacteria were
always observed in a single domain on the surface of the
bacteria (Fig. 1B). Sixty-four percent (37/58) of labeled bacte-
ria grown to mid-log phase displayed focal SrtA localization.
The majority (96%) of log-phase cells in which SrtA was ob-
served to be localized displayed single SrtA foci (54/56), and
4% of the bacteria (2/56) had two SrtA foci each. Quantitative
analysis of the location of SrtA foci on single bacteria was
performed by dividing each bacterium into three equal regions
spanning from the most recent visible septum to the pole, a
method previously established for the localization of M protein
and protein F on the surface of S. pyogenes (5). Region 1
corresponded to the equatorial region, which spanned the site
of the current division plane. Region 2 was located where the
next division plane would arise, which in enterococci is called
the midcell, since this class of organisms divides in parallel
chains. Region 3 included the polar region which corresponds
to the septal area from the previous round of cell division (Fig.
1C). A significant majority, 79.4% (P ? 0.0001), of SrtA foci
observed in both log- and stationary-phase cultures were found
either in region 1 (41.3% or 32.4%, respectively, for log- or
stationary-phase cultures) or in region 3 (38.3% or 47.1%,
respectively, for log- or stationary-phase cultures). Only 21%
were localized to region 2. Together these data suggest that
SrtA localizes to the active division plane but also remains
associated with polar regions, the sites of previous cell division.
This pattern of localization was not altered when actively di-
viding cells in mid-logarithmic growth were examined, indicat-
ing that our observations of SrtA foci were not growth stage
dependent (Fig. 1C).
SecA localizes to a distinct membrane domain of Enterococ-
cus faecalis. Frequent localization of SrtA to the equatorial
region, the site of the nascent cell division septum, suggested a
link between secretion, cell division, and cell wall synthesis. To
address whether localized sites of Sec-mediated membrane
translocation occur in E. faecalis, we first examined the distri-
bution of SecA translocons in the membrane. The SecA anti-
body used in these studies was raised against Bacillus subtilis
SecA and was shown to cross-react in immunoblots with a band
of the size expected for SecA in both S. pyogenes (35) and E.
faecalis (data not shown) whole-cell lysates. When examined by
immunogold electron microscopy of thin-sectioned bacteria,
SecA-specific staining of E. faecalis grown to early stationary
phase was observed in single membrane domains in 3% of
sections; the remaining 97% of cells were unlabeled. As thin
sections reflect only a fraction of the cell, this frequency does
not reflect the actual percentage of cells in which foci occur. To
examine the site of SecA localization in each bacterium, we
collected an additional 50 bacterial cells from the population
labeled with three or more gold particles corresponding to
SecA. Fifty-four percent (30/56) of SecA foci were located in
the equatorial region 1 of the bacteria, compared to 30.4%
(17/56) and 16.1% (9/56) in regions 2 and 3, respectively (Fig.
2B). Multiple foci were never observed (0/56) under the early-
stationary-phase growth conditions examined here.
To address the possibility that SecA localization was depen-
dent on growth phase, we next directly compared SecA local-
ization in mid-logarithmic phase versus stationary phase.
Among bacteria positively labeled with three or more gold
particles, single SecA foci were observed at approximately the
same frequency in a log-phase population (72.2%, 39/54) as in
a stationary-phase population (68.1%, 32/47). In the remaining
15/54 and 15/47 SecA-labeled bacteria from each growth
phase, respectively, the gold particles were not clustered.
These observations indicate that SecA focus formation is not
dependent on growth phase. Localization of SecA resembled
the pattern observed for SrtA. Thus, we tested the hypothesis
that both SrtA and SecA are localized at the same membrane
FIG. 1. Sortase A is focally localized in E. faecalis. (A) Broth
clumping assay on OG1SS/pCF10-derived strains. Proper sorting of AS
and subsequent clumping of E. faecalis is dependent on SrtA but not
SrtC. In strains containing SrtA (first, third, and fifth tubes) the bac-
teria form large aggregates that settle to the bottom of the tube. Strains
that lack SrtA (second and fourth tubes) remain turbid. (B) Anti-HA
immunoelectron microscopy on OG1SS/pCF10 ?SrtA complemented
with an HA epitope-labeled SrtA expressed under its native promoter.
SrtA-HA localizes to single domains on the surface of the bacterium.
(C) Quantitative localization analysis of SrtA immunoelectron micro-
graphs. Bacteria were equally divided into three regions from youngest
visible septum to pole. Bacteria without a visible septum were excluded
from this analysis. Dark gray bars represent log-phase bacteria, and
light gray bars represent stationary-phase bacteria.
3240 KLINE ET AL.J. BACTERIOL.
microdomain in colabeling experiments. We observed consis-
tent colocalization of both SrtA-HA and SecA to a single
membrane site (Fig. 2C).
Disruption of sorting leads to focal substrate accumulation.
Following translation, sortase substrates such as AS are tar-
geted to the membrane for Sec-mediated translocation across
the membrane in a process facilitated by the Sec signal se-
quence. Current models predict that after translocation, sor-
tase substrates are retained in the membrane by their trans-
membrane helix and flanking positively charged tail until
sortase cleavage removes the helix and tail and covalently
couples the substrate to the cell wall (41). Once secreted
through the Sec pathway and processed by sortase, the distri-
bution of sortase substrates around the cell is driven by incor-
poration into nascent cell wall components used in peptidogly-
can biogenesis. Colocalization of SecA with SrtA at a single
domain suggested that the secretion and processing machiner-
ies for CWS proteins are spatially coupled. This hypothesis was
investigated by studying the localization pattern of AS using
immunogold electron microscopy. In wild-type (30) or in
?SrtA ? SrtA-HA E. faecalis, AS was distributed around the
bacterial surface (Fig. 3A). In contrast, in E. faecalis ?SrtA, AS
was no longer assembled around the periphery of the cell wall
but instead localized to a single site (Fig. 3B), as was observed
for SecA and SrtA (Fig. 2C). These results suggested that AS
is retained at membrane microdomains when it is not properly
incorporated into the cell wall by SrtA.
Pilus-associated sortase C and substrates localize to single
sites. While many SrtA enzymes are considered “housekeep-
ing” sortases responsible for the surface attachment of most
cell wall proteins, a subset of sortases function in pilus biogen-
esis (6, 9). E. faecalis SrtC is required for the polymerization of
pili that consist of three proteins: EbpA, EbpB, and EbpC (27).
We hypothesized that efficient pilus biogenesis is facilitated by
focal localization of SrtC. To assess SrtC localization, we con-
structed a strain in which srtC is deleted and complemented
with a plasmid expressing an HA-tagged SrtC (?SrtC ? SrtC-
HA). The HA epitope tag on SrtC did not alter its function in
pilus production, since immunolabeling of negatively stained
bacteria for the major pilus subunit EbpC showed that approx-
imately 35% of the wild-type cells expressed pili, similar to
levels for ?SrtC ? SrtC-HA (Fig. 4A) and similar to previously
reported levels for the same growth conditions (27).
To determine if SrtC, like SrtA, is found at sites of secretion,
we examined localization of SrtC-HA in early stationary phase.
We found that SrtC-HA colocalized with SecA in discrete foci
at the membrane (Fig. 4B). Examination of immunolabeled
thin sections revealed focal SrtC localization on 18.4% (109/
593) of these cells, while the rest were unstained. Of the 109
stained cells, 95.4% (104 bacteria) had a single focus, 3.7% (4
bacteria) had two foci, and 0.9% (1 bacterium) had three foci
(Fig. 4C). This last class represented 0.2% (1/593) of all cells
expressing SrtC-HA and was below the background frequency
for single foci of 0.7% (4/577) observed in wild-type cells that
do not express SrtC-HA (data not shown). Since a ring struc-
ture would be expected to produce a majority of cells with two
FIG. 2. SecA localizes at single domains in E. faecalis. (A) Anti-SecA immune electron microscopy of OG1SS/pCF10. SecA localizes to a single
domain on the surface of the bacterium near the equatorial region. (B) SecA focal localization, quantified as described for Fig. 1C.*, P ? 0.005
by Fisher’s exact test. (C) Double-label immunoelectron microscopy using anti-SecA (large particles) and anti-HA (small particles) antibodies.
SecA and SrtA colocalize to the same region on the surface of E. faecalis. Scale bars, 0.5 ?m. Inset, twofold magnification of a representative area
FIG. 3. Sortase A substrate accumulates focally in the absence of
sortase. Immunolocalization of AS in OG1SS/pCF10 ?SrtA exposed to
pheromone in the presence (A) or absence (B) of SrtA-HA is shown.
E. faecalis was induced to express AS as described in Materials and
Methods. Scale bar, 0.5 ?m.
VOL. 191, 2009 ENTEROCOCCAL SORTASE AND SUBSTRATE LOCALIZATION3241
foci and since the frequency of single foci is consistent with that
predicted for 0.5-?m cells cut in 70-nm sections (17.5% ob-
served versus 14.3% predicted; see Materials and Methods),
these data support the conclusion that SrtC is localized to a
single membrane domain rather than organized into a circum-
ferential ring; however, we cannot exclude the possibility that
more than one focus of localized protein exist on a cell. In
addition, quantitative analysis of the locations of foci indicated
a tendency toward localization in the vicinity of the septum
versus the periphery or poles (Fig. 4D, region 1).
To examine whether other sortase substrates accumulate in
foci in the absence of their cognate sortase, we analyzed the
fate of the pilin subunit EbpA in the absence of SrtC. In a
?SrtC strain, EbpA is not incorporated into pilus fibers in
wild-type cells (27), and immunoblot analysis showed that
EbpA monomers are instead both cell wall associated and also
secreted into the culture supernatant in the absence of SrtC
(see Fig. S1 in the supplemental material). Similar to the ac-
cumulation of AS in the absence of SrtA (Fig. 3B), immuno-
electron microscopy showed that the cell wall-associated EbpA
monomers formed foci in 53% of the EbpA-labeled cells in the
absence of SrtC (Fig. 5A and C, left panel) while no foci were
observed in wild-type cells (Fig. 5A and D, left panel), a sta-
tistically significantly enrichment (P ? 0.00001). One explana-
tion for the incomplete focal localization of EbpA in the ?SrtC
strain is that SrtA is able to attach a subset of EbpA subunits
to the cell wall as has been described for Corynebacterium
diphtheriae and Streptococcus pneumoniae (22, 28). Thus, to
further validate focal pilin subunit accumulation in the absence
of SrtC, immunofluorescence microscopy on whole bacterial
cells was performed. EbpA and EbpC were shown to be dis-
tributed in a uniform pattern on the surface of wild-type cells
(Fig. 5A, B, and C, right panel). Similarly, in ?SrtA cells, pilin
subunits exhibited uniformly distributed staining (Fig. 5A and
B). In contrast, both the ?SrtC and ?SrtA ?SrtC double mu-
tant strains were significantly enriched for EbpA staining of
singular foci (P ? 0.0001) (Fig. 5A). Similar observations were
made for EbpC (Fig. 5B). Thus, we conclude that the surface
distribution of pilin subunits is altered in strains lacking the
SrtC enzyme necessary for pilus polymerization.
A positively charged sequence is important for focal sortase
localization retention. The clustering of SrtA and SrtC in sin-
gular foci, as well as sortase substrate clustering in the absence
of the cognate sortase, suggested that sortases and CWS pro-
teins may contain a signature domain that retains them within
membrane microdomains prior to processing. Sequence com-
parisons revealed that each of these proteins has a single trans-
FIG. 4. Sortase C localizes to single foci in E. faecalis. (A) Piliation
levels of wild-type OG1X and sortase mutants. Results are from a
representative experiment in which ?100 cells/strain/experiment were
counted. Statistical significance measured by chi-square test:*, P ?
0.001.**, P ? 0.0001. (B) Coimmunolocalization of SecA (large par-
ticles) and SrtC (small particles) found together in foci. Inset, close-up
of colocalized SecA and SrtC. Scale bar, 0.5 ?m. (C) Quantitative
analysis of SrtC immunoelectron micrographs.*, P ? 0.0001 by chi-
square test. (D) Location of SrtC foci in bacteria equally divided into
three regions from youngest visible septum to pole. Statistical signifi-
cance measured by Fisher’s exact test:*, P ? 0.05;**, P ? 0.001.
3242KLINE ET AL. J. BACTERIOL.
membrane domain that is flanked by a highly positively
charged region. Positive charges flanking a transmembrane
helix have been well characterized as a determinant of mem-
brane topology (49). In SrtA, this positively charged domain is
in the uncleaved secretion sequence of the protein (which also
serves as the membrane-spanning domain), whereas in SrtC
this domain is at the C terminus. Sequence comparisons of
sortase substrates show that cell wall sorted proteins, including
AS and the Ebps, contain a positively charged tail as part of
their CWS (Fig. 6A and data not shown) (40, 41). Therefore,
we hypothesized that in these proteins, a higher positive charge
not only determines topology but also mediates localization to
and retention within membrane microdomains.
Since the SrtC charged domain is at the C terminus and thus
is not part of the signal peptide, we used it as a model protein
to investigate whether the positively charged residues play a
role in localizing membrane proteins to discrete foci, distinct
from the signal peptide. This was examined by mutating the
positively charged C-terminal domain in SrtC. Three different
mutants were constructed: a mutant with a deletion of all
positively charged residues from the C terminus, a mutant with
every other positively charged amino acid replaced by a nega-
tively charged residue, and a mutant with every positively
charged residue replaced by a negative one. The effect of these
mutations on SrtC localization was determined by immunogold
electron microscopy. In contrast to the discrete localization
observed for wild-type SrtC (Fig. 4B and 6D), all three mutants
displayed an altered pattern of localization (Fig. 6E to G)
corresponding to a decrease in the frequency of focal localiza-
tion of gold particles and an increase in random gold labeling.
Quantifying these results, we observed 36% fewer foci for
SrtC?tail, 72% fewer for SrtC(?/?)tail, and 70% fewer for
SrtC(?)tail compared to wild-type SrtC (Fig. 6B). Mislocal-
ized SrtC typically appeared as multiple smaller SrtC clusters
along with several single gold particles. SrtC mislocalization is
not due to instability of the mutated proteins, as verified by
Western blot analysis (Fig. 6C). The clustering pattern of
SrtC is unlikely to be mediated by the HA tag, since the
clustering is dispersed in the SrtC mutants (which also have
an HA tag). Thus, we conclude that the positively charged
domain flanking the transmembrane helix is necessary for
Focal localization of SrtC is necessary for efficient pilus
formation. If SrtC localization to single foci is important for
coupling secretion of pilus subunits and their subsequent process-
ing into pilus fibers, then disruption of SrtC localization should
also disrupt pilus biogenesis. We examined the location and ex-
tent of pilus expression on negatively stained bacteria with anti-
EbpC antibodies. As reported for pili of other gram-positive or-
pili, and for those cells that did express pili, no differences were
observed in either the structure of the pili or their localization
pattern on the cell surface for the wild-type, ?SrtC ? SrtCWT,
and ?SrtC ? SrtC?tail strains (see Fig. S2 in the supplemental
material). In contrast, the overall proportion of piliated cells in
each culture differed markedly. Complementation of ?SrtC with
FIG. 5. Pilus subunits accumulate focally in the absence of SrtC. (A and B) Quantification of EbpA (A) or EbpC (B) immunofluorescent
labeling of whole E. faecalis OG1X wild-type or sortase mutant cells grown to stationary phase.*, P ? 0.0000001 by Fisher’s exact test. (C and
D) EbpA labeling of wild-type (WT) (C) or ?SrtC bacteria (D) and localization by electron microscopy (left panels). Representative images of
whole-cell immunofluorescence labeling of EbpA (red), DNA (blue), and cell wall (green) are also shown (right panels).
VOL. 191, 2009ENTEROCOCCAL SORTASE AND SUBSTRATE LOCALIZATION3243
a plasmid-encoded copy of srtC restored piliation to wild-type
sion of SrtC?tail, resulted in a significantly reduced level of pili-
ation (15% piliation for ?SrtC ? pSrtC?tail versus 55% piliation
for ?SrtC ? pSrtCwt, P ? 0.0001) (Fig. 4A), suggesting that focal
subcellular localization of SrtC is required for efficient pilus bio-
genesis. Taken together, these data indicate that while SrtC mis-
localization has no effect on the final destination of the sortase
FIG. 6. Sortase C localization is dependent on a positively charged cytoplasmic tail. (A) Alignment of proteins observed in localized foci in E.
faecalis. The cartoon depicts amino acids adjacent to the transmembrane helices (TMH) of E. faecalis SrtA, SrtC, AS, and Ebp pilus subunits. NH3
and CO2indicate the N and C termini of the proteins, respectively. Boldface amino acids are positively charged. (B) Quantitative analysis of SrtC
immunoelectron micrographs. Bacteria labeled with three or more gold particles were assessed for the presence or absence of focal localization.
The percentage of cells displaying SrtC foci is expressed relative to wild-type (WT) value. Statistical significance measured by Fisher’s exact test:
*, P ? 0.05;**, P ? 0.001. (C) Anti-HA immunoblot of whole OG1RF, demonstrating stability of SrtC tail mutants. (D) Expression of SrtC-HA
under control of the RofA promoter results in localization to single domains on the surface of the bacterium in wild-type E. faecalis strain OG1RF.
(E to G) Immunolocalization of SrtC tail mutants (the amino acid sequence of the mutagenized tail is indicated). Scale bar, 0.5 ?m.
3244 KLINE ET AL.J. BACTERIOL.
substrate after attachment to the cell wall, proper SrtC placement
in the cell membrane facilitates its efficient function.
Bacterial cells display exquisite subcellular organization in
the processing of virulence factors for display on the cell sur-
face. Localization of presecretory proteins facilitates efficient
secretion, processing, and assembly of macromolecular struc-
tures necessary for pathogenic interactions. This process has
been enigmatic in gram-positive organisms due to incomplete
understanding of basic molecular secretion mechanisms. Re-
cently, two models have been proposed for how proteins are
secreted in gram-positive cocci. In the first model, components
of the general secretory pathway (Sec machinery) have been
shown to localize to distinct domains in S. pyogenes and Strep-
tococcus mutans (17, 35), leading to the proposal that protein
secretion and processing may be spatially coupled. Supporting
this model, two secreted proteins from S. pyogenes, SpeB and
HtrA, colocalize with this secretion domain, termed the Ex-
Portal. In the second model, domains within the secreted pro-
teins themselves, and not the location of the Sec machinery,
are proposed to direct the localization of secreted proteins in
gram-positive cocci. A domain of the N-terminal secretion
signal was shown to differentially influence the sites of protein
F and M protein appearance on the cell surface of S. pyogenes,
as well as that of a number of LPXTG-containing proteins in S.
aureus (5, 7). Consistent with the model for localized secretion
machinery, Sec components were found to localize in a helical
pattern along the lengths of both gram-positive B. subtilis and
gram-negative E. coli rods (3, 43), suggesting that secretion
localization may be a conserved phenomenon in similarly
shaped bacteria. Nevertheless, the molecular details governing
the spatial subcellular distribution of the Sec apparatus in
gram-positive cocci is not yet understood. Reconciliation of
these models awaits careful examination of the entire secretion
apparatus locale, including the SecYEG translocation channel,
throughout the cell cycle.
Here we show that both secretion and sortase processing are
spatially coupled in E. faecalis. The observation that SecA can
localize to single domains in both log and stationary phases is
consistent with the ExPortal model of localized protein secre-
tion and indicates that sortase proteins are also found at this
subcellular location. The significant enrichment of SecA and
sortase enzyme domains in the vicinity of the cell division plane
is consistent with cell wall synthesis in enterococci and strep-
tococci occurring at the midcell (reviewed in reference 51).
Localization of SrtC was facilitated by a positively charged
sequence within the C terminus of the protein, distinct from
the N-terminal secretion signal. Efficient assembly of sortase-
dependent pili required proper subcellular localization of sor-
tase. Our results suggest a model of coordinated localized
secretion and sorting of cell wall proteins and are consistent
with a model that in E. faecalis, proteins do not always traverse
the cell membrane in a random manner but instead have at
least one pathway that coordinates protein secretion and sub-
sequent processing in localized regions across the cell mem-
brane. It is likely that gram-positive cocci have evolved multi-
ple mechanisms for subcellular localization. It will be
interesting to determine whether signal sequence domains,
analogous to YSIRK motifs identified in S. pyogenes and S.
aureus (5, 7), also play a role in cell wall protein deposition in
E. faecalis. Notably, none of the enterococcal proteins exam-
ined in this study and only one of 57 predicted CWS proteins
in the sequenced E. faecalis V583 genome possesses a signal
sequence bearing a canonical YSIRK motif (reference 32 and
data not shown).
The cell wall of gram-positive bacteria is responsible for
scaffolding its surface-exposed proteins but is also a significant
barrier to secretion (44). This likely requires unique mecha-
nisms for efficient transit and processing of virulence factors
across the membrane and into the extracellular space. The
assembly and attachment of gram-positive pili to the cell wall
constitute an excellent model system for study of such com-
plexity. The genes encoding pilus subunits are found geneti-
cally clustered with a sortase that is involved in the covalent
polymerization of subunits during pilus biogenesis. After pilus
polymerization, anchorage of the pilus to the growing cell wall
is facilitated either by a “housekeeping” sortase encoded else-
where on the chromosome (2, 29, 45) or by the pilus-associated
sortase enzyme itself, as has been reported for S. pneumoniae
(20). We show that the enterococcal pilus subunits EbpA and
EbpC are significantly enriched in single foci in the absence of
SrtC. These observations suggest a model in which a microdo-
main for secretion and sortase action may facilitate a high local
concentration of subunits that are primed for pilus assembly.
Interestingly, recent findings with S. pneumoniae show pilus
localization at multiple, symmetric foci in wild-type cells (14).
Pilus biogenesis in pneumococci appears to be a more complex
process than in enterococci, with three pilin-associated sortase
enzymes that not only display substrate specificity but also are
required for focal presentation of pili (14). These findings are
consistent with a model in which pilus formation in gram-
positive bacteria involves the coordination of subunit secre-
tion, processing by multiple sortase enzymes, and cell wall
synthesis. For secreted proteins such as pilin subunits, the
enrichment of SecA and SrtC foci at or near the division
septum where peptidoglycan synthesis is occurring may also
reflect the most energetically favorable site for secretion and
processing, where the cell wall barrier is thinnest. Organization
of multiple cellular processes at a single site not only facilitates
spatial and temporal coordination of these processes but also
promotes efficiency in their function.
An interesting prediction of any localized secretion model is
that the membrane contains an asymmetric distribution of
proteins, with one subset retained in foci while another be-
comes routed into the peripheral membrane. Thus, the mem-
brane proteins themselves should contain specific motifs that
are responsible for their trafficking to their appropriate desti-
nation following insertion into the membrane. Our data sug-
gest that a high positive charge flanking a membrane-spanning
region can function as a retention sequence for SrtC localiza-
tion. Interestingly, the CWS sequences of sortase substrates in
S. aureus and E. faecalis also possess a positively charged tail
that is necessary for efficient cell wall sorting (19, 41). We
hypothesize that this positively charged region may act to re-
tain a sortase substrate within membrane microdomains fol-
lowing its translocation by the Sec pathway in order to promote
interaction with the similarly localized sortase enzyme. Thus,
in the absence of their cognate sortases, the sortase substrates
VOL. 191, 2009ENTEROCOCCAL SORTASE AND SUBSTRATE LOCALIZATION3245
AS and Ebp pilin subunits were retained in membrane mi-
Understanding how positive charge mediates localization of
sortase C in E. faecalis will lead to important insights into the
molecular underpinnings of gram-positive pathogen secretion
and protein processing. One explanation is that cytoskeletal
proteins or other subcellularly localized proteins form a scaf-
fold for protein localization. Currently, the streptococci and
enterococci lack any of the known bacterial cytoskeletal pro-
teins described to date. Alternatively, a lipid-stabilized domain
similar to lipid rafts in eukaryotic cells (34) may facilitate the
retention of proteins by protein-lipid interactions. The latter
possibility is consistent with findings that negatively charged
lipids are necessary for SecA localization in B. subtilis and that
anionic lipid domains occur at single sites that are consistent
with the location of the ExPortal in S. pyogenes (3, 37).
In this study, we show that secretion and sortase processing
occur together in E. faecalis. Sortase localization is facilitated
by a positive charge that is necessary for efficient pilus biogen-
esis. These findings present a novel mechanism for coordinate
secretion and processing of cell wall proteins in gram-positive
cocci. Together, these data increase our understanding of basic
molecular processes in this important category of pathogens
and could lead to the identification of novel targets for thera-
We thank members of the Hultgren, Caparon, and Normark labs for
helpful discussion, critique, and critical reading of the manuscript;
Darcy Gill for assistance with negatively stained immunoelectron mi-
croscopy; and G. Dunny and D. Manias for providing Asc10 antibody.
This work was supported by National Institutes of Health and Office
of Research on Women’s Health Specialized Center of Research grant
P50DK6454002 with the Food and Drug Administration (to S.J.H.);
NIH grants AI46433 (M.G.C.), AI47923 (B.E.M.), and AI068362
(S.L.C.); Medical Scientist Training Program grant T32 GM07200
(A.L.K.); and AHA postdoctoral fellowship 0625736Z (K.A.K.).
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