JOURNAL OF BACTERIOLOGY, Oct. 2009, p. 6203–6210
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 20
Capsular Polysaccharide Production in Enterococcus faecalis and
Contribution of CpsF to Capsule Serospecificity?†
Lance R. Thurlow, Vinai Chittezham Thomas, and Lynn E. Hancock*
Division of Biology, Kansas State University, Manhattan, Kansas 66506
Received 6 May 2009/Accepted 24 July 2009
Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of
the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to
produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes
of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production
did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa
type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system,
we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating
that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that
serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies.
Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and
D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed
amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosy-
lation of serotype C capsular polysaccharide in E. faecalis.
Enterococcus faecalis is a gram-positive bacterium commonly
found as a commensal organism in the gastrointestinal tracts of
most mammals. E. faecalis is one of the leading causes of
hospital-acquired urinary tract infections, bacteremia, and sur-
gical-site infections (29). The development of multiple antibi-
otic resistances, including resistance to vancomycin, makes
treatment of enterococcal infections difficult (11). The 2004
National Nosocomial Infections Surveillance report indicated
that nearly 30% of enterococci isolated from clinical settings
were resistant to vancomycin, constituting a 12% rise from the
previous 5 years (26). The development of alternative thera-
pies to treat enterococcal infections has frequently been sug-
gested due to rising percentages of antibiotic-resistant entero-
coccal strains (13–15, 19).
Capsular polysaccharides are major contributors to the
virulence of many microorganisms. The presence of capsule
allows these microbes to escape detection and clearance by the
host immune system (9, 27, 30, 41). There have been several
publications regarding the role of cell wall polysaccharides in
the pathogenesis of enterococcal infections (10, 13, 17, 37, 43).
Several attempts have been made to establish a serotyping
system for E. faecalis capsular polysaccharides (16, 23, 35, 36).
These serotyping schemes include differences in capsular poly-
saccharide antigens but are also based on differences in surface
antigens, including lipoteichoic acid (16, 38). To date, only one
study has linked genetic evidence with capsule production (12).
Two loci that have been reported to contain putative genes for
capsule production are the epa and cps operons (10, 42). The
polysaccharide produced by the epa locus is thought to be the
cell wall rhamnopolymer (10), but it cannot be detected on the
surface of the bacterium (43). Although rhamnopolymer pro-
duction is reported to be abrogated by mutation (43), the full
nature of rhamnopolymer production is yet to be determined
for many E. faecalis strains. Probing the genomes of serotype A
and B strains with a probe specific to the cps locus, including
the genes cpsA and cpsB, identified a single ClaI restriction
fragment for serotypes A and B (16). However, multiple ClaI
restriction fragments were identified in serotypes C and D (16),
suggesting that the genes responsible for capsule production in
serotypes C and D were absent in serotypes A and B. Further-
more, the hybridization pattern between serotype C and D
strains indicated a single restriction fragment polymorphism,
but the basis on which genes were different between the two
serotypes was not fully characterized (16). Studies based on the
serotyping scheme proposed by Hufnagel et al. (17) have
shown that serotype C and D strains are much more resistant
to opsonophagoctyosis by neutrophils in the presence of nor-
mal human serum. More recently, a study by McBride et al.
indicated that serotype C clinical isolates harbored a greater
repertoire of antibiotic resistance cassettes and were more
likely to possess multiple virulence factors than the other se-
rotypes, suggesting that the presence of the capsule is associ-
ated with pathogenic lineages of E. faecalis (17, 24).
It is essential to understand the underlying mechanisms of
capsule production in E. faecalis because of ongoing efforts to
develop alternative therapies targeting capsule. Here, we used a
novel vector system for creating isogenic, in-frame deletion mu-
tants to analyze the genetic basis for capsule production and
serotype specificity. Our results show that only serotype C and D
strains of E. faecalis produce capsular polysaccharides, based on
the observation that deletions of cpsC, cpsD, cpsE cpsG, and cpsI
abolish the production of capsule. In conjunction with these ob-
servations, we also demonstrated that the presence of capsule
prevents detection of lipoteichoic acid on the surface of serotype
* Corresponding author. Mailing address: Division of Biology, Kan-
sas State University, 119 Ackert Hall, Manhattan, KS 66506. Phone:
(785) 532-6122. Fax: (785) 532-6653. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 14 August 2009.
C and D strains but not on unencapsulated strains. Our data also
show that CpsF is responsible for the difference in serospecificity
between serotype C and D strains.
MATERIALS AND METHODS
Bacterial strains and growth conditions. All relevant bacterial strains are
listed in Table 1. Escherichia coli EC-1000 (20) and Electro-10 Blue (Stratagene)
were used for plasmid construction. E. coli clones were grown in Luria-Bertani
(LB) broth supplemented with the appropriate antibiotics when required (32). E.
faecalis strains were cultivated in Todd-Hewitt broth supplemented with the
appropriate antibiotics when needed (THB; Becton, Dickinson and Company,
Sparks, MD). When required for selective growth of E. coli, chloramphenicol
(Cm) was used at 10 ?g/ml and spectinomycin was used at 150 ?g/ml. When
required for the selective growth of E. faecalis, Cm was used at 15 ?g/ml and
spectinomycin was used at 750 ?g/ml. For detection of ?-galactosidase activity,
5-bromo-4-chloro-3-indolyl-a ˆ-d-galactopyranoside (X-Gal) was used at 80 ?g/ml
for E. coli and 120 ?g/ml for E. faecalis.
Dot blot analysis. We performed dot blots with DNA from representative E.
faecalis strains, including FA2-2, V583, MMH594, Maekawa types 1, 2, 4, 5, 7, 8,
11, and 18, and strains OG1RF, 12030, 12107, and E-1 to determine the presence
of cps operon genes. Purified DNA from each strain was denatured in 0.4 M
NaOH to a concentration of 1 ?g/ml and spotted onto nylon membranes. The
membranes were rinsed several times with Tris-EDTA buffer, pH 8.0. DNA was
cross-linked to the membrane using UV irradiation. Gene-specific radiolabeled
probes were generated by PCR using primers (see Table S1 in the supplemental
material) for each of the cpsA through cpsK genes and the downstream gene, hcp.
Membrane strips were placed in 12 hybridization tubes to be probed indepen-
dently by each gene-specific probe. Following hybridization, membrane strips
were aligned adjacent to one another beginning with the strip probed by the
cpsA-specific probe and continuing through to hcp. These membranes were then
exposed to X-ray film for autoradiography.
Construction of pLT06. For descriptions of all primers and plasmids, see Table
S1 in the supplemental material. pLT06 is a combination of pCJK47 (20),
pGB354 (3), and pCASPER (6) (see Fig. 2). The ermC cassette in pCJK47 was
replaced with the Cm acetyltransferase (cat) gene from the Streptococcus aga-
lactiae plasmid pGB354. The vector pCJK47 was digested with the restriction
enzymes BglII and NsiI, resulting in 5.8-kb and 0.9-kb fragments. The cat gene
from pGB354 was amplified by PCR with the primers Cat5? and Cat3?. The
resulting PCR product was cloned as a blunt-end fragment into a 5.8-kb fragment
of pCJK47 (T4 DNA polymerase treated). The resulting construct was called
pKS05. pKS05 was subsequently digested with SmaI and EcoNI, and the 5.7-kb
fragment containing P-pheS, cat, and lacZ was gel extracted (QIAquick gel
extraction kit; Qiagen), followed by Klenow treatment (Bioline). pCASPER was
digested with EcoRV and PshAI, and the 2.15-kb product containing orfB, orfC,
repA(Ts), and orfD was gel extracted. The 5.75-kb pKS05 product and the 2.15-kb
product of pCASPER were blunt end ligated, resulting in pLT06.
Construction of markerless exchange vectors. Vector pLT06 was used to
create in-frame deletions of cpsC, cpsD, cpsE, cpsF, cpsG, cpsH, and cpsI in E.
faecalis strains V583 and FA2-2. Relevant primers are listed in Table S1 in the
supplemental material. Fragments (1.0 kb each) were PCR amplified upstream
and downstream of the gene targeted for mutation. The PCR products were
ligated and reamplified, resulting in a 2.0-kb product. The 2.0-kb PCR product
was digested by restriction enzymes as described in Table 2 and ligated with
pLT06. The ligated products were electroporated into E. coli Electro 10 Blue
(E10B) for propagation and grown on LB plates containing Cm and X-Gal at
30°C. Blue colonies were screened for the presence of the ?2.0-kb inserts using
primers OriF and KS05SeqR. Positive clones were grown overnight in liquid LB
medium containing Cm at 30°C. The plasmid was purified using the QIAprep
spin miniprep kit (Qiagen). The ?2.0-kb inserts from each construct were se-
quenced using the primers OriF and KS05SeqR to ensure that no mutations
arose during cloning. The resulting deletion constructs, pLT08, pLT13, pLT16,
pLT18, pLT22, pLT23, and pLT24, were used to generate the cpsF, cpsH, cpsD,
cpsE, cpsC, cpsG, and cpsI deletions, respectively.
Generation of deletion mutants. E. faecalis V583 and FA2-2 were used for the
generation of isogenic, in-frame cps deletion mutants. Both V583 and FA2-2 are
classified as serotype C strains and contain cpsF (16). Deletion constructs were
transformed by electroporation into V583 and FA2-2 as described previously (8).
Transformed bacteria were grown on Todd-Hewitt broth (THB) plates containing
Cm and X-Gal at 30°C. Blue colonies were screened for the presence of the engi-
neered deletion constructs by colony PCR using the primers OriF and KS05SeqR.
Colonies that were positive for the deletion constructs were inoculated into 5.0 ml of
THB containing Cm and grown overnight at 30°C. The cultures were back diluted
1:1,000 in fresh THB with 15 ?g/ml Cm and grown for 2.5 h at 30°C, followed by
shifting to 42°C for 2.5 h to force single-site integration by homologous recombina-
tion. Following incubation at 42°C, the cells were serially diluted and plated on THB
containing Cm and X-Gal. Blue colonies growing at 42°C were screened for the
targeted integration using PCR with primers flanking the site of integration. Positive
integration clones were serially passaged from overnight cultures for two successive
days in THB with no selection at 30°C to force the second site recombination event.
Following serial passage at 30°C, the cultures were plated by serial dilution on
The resulting white colonies were screened for the deletion of the target genes by
PCR. Genomic DNA from colonies containing the deletions were purified and
sequenced to confirm gene deletions. The resulting deletion mutants are listed in
Complementation of deletion mutants. The markerless gene deletions were
complemented in trans by cloning target genes in a pAT28 plasmid background
(39). The promoter region for the cps operon (cpsC promoter) was PCR ampli-
fied from the plasmid pCPSC2 using the primers Vlac1 and Vlac2 (12, 28). The
amplified product was cloned as an EcoRI/BamHI fragment into pAT28, gen-
erating pLT09 (Table 2). PCR-amplified gene products were generated for cpsC,
cpsD, cpsE, cpsF, cpsG, cpsH, cpsI from purified V583 genomic DNA using
primers listed in Table S1 in the supplemental material. The amplified products
were cloned into pLT09, generating the complementation plasmids pLT10
(cpsF), pLT14 (cpsH), pLT25 (cpsD), pLT32 (cpsE), pLT33 (cpsG), pLT34
(cpsC), and pLT35 (cpsI) (Table 2). The complementation vectors were trans-
formed by electroporation into the corresponding deletion mutants (Table 1),
resulting in strains LT03, LT04, LT07, LT08, LT25, LT27, LT29, LT31, and
LT33. The serotype D strains T-5 and T-18 and the serotype B strain OG1RF
were complemented with pLT10, generating strains LT09, LT10, and LT11,
respectively (Table 1).
TABLE 1. E. faecalis strains used in this study
Maekawa type 1
Maekawa type 2
Maekawa type 5
Maekawa type 7
Maekawa type 8
Maekawa type 11
Maekawa type 18
LT01 ? pLT10
LT02 ? pLT10
LT05 ? pLT34
LT06 ? pLT34
T-5 ? pLT10
T-18 ? pLT10
OG1RF ? pLT10
LT15 ? pLT25
LT17 ? pLT32
LT19 ? pLT33
LT21 ? pLT14
LT23 ? pLT35
aStrains marked “NT” were nontypeable by conventional serotyping methods
6204THURLOW ET AL.J. BACTERIOL.
Determination of serospecificity by ELISA and slide agglutination. Serotype C
strains, including FA2-2 and V583, can be detected by enzyme linked immu-
nosorbent assay (ELISA) or agglutination using the Maekawa type 2 (MT2)
antibody (12, 23). However, serotype D strains, such as Maekawa serotypes T-5,
T-6, and T-18, cannot be detected by ELISA or agglutinated by MT2 antibodies
(16, 23). We used the MT2 antibodies to compare the serospecificities of V583,
FA2-2, LT01, LT03, T-5, LT09, T-18, LT10, OG1RF, and LT11.
Overnight cultures were diluted 1:100 in fresh THB supplemented with the
appropriate antibiotics and were allowed to grow to mid-log phase (optical
density at 600 nm of 0.6). Log-phase cells were washed three times with equal
volumes of phosphate-buffered saline (PBS), aliquoted (50 ?l) into wells of a
high-binding 96-well Costar plate (Corning), and allowed to adhere overnight at
4°C. Simultaneously, MT2 antibodies were diluted 1:1,000 in PBS and were
absorbed against T-5 cells in PBS overnight at 4°C to remove any cross-reactivity.
Following overnight incubation, the ELISA plates were washed three times in
PBS-Tween 20 (PBS-T) (0.05%) and blocked with 5.0% skim milk in PBS for 2 h.
Plates were subsequently washed three times with PBS-T, and the primary MT2
antibodies were added at a dilution of 1:1,000 and allowed to bind overnight at
4°C. The plates were washed again with PBS-T, and goat antirabbit secondary
antibodies conjugated to horse-radish peroxidase (Jackson ImmunoResearch,
West Grove, PA) were added to the wells. The plates were incubated at room
temperature for 2 h, followed by washing with PBS-T three times, followed by
washing with PBS three times to remove residual detergent. The ELISA was
developed in the presence of the o-phenylenediamine dihydrochloride (Sigma)
substrate for 30 min in the dark. The ELISA plates were analyzed using a
PowerWave XS 96-well plate reader (Bio-Tek instruments) at an optical density
of 490 nm.
Slide agglutination assays were performed as described previously (23). Sero-
type A antiserum contains antibodies directed toward E. faecalis lipoteichoic acid
(LTA) (16). Briefly, 5.0 ?l of serum was added to 15.0 ?l of test cells on a glass
slide and gently rotated for 1 min. Agglutination was determined by visual
clumping of the cells. Sterile PBS was used in place of antiserum as a negative
Preparation and purification of cell wall carbohydrates. Cell wall carbohy-
drates and capsular polysaccharides were isolated and purified as described
previously with slight modifications (10, 14). Briefly, bacteria were grown in two
or four liters of THB supplemented with 1% glucose at 37°C to mid-log phase.
Cells were washed in 300 ml of Tris-sucrose solution (10 mM Tris-Cl [pH 8.0],
25% sucrose), and the resulting cell pellets were resuspended in Tris-sucrose
solution with lysozyme (1 mg/ml), mutanolysin (10 U/ml), and 0.05% sodium
azide and incubated with gentle rocking at 37° for 16 h. Following incubation, the
samples were centrifuged and the supernatants were treated with RNase A (100
?g/ml) and DNase (10 U/ml) and incubated for 4 h at 37°C with gentle agitation.
Pronase (50 ?g/ml) was added to the samples and additionally incubated at 37°C
for 16 h. The supernatants were collected and passed through a 0.2-?m filter,
followed by extensive dialysis against distilled water. The samples were then
lyophilized and resuspended in a minimal volume of gel filtration buffer (50 mM
Tris base–150 mM NaCl–0.05% sodium azide, pH 7.0) and were run over an
S-400 size exclusion column (GE Healthcare Bio-Sciences, Uppsala, Sweden).
Collected fractions were analyzed for capsular polysaccharide content using
acrylamide gel electrophoresis and the cationic dye Stains-All for detection as
described previously (10). Fractions containing capsular polysaccharide were
pooled, extensively dialyzed against distilled water, lyophilized, and resuspended
in a minimal volume of 50 mM Tris buffer (pH 8.0). The sample was applied to
an anion exchange Q-Sepharose column for further purification (GE Healthcare
Bio-Sciences, Uppsala, Sweden). Bound capsular polysaccharide was eluted us-
ing a stepwise gradient starting with 50 mM Tris (pH 8.0) and ending with 50 mM
Tris–1 M NaCl (pH 8.0). Determination of fractions containing capsular poly-
saccharide was carried out as described above. Capsular polysaccharide contain-
ing fractions were pooled, extensively dialyzed against distilled water, lyophi-
lized, and used for downstream applications.
Small-scale cell wall carbohydrate preparations for determining production of
capsular polysaccharide were performed as stated above with slight modifica-
tions. Cells were grown in 25 ml of THB supplemented with 1% glucose until
they reached an optical density at 600 nm of 0.6 to 0.8. The cells were harvested,
washed with 2.0 ml of Tris-sucrose solution, and treated with lysozyme and
mutanolysin at the same concentrations listed above for 16 h at 37°C. The cell
suspensions were centrifuged, and the pellets were discarded. The remaining
supernatants were treated with RNase (100 ?g/ml) and DNase (10 U/ml) for 4 h
before final treatment with pronase as described above. Remaining impurities
were extracted with 500 ?l of chloroform, and the remaining carbohydrates were
precipitated with ethanol at a final concentration of 75% at ?80°C for 30 min.
The resulting pellets were air dried and resuspended in 100 ?l of sterile distilled
water, and 25 ?l was loaded onto an acrylamide gel as described previously. The
gels were stained in Stains-All following electrophoresis. Stained gels showed the
presence of three distinct staining regions, with the highest-molecular-weight
band corresponding to capsular polysaccharide (10).
Carbohydrate compositional analysis. Analysis of purified capsular polysac-
charide was performed at the Glycotechnology Core Resource at the University
of California, San Diego, using high-pH anion-exchange chromatography using a
Dionex DX 500 high-performance liquid chromatography system (Dionex,
Sunnyvale, CA) with pulsed amperometric detection (ED40; Dionex) (HPAEC-
PAD). Samples were hydrolyzed with 2 M trifluoroacetic acid at 100°C for 5 h,
dried, and resuspended in 25 ?l distilled water. Sugars were eluted with 120 mM
sodium hydroxide at a flow rate of 0.4 ml/min. The carbohydrate composition of
each polysaccharide was determined by comparison to known carbohydrate
standards that were prepared under identical conditions.
TABLE 2. Plasmid constructs used in this study
Conjugative donor plasmid, carries OriTpCF10, lacZ, and pheS, used in pLT06 construction
Contains Catr, used in the construction of pLT06
Contains orfB, orfC, RepA(Ts), and orfD, used in pLT06
Broad-range shuttle vector, spectinomycin resistant
Source of the CpsC promoter used in pLT09
pCJK47 derivative containing Catr
Deletion construct used for making mutants
pLT06 containing a 2.0-kb EcoRI/PstI fragment containing engineered cpsF deletion
pAT28 containing a 398-bp EcoRI/BamHI fragment containing the native CpsC promoter
pLT09 containing 851-bp SalI/SphI fragment containing cpsF
pLT06 containing a 2.0-kb EcoRI/PstI fragment containing engineered cpsH deletion
pLT09 containing a 447-bp BamHI/SphI fragment containing cpsH
pLT06 containing a 2.0-kb BamHI/SmaI fragment containing engineered cpsD deletion
pLT06 containing a 2.0-kb EcoRI/PstI fragment containing engineered cpsE deletion
pLT06 containing a 2.0-kb EcoRI/PstI fragment containing engineered cpsC deletion
pLT06 containing a 2.0-kb SmaI/SphI fragment containing engineered cpsG deletion
pLT06 containing a 2.0-kb EcoRI/PstI fragment containing engineered cpsI deletion
pLT09 containing a 1,418-bp BamHI/SphI fragment containing cpsD
pLT09 containing a 2,562-bp BamHI/SalI fragment containing cpsE
pLT09 containing a 2,555-bp SalI/SphI fragment containing cpsG
pLT09 containing a 1,319-bp BamHI/SphI fragment containing cpsC
pLT09 containing a 1,192-bp BamHI/SphI fragment containing cpsI
VOL. 191, 2009CAPSULE PRODUCTION IN ENTEROCOCCUS FAECALIS6205
Dot blot analysis of the capsule locus from serotype A, B, C,
and D strains. Dot blot analysis was performed for represen-
tatives of the four E. faecalis serotypes. E. faecalis serotype A
or B strains E-1, OG1RF, type 1, type 4, type 7, 12030, and
12107, serotype C strains FA2-2, V583, MMH594, type 2, type
8, and type 11, and serotype D strains type 5 and type 18 were
used. Blotting was performed to determine the presence or
absence of specific capsule operon genes (cpsC to cpsK), as
well as the conserved flanking genes cpsA, cpsB, and hcp, which
are known to reside adjacent to the capsule operon. All sero-
types contained the genes cpsA, cpsB, and hcp (Fig. 1). Only
serotypes C and D contained the genes cpsC to cpsK, with the
only identifiable difference between the two serotypes being
that serotype D strains lacked cpsF (Fig. 1).
Construction of pLT06 and generation of cps operon dele-
tion mutants. The development of pCJK47 by Kristich et al.
was one of the first vector systems for generating gene deletion
mutations in E. faecalis. (20). Limitations of this system in-
volved the necessity of conjugally mating the plasmid construct
from a donor strain (20). This delivery method is inefficient for
delivery of cloned DNA into target strains that harbor endog-
enous plasmids, such as the vancomycin-resistant strain V583.
Another noted obstacle associated with this system is the mo-
bilization and unwanted transfer of genomic DNA from the
donor strain into the recipient strain. The erm resistance cas-
sette used in pCJK47 for selection was also unsuitable for work
with V583 due to inherent resistant to erythromycin.
To counter these limitations, we constructed an improved
vector system, pLT06, to generate markerless in-frame dele-
tions of cps operon genes (Fig. 2). Insertional inactivation
techniques would not have been suitable to assess the contri-
butions of the individual cps operon genes to capsule produc-
tion or serospecificity. The pLT06 vector contains components
of pCJK47, including lacZ and the counterselectable marker
P-pheS (Fig. 2). pLT06 also contains the chloramphenicol
acetyltransferase (cat) marker from pGB354 for selection pur-
poses and orfB, orfC, repA(Ts), and orfD from pCASPER. The
combination of genes comprising pLT06 allowed for direct
transformation by electroporation of cloned DNA into target
E. faecalis strains. The plasmid can replicate in E. faecalis at
permissive temperatures of 30°C but cannot replicate at the
nonpermissive temperature of 42°C due to the temperature-
sensitive nature of the repA gene. Flanking regions of the gene
targeted for deletion were cloned into pLT06 to serve as tem-
plates for targeted recombination. Derivatives of pLT06 de-
signed to delete the targeted genes are forced to integrate into
the host genome through single-site homologous recombina-
tion when grown at nonpermissive temperatures in the pres-
ence of Cm. If recombination does not occur, then the subse-
quent clones of the host cell harboring pLT06 will not survive
since they will not carry the cat cassette for resistance to chlor-
amphenicol. Clones containing properly integrated pLT06
constructs were serially passaged at the permissive tempera-
ture in THB without selection to induce the second site re-
combination event and subsequent loss of pLT06. Bacteria
harboring integrated or circularized pLT06 constructs should
not grow on MM9YEG agar due to the presence of the p-
chloro-phenylalanine substrate and the P-pheS cassette (20).
White colonies from the MM9YEG plates were screened by
PCR to confirm deletion of the target gene. Approximately
50% of the screened colonies harbored the desired mutation.
PCR amplification from the cpsE-cpsG junction in serotype C
strains (FA2-2 and V583), the corresponding cpsF mutants
(LT01 and LT02), and serotype D strains (T-5 and T-18) shows
a 2.8-kb amplicon in strains containing cpsF and a 2.0-kb am-
plicon in strains lacking cpsF. DNA sequence analysis of LT01
and LT02 and complementation with cpsF with pLT10 showed
that a nonpolar deletion was generated using pLT08.
Determination of capsular polysaccharide production in se-
rotypes A, B, C, and D. Cell wall polysaccharides were purified
from parental and mutant strains to assess capsule production.
This method of detection allows for the most direct and solid
evidence of the presence of a capsule as opposed to an anti-
body-based method, which could falsely detect other cell wall
antigens (38). Small-scale cell wall polysaccharide preparations
were loaded on polyacrylamide gels, electrophoresed, and
stained with the cationic dye Stains-All. The high-molecular-
weight dark blue band corresponds to capsular polysaccharide
and correlates with previously described high-molecular-
weight E. faecalis capsule (10). The light blue band immedi-
ately below the capsule corresponds to the rhamnopolymer,
and the low-molecular-weight dark blue smear corresponds to
teichoic acid as described previously (10). From results shown
in Fig. 3, it is clear that serotype A and B strains (lanes B and
C, showing 12030 and OG1RF, respectively) do not produce
the high-molecular-weight capsular polysaccharide. Interest-
ingly, the serotype A strain 12030 did not appear to produce
detectable rhamnopolymer; however, the basis for this obser-
vation is not known at the present time. Consistent with ge-
netic data (Fig. 1), all serotype C and D strains produced the
high-molecular-weight band corresponding to capsular poly-
saccharide (Fig. 3).
Determination of serospecificity between serotype C and D
strains. Given that the only genetic difference between sero-
type C and D strains is the presence of cpsF in serotype C
strains, we hypothesized that CpsF was the sole contributor to
differences in antigenicity between serotype C and D strains.
We performed an ELISA with MT2 antiserum to detect the
serotype C antigenic determinant (23). MT2 antiserum has
FIG. 1. Dot blot analysis of the four putative serotypes of E. fae-
calis. Serotypes A and B (top) hybridize only to cpsA, cpsB, and the
control gene hcp, which sits outside of the capsule locus. The serotype
C strains (middle) hybridize to all the genes in the cps locus (cpsC to
cpsK), as well as the cpsA, cpsB, and hcp genes. Serotype D strains
(bottom) hybridize to all genes of the cps locus except cpsF.
6206THURLOW ET AL. J. BACTERIOL.
FIG. 2. (a) Strategy for the construction of plasmid pLT06, used in this study for construction of isogenic, in-frame deletion mutants of E.
faecalis. See Materials and Methods for details. The erm marker from pCJK47 was replaced with the cat marker from pGB354, resulting in pKS05.
oriT from pKS05 was replaced with an enterococcal origin of replication and the temperature-sensitive repA gene, resulting in pLT06. pLT06 was
subsequently used to engineer all of the isogenic, in-frame deletion mutants used in this study. (b) Diagram of the generation of the in-frame,
isogenic cpsF mutation using pLT08. Integration through homologous recombination of pLT08 into the E. faecalis genome took place at the
nonpermissive temperature of 42°C. Strains harboring the integrated plasmid were serially passaged at the permissive temperature of 30°C in the
absence of the selecting antibiotic Cm. Serial passaging induced the second site homologous recombination event and the excision of the plasmid.
Bacteria were plated on medium containing ?-chlorophenylalanine and X-Gal to screen for isolates that lost the plasmid. White colonies were
screened by PCR for the deletion event, and isolated DNA was sequenced to confirm that an in-frame deletion had occurred.
VOL. 191, 2009CAPSULE PRODUCTION IN ENTEROCOCCUS FAECALIS 6207
been shown to be specific for the serotype C antigen (12).
While the serotype C strain FA2-2 was detected by the MT2
antiserum, LT01 (FA2-2 ?cpsF) and the serotype D strains T-5
and T-18 were not detected by the MT2 antiserum (Fig. 4).
Strain LT03 (FA2-2 ?cpsF pLT10), along with the serotype D
strains LT09 (T-5) and LT10 (T-18), containing the comple-
mentation vector pLT10, were detected by the MT2 antiserum.
As expected, the serotype B strain OG1RF was not detected by
the MT2 antiserum after transformation with pLT10 (LT11)
Capsule production alters detection of lipoteichoic acid by
slide agglutination. Recently it was discovered that agglutinat-
ing antibodies generated to the serotype A strain 12030 were
directed toward LTA and not toward capsule as described
previously (16, 38). This suggests that sera developed for sero-
typing and detecting serotype A strains should recognize other
strains with exposed LTA. We used serotype A antisera in agglu-
tination assays to determine if our mutant strains could be agglu-
tinated. No agglutination was observed for FA2-2 and LT01
(FA2-2 ?cpsF), but 12030 and LT05 (FA2-2 ?cpsC) agglutinated
in the presence of these antibodies (Table 3). This suggests that
the presence of capsule in serotype C and D strains protects LTA
from detection by agglutinating antibodies.
Comparison of serotype C and D capsule polysaccharides by
HPAEC-PAD. We used purified capsular polysaccharide from
FA2-2 (serotype C) and LT01 (FA2-2 ?cpsF/serotype D) to
determine the contribution of cpsF to the difference in antige-
nicity between serotype C and D strains. Capsular polysaccha-
rides were purified as described in Materials and Methods.
Analysis comparing the FA2-2 capsule to the LT01 capsule
indicated a difference in the ratio of glucose to galactose be-
tween the two capsule serotypes (for strains FA2-2 and FA2-2
?cpsF, galactose values were both 1.0 while glucose values
were 4.4 and 3.0, respectively), indicating that CpsF could be a
Contributions of cps operon genes to capsule production.
We generated in-frame deletions of cpsCDEFGHI in the se-
rotype C strain V583 to determine their contribution to cap-
sule production. Figure 5 clearly shows that genes cpsC, cpsD,
cpsE, cpsG, and cpsI are essential for production of the high-
molecular-weight capsular polysaccharide. Further, these phe-
notypes were not due to polar effects on downstream genes,
since complementation of each gene in trans restores capsule
production. The genes cpsF and cpsH are the only genes in the
cps operon that are not essential for capsule production
FIG. 3. Acrylamide gel stained with Stains-All, showing the pres-
ence/absence of capsule production in serotype A to D strains. The
high-molecular-weight bands correspond to capsular polysaccharide as
described previously (10). The serotype A strain 12030 (B) and the
serotype B strain OG1RF (C) do not produce the capsule band. The
serotype C strains V583 and FA2-2 (D and E) and the serotype D
strains T-5 and T-18 (F and G) produce the high-molecular-weight
FIG. 4. CPS ELISA using MT2 antibodies to detect serotype C
capsule. Serotype C strains V583 and FA2-2 show reactivity with the
MT2 antibody. The cpsF deletion mutant LT01 is not detected by the
antibody, but complementation of LT01 (FA2-2 ?cpsF) with pLT10
(LT03) restores reactivity to the antibody. The serotype D strains T-5
and T-18 are not detected by the serotype C antibody. However, LT09
(T-5 plus pLT10) and LT10 (T-18 plus pLT10) are seroconverted to
serotype C strains when complemented with cpsF. The serotype B
strain OG1RF is not detected by the MT-2 antibody before or after
(LT11) complementation with pLT10, indicating that serotype conver-
sion cannot occur in a strain that does not produce capsular polysac-
charide. O.D. 490, optical density at 490 nm.
FIG. 5. Polyacrylamide gel stained with Stainz-all, showing the
high-molecular-weight capsule bands of capsule mutants and comple-
mented mutants. Lanes: A, V583; B, LT06 (V583 ?cpsC); C, LT08
(V583 ?cpsC plus pLT10); D, LT15 (V583 ?cpsD); E, LT25 (V583
?cpsD plus pLT25); F, LT17 (V583 ?cpsE); G, LT27 (V583 ?cpsE plus
pLT32); H, LT02 (V583 ?cpsF); I, LT04 (V583 ?cpsF plus pLT10); J,
LT19 (V583 ?cpsG); K, LT29 (V583 ?cpsG plus pLT33); L, LT21
(V583 ?cpsH); M, LT31 (V583 ?cpsH plus pLT14); N, LT23 (V583
?cpsI); O, LT33 (V583 ?cpsI plus pLT35). Only the genes cpsF and
cpsH are not essential for capsule production. Deletion of the genes
cpsC, cpsD, cpsE, cpsG, and cpsI completely abrogates capsule pro-
duction. This observation supports the evidence that serotypes A and
B do not produce capsule based on the absence of essential genes for
capsule production in these strains. Complementation of these dele-
tions restores capsule production.
TABLE 3. Slide agglutination using serotype A antiserum
FA2-2 ........................................................................................... Negative
FA2-2 ?cpsF................................................................................ Negative
FA2-2 ?cpsC ............................................................................... Positive
12030 ............................................................................................ Positive
6208 THURLOW ET AL.J. BACTERIOL.
Previous reports of capsule production in E. faecalis have
focused on differences in antigenicity between cell surface
polymers (16, 22). One study divided E. faecalis into 21 differ-
ent serogroups based on differences in agglutination to poly-
clonal antibodies generated to heat-killed cells (23). The anti-
serum used in this study was possibly detecting capsule as well
as other surface antigens (16). A more recent study grouped
strains of E. faecalis into four capsular serotypes (A to D)
based on serospecificity (16). This study inferred that serotypes
A and B shared a locus similar to that of serotypes C and D
that was responsible for capsule production in all four sero-
types. Accordingly, the capsular antigen of serotype A was
purified and compositionally analyzed and the structure de-
duced by nuclear magnetic resonance analysis (14, 40). How-
ever, it was recently reported that the serotyping antibody used
to classify serotype A isolates actually recognized LTA and
that the determined structure of the serotype A capsule cor-
responded to LTA (38). To date, only one genetic locus had
been determined to be responsible for capsule production in E.
faecalis (10). The capsule locus described by Hancock et al. is
comprised of nine genes (cpsC-cpsK) that directly contribute to
the expression of a capsular polysaccharide in E. faecalis (10).
We have shown that serotype C and D strains contained all
genes of the cps locus described by Hancock et al. (preceded by
cpsA and cpsB), with the variation between serotypes C and D
being attributed to the presence (serotype C) or absence (se-
rotype D) of cpsF.
Previous studies have shown that the genes cpsA and cpsB
are not part of the capsule operon since they are transcribed
from a different promoter (12). Attempts to mutate these
genes never resulted in the recovery of viable isolates (12).
However, reactive capsule antigen could be produced in a
heterologous host (E. coli) by complementation with the cpsC-
cpsK operon (12). The absence of capsule production by sero-
types A and B (Fig. 3) highlights the fact the CpsA and CpsB
play no role in capsule production. Therefore, based on se-
quence homology, we propose renaming cpsA to uppS, consis-
tent with its function as an undecylprenyl pyrophosphate syn-
thetase. We also propose to rename cpsB to cdsA since it
shares strong sequence similarity with known cytidyl trans-
ferase proteins. Both UppS and CdsA are known to be essen-
tial proteins in other bacterial systems (1), which explains the
inability to recover such mutants for E. faecalis (12).
We have demonstrated that the production of capsule pre-
vents detection of LTA by agglutinating antibodies (Table 3).
This observation is consistent with the argument that LTA is
shielded from agglutinating antibodies by capsule. Our obser-
vations support a role for CpsF in determining serospecificity
between serotype C and D strains. Compositional analysis sug-
gests that CpsF is responsible for the altered ratios of glucose
to galactose present in the capsules of serotypes C and D
(Table 4). Additionally, we propose that serotypes C and D are
the only E. faecalis serotypes that produce a capsular polysac-
charide, which is supported with the data in Fig. 3 and the
underlying genetics known to contribute to capsule production
(Fig. 1 and 5).
CpsF has no known sequence similarity to any characterized
protein, thus making it difficult to predict a possible contribu-
tion to serotype differences. Purified capsular polysaccharide
extracts from FA2-2 (serotype C) and LT01 (serotype D) were
analyzed by HPAEC-PAD to determine the possible contribu-
tion of CpsF. HPAEC-PAD analysis revealed a difference in
glucosylation of the polysaccharides, with FA2-2 containing an
extra glucose relative to galactose compared to data for LT01
(FA2-2 ?cpsF) (Table 4). The ratio of glucose to galactose for
the serotype C strain FA2-2 is identical to results of previous
compositional analysis (10). This result indicates that CpsF is a
putative glucosyltransferase, but ongoing studies to reveal the
structure of the repeating unit will provide solidifying evidence
for the functional role of CpsF.
Opsonophagocytic killing of both serotype C and D strains
by healthy human sera is drastically reduced compared to that
of the unencapsulated serotype A and B strains (17). Addi-
tional studies with the serotype B strain, OG1RF, demon-
strated the presence of protective antibodies in normal serum,
leading to clearance of E. faecalis (2). This could be due by the
presence of opsonizing anti-LTA antibodies present in normal
human serum (17). Presumably, the presence of capsule in
serotype C and D strains masks LTA from detection by the
circulating anti-LTA antibodies. Serotype A antibodies that
recognize LTA (38) cannot recognize or agglutinate the en-
capsulated serotype C and D strains (Table 3). However, these
same antibodies readily recognize and agglutinate the unen-
capsulated strain LT05 (V583 ?cpsC) and the serotype A
strain 12030. These observations are consistent with the in-
creased virulence associated with serotype C and D strains
(24). Furthermore, LTA is a pathogen-associated molecular
pattern that is recognized by the pattern recognition receptor
Toll-like receptor 2 (33, 34). Recognition of LTA results in
increased cytokine production and neutrophil recruitment to
the site of infection (5, 21). Presumably, the presence of cap-
sule would attenuate the host innate immune response. Cur-
rently, we are conducting studies to determine the effects of
capsule on innate immune system evasion.
In summary, the results presented in this study argue that
only E. faecalis serotypes C and D produce a true capsular
polysaccharide while serotypes A and B do not. We provide
empirical proof that CpsF is the basis for the difference in
antigenicity between serotype C and D strains. Finally, the
inability to detect LTA on the surface of encapsulated strains
indicates that the capsule of E. faecalis may play a role in
evasion of the host innate immune response. Future studies
will aim to address such questions in order to develop targeted
therapies to treat infections caused by multidrug-resistant E.
We thank Johannes Huebner for providing serotype A antisera. We
also thank Anup Datta at the Glycotechnology Core Resource at the
University of California, San Diego, for the compositional analysis of
the capsular polysaccharides.
This work was supported in part by a Faculty Enhancement award
(to L.E.H.) from the NIH COBRE program of the National Center for
Research Resources (NCRR), no. P20-RR016443.
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