JOURNAL OF BACTERIOLOGY, Mar. 2008, p. 1649–1657
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 5
Structural and Biological Characterization of a Capsular Polysaccharide
Produced by Staphylococcus haemolyticus?
Sigrid Flahaut,1† Evgeny Vinogradov,2Kathryn A. Kelley,1Shannon Brennan,1
Keiichi Hiramatsu,3and Jean C. Lee1*
Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 021151; Institute for
Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A 0R62; and Department of
Microbiology and Infection Control Science, Juntendo University, Tokyo 113-8421, Japan3
Received 11 October 2007/Accepted 17 December 2007
The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule
operon composed of 13 genes in tandem. The first seven genes (capABCDEFGSh) showed >57% similarity
with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLMShgenes are unique to S.
haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltrans-
ferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron
microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a
JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small
amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus
cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of
these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demon-
strated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to
complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of
capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: ?3-?-L-
FucNAc-3-(2-NAc-4-N-Asp-2,4,6-trideoxy-?-D-Glc)-4-?-D-GlcNAc-. This structure is unique among staph-
ylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as
an N-acyl substituent.
Among the coagulase-negative staphylococci (CoNS), Staph-
ylococcus haemolyticus plays an important role in hospital-ac-
quired opportunistic infections. S. haemolyticus is second only
to Staphylococcus epidermidis in its frequency of isolation from
human blood cultures (7, 9), and it may also cause peritonitis,
otitis, urinary tract infections, and septicemia. Methicillin-re-
sistant S. haemolyticus strains have been associated with for-
eign body infections (7), and S. haemolyticus is notorious for its
multidrug-resistant phenotype, with decreased susceptibility to
methicillin, teicoplanin, and vancomycin (24). S. haemolyticus
was the first species of CoNS sharing teicoplanin and vanco-
mycin resistance (23).
The genome of the human-pathogenic S. haemolyticus strain
JCSC1435 was sequenced in 2005 (25). At least 57 open read-
ing frames (ORFs) associated with virulence were reported. As
suggested by its species name, S. haemolyticus carries three
candidate ORFs encoding hemolysins; other putative virulence
factors include adhesins, exonucleases, proteases, and genes
encoding a capsular polysaccharide (CP). Although slime
production by S. haemolyticus has been reported (4), biofilm
formation and adherence to acrylic by S. haemolyticus are
significantly reduced compared to those of Staphylococcus epi-
dermidis (2, 3). Moreover, the biofilm-associated ica locus,
present in S. epidermidis and Staphylococcus aureus, is absent
from strain JCSC1435 and other clinical isolates of S. haemo-
lyticus (2, 6).
Previous reports have indicated that certain bovine strains of
S. haemolyticus (21, 26) produced a CP that was serologically
cross-reactive with the S. aureus type 5 CP (CP5). However,
probes specific for the S. aureus cap5 or cap8 locus showed
little hybridization to chromosomal or plasmid DNA prepared
from the bovine strains of S. haemolyticus (26).
The purpose of this study was to purify and characterize the
CP produced by JCSC1435, an S. haemolyticus strain of human
origin that is resistant to multiple antibiotics (25). We have
visualized the CP by immunoelectron microscopy and shown
that it is produced by other clinical isolates of S. haemolyticus.
Antibodies specific for the CP neutralized the antiphagocytic
effect of the capsule in a complement-dependent opsonoph-
agocytic killing assay.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The S. haemolyticus strains used in
this study are listed in Table 1. Strains JCSC1435 and 8HT4 were previously
described (25); mutant 8HT4 has a 91.9-kb chromosomal deletion within the oriC
environ that includes the entire capsule operon. Unless otherwise indicated, S.
haemolyticus strains were cultivated in tryptic soy broth (TSB) with 1% (wt/vol)
glucose at 37°C with vigorous shaking.
CP antibodies and serotyping. Antiserum to the S. haemolyticus CP was
prepared by immunizing a rabbit with heat-killed (70°C for 1 h) cells of strain
JCSC1435 as described previously (30). The antiserum was adsorbed with the
acapsular mutant 8HT4 to render the serum capsule specific. The reactivity of S.
haemolyticus clinical isolates with JCSC1435 CP antiserum was determined by a
colony immunoblot method (16). In addition, capsule extracts (26) from S.
haemolyticus strains were reacted with polyclonal antibodies specific for the S.
* Corresponding author. Mailing address: Channing Laboratory,
181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-2652. Fax:
(617) 731-1541. E-mail: firstname.lastname@example.org.
† Present address: LBCM/LR2B, Universite ´ du Littoral-Co ˆte
d’Opale, BP120, 62 327 Boulogne/mer cedex, France.
?Published ahead of print on 28 December 2007.
haemolyticus CP and S. aureus CP1, CP2, CP5, and CP8 by double immunodif-
DNA and RNA manipulations. S. haemolyticus strains were harvested from
TSB cultures incubated overnight with shaking at 37°C. The bacterial cells were
lysed with lysozyme and lysostaphin (1 mg/ml each) for 1 h at 37°C, and genomic
DNA was isolated with the Wizard Genomic Purification kit (Promega Corp.,
Madison, WI). PCRs were performed with Taq DNA polymerase (Invitrogen
Corp., Carlsbad, CA) and 1.5 mM MgCl2. Primers for PCR and reverse tran-
scription-PCR (RT-PCR) and the lengths of the amplicons are indicated in
Table 2. Amplification of the S. haemolyticus recA gene served as a positive PCR
Total RNA was prepared from S. haemolyticus cells collected in the exponen-
tial, postexponential, and stationary phases of growth. The bacteria were lysed
with 0.5 ml zirconia-silicon beads (Fisher Scientific, Pittsburgh, PA) in a dental
amalgamator (32), and RNA was purified with the RNeasy Mini kit (Qiagen,
Valencia, CA). RNA was treated with 80 U RNase OUT (Invitrogen), and
contaminating DNA was removed by treatment for 30 min with 10 U DNase I
(Invitrogen). RT-PCR amplification was carried out with the Access RT-PCR
System kit (Promega) following the manufacturer’s recommendations. A control
sample without reverse transcriptase was included for each reaction to confirm
the absence of contaminating DNA.
Transmission electron microscopy. S. haemolyticus cells were harvested from
TSB cultures incubated with shaking until the postexponential growth phase. The
bacterial cells were mixed for 2 h at room temperature with 0.2 ml of preimmune
rabbit serum or JCSC1435 CP antiserum. Samples were fixed and sectioned as
described previously (29).
Immunogold labeling of S. haemolyticus surface antigens was performed on
Formvar carbon-coated copper grids inoculated with ?107CFU in phosphate-
TABLE 1. Presence of S. haemolyticus capsule genes in strains from humans and cows
Strain Source (reference)
Presence of capsule gene as determined by PCR assay
Clinical isolate from Japan (25)
JCSC1435 mutant with a 91.9-kb chromosomal
Peritoneal fluid (3)
Bovine strain from Norway (26)
Bovine strain from Norway (26)
Bovine strain from Norway (26)
Bovine strain from Norway (26)
Bovine strain from Norway (26)
Bovine strain from Norway (26)
aNRS strains were clinical isolates from the United States that were obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus.
bUnlike the other clinical isolates that carried the capShgenes, strain NRS116 showed negative RT-PCRs with primers derived from capHIShand capKLSh(see text).
TABLE 2. Primers used in this study
Primer namePosition in Fig. 1 Amplicon (length in bp) Sequence (5?–3?)
1650FLAHAUT ET AL.J. BACTERIOL.
buffered saline (PBS). The samples were blocked for 10 min in 0.5% fish skin
gelatin in PBS with 0.1% Tween 20. A 1:10 dilution of JCSC1435 CP antiserum
or antibodies to poly-?-D-glutamic acid (PGA) (1) (kindly provided by Julia
Wang, Channing Laboratory) was added, and the samples were incubated for 30
min at ambient temperature. The grids were washed four times in PBS and
incubated with protein A-gold particles (20 nm). After four washes in deionized
water, the samples were air dried and examined with a JEOL 1200EX micro-
In vitro opsonophagocytic killing assay. The opsonophagocytic killing assay
was performed as described by Xu et al. (30) with the following modifications. S.
haemolyticus strains were cultivated with shaking for 16 h at 37°C in TSB. The
bacterial cells were washed once and diluted in a minimal essential medium
(Invitrogen) containing 1% bovine serum albumin (MEM-BSA; Sigma Chemical
Co., St. Louis, MO). The assay was performed in polypropylene tubes containing
?3.2 ? 106human polymorphonuclear leukocytes (PMNs), ?7.2 ? 105CFU S.
haemolyticus, 0.25% heat-inactivated rabbit antiserum, and 1% guinea pig serum
(as a complement source) in a total volume of 500 ?l MEM-BSA. Control
samples contained S. haemolyticus and PMNs with either complement or
antiserum or serum and complement with no added PMNs. The tubes were
rotated end over end (12 rpm) for 2 h at 37°C. Samples were vortexed and
diluted in sterile deionized water, and bacterial killing was estimated by
plating the diluted samples in duplicate on tryptic soy agar. The percentage
killing was defined as the reduction in CFU/ml after 2 h compared with that
at time zero (30).
Biofilm formation. S. haemolyticus biofilm formation was measured using a
standard microtiter assay, as described previously (2, 6). Bacteria were grown
overnight in TSB plus 1% glucose and diluted 1:100 in the same medium, and
200-?l aliquots were transferred into wells of tissue culture-treated polystyrene
microtiter plates. The plates were incubated for 24 h at 37°C without shaking,
and culture turbidities were measured in each well at 650 nm. To remove the
medium and nonadherent bacterial cells, the microtiter plates were washed twice
with 0.9% NaCl, and 200 ?l of 0.15% safranin per well was added. After 10 min
at ambient temperature, the safranin solution was removed by washing, and the
biofilms were air dried for 45 min at 50°C. One hundred microliters H2O was
added to each well before measurement of optical density at 540 nm. The
absorbance reading of each sample was normalized by dividing the optical den-
sity at 540 by the culture turbidity at 650 nm. Differences in biofilm production
were evaluated by the Student t test.
CP purification. CP was purified by methods similar to those we have de-
scribed previously (27). Briefly, S. haemolyticus JCSC1435 was cultivated in 20
liters of TSB to an optical density at 600 nm of 2.0. Autoclaved aqueous bacterial
extracts were treated with DNase and RNase at 37°C overnight, followed by
treatment for 8 h with pronase. The dialyzed material was passed over a column
of DEAE-Sephacel equilibrated with 0.05 M sodium acetate-0.05 M NaCl, pH
6.0. Fractions that eluted in a 2-liter gradient (0.05 M sodium acetate with 0.05
to 0.5 M NaCl) were analyzed for absorbance at 206 nm and for serologic activity
with antibodies to S. haemolyticus CP. Serologically active fractions were pooled,
dialyzed, and lyophilized. The CP was chromatographed on a Sephacryl S-300
column equilibrated in 0.2 M NaCl, and the fractions were monitored as de-
scribed above. Serologically active fractions were pooled, dialyzed, and lyophi-
lized. The purity of the polysaccharide was assessed by UV (260- and 280-nm)
absorption spectra, chemical assays for protein and phosphate, and nuclear
magnetic resonance (NMR) spectroscopy.
Structural characterization of the S. haemolyticus CP.
spectra were recorded using a Varian Inova 500 MHz spectrometer in D2O
solutions at 60°C with an acetone standard (2.225 ppm for1H and 31.5 ppm for
13C) using standard pulse sequences for correlation spectroscopy, total correla-
tion spectroscopy (mixing time, 120 ms), nuclear Overhauser effect spectroscopy
(NOESY) (mixing time, 200 ms), and heteronuclear single quantum correlation
and heteronuclear multiple-bond correlation (HMBC) (optimized for an 8-Hz
coupling constant). Spectra in 10% D2O were recorded at 25°C with WET water
suppression (water suppression enhanced through T1 effects).
For monosaccharide analysis and identification of the aspartic acid residue, the
S. haemolyticus CP (0.5 mg) was hydrolyzed (0.2 ml of 3 M trifluoroacetic acid,
120°C, 2 h) and evaporated to dryness under a stream of air. The residue was
dissolved in water (0.5 ml), reduced with NaBH4(?5 mg, 1 h), neutralized with
acetic acid (0.3 ml), and dried, and methanol (1 ml) was added. The mixture was
dried twice with the addition of methanol, and the residue was acetylated with
acetic anhydride (0.5 ml, 100°C, 30 min), dried, and analyzed by gas-liquid
chromatography (GLC) on an HP1 capillary column (30 m by 0.25 mm) with a
flame ionization detector (Agilent 6850 chromatograph) in a temperature gra-
dient of 170 (4 min) to 260°C at 4°C/min and by gas chromatography (GC)-mass
spectrometry on a Varian Saturn 2000 system with the same column and an
ion-trap mass spectral detector.
For determination of the absolute configuration of the monosaccharides, CP
(1 mg) was treated with (S)-2-butanol/AcCl (0.25 ml, 10:1 [vol/vol], 2 h, 85°C),
dried under a stream of air, acetylated, and analyzed by GC in comparison with
authentic standards prepared from respective monosaccharides with (S)- and
(R)-2-butanol. The absolute configuration of L-aspartic acid was determined in
the hydrolysate of S. haemolyticus CP by high-pressure liquid chromatography on
a penicillamine chiral column (Phenomenex) in 2 mM CuSO4in 15% methanol
Expression of the S. haemolyticus capsule genes. The capSh
operon in the genome of S. haemolyticus JCSC1435 (25) is
comprised of 13 ORFs (SH0388 to SH0400) in a 14,652-bp
region (Fig. 1) spanning nucleotides 402008 to 416209. The
first seven genes (capAShthrough capGSh) are homologous to
S. aureus cap5A (or cap8A) through cap5G (or cap8G), with
DNA homologies between 65% and 81% and amino acid iden-
tities between 57% and 89%. CapHShthrough CapKShshare
FIG. 1. S. haemolyticus capsule locus and flanking genes. Arrowheads denote sites where primers anneal. Primer names and sequences are
shown in Table 2. The percent G?C content for individual genes is shown above each ORF. The predicted functions of each gene product based
on protein homologies are shown.
VOL. 190, 2008 S. HAEMOLYTICUS CAPSULAR POLYSACCHARIDE1651
only limited homology with other gene products and probably
include putative glycosyltransferases, as well as genes involved
in CP transport and polymerization. CapLShis homologous to
bacterial aminotransferases, and CapMShis a putative tran-
scriptional regulator (Fig. 1). The overall G?C content in the
coding regions of the S. haemolyticus cap region is 28.8%,
which is lower that that of the total genome (32.8%). The G?C
content of the individual capShgenes is shown in Fig. 1. The
capHIJShgenes in the center of the capShlocus showed the
lowest G?C content, ranging from 22.5% to 23.3% G?C. This
suggests that this central DNA region, specific to S. haemolyti-
cus, represents the genes most recently acquired by the bacte-
rium via horizontal gene transfer. A putative Rho-dependent
transcriptional terminator was located at the end of the capMSh
Previous serologic studies indicated that certain bovine
strains of S. haemolyticus produce a CP that reacts serologically
with polyclonal and monoclonal antibodies to S. aureus CP5
(21, 26). We prepared capsule extracts from strain JCSC1435
and seven S. haemolyticus clinical isolates of human origin
(Table 1). None of the extracts reacted with antibodies specific
to S. aureus CP1, CP2, CP5, or CP8 (not shown).
To determine whether clinical isolates of S. haemolyticus
carried capsule genes similar to those within the JCSC1435
genome, we designed primers (Table 2) to amplify specific
regions of the S. haemolyticus capsule locus: capABCDSh,
capEFGSh, capHIJSh, and capKLMSh. Genomic DNA from S.
haemolyticus JCSC1435 yielded amplicons of the expected size
with each primer pair, whereas none of the genomic DNA
preparations from six CP5?bovine isolates (26) or mutant
8HT4 were positive by PCR. Of the seven clinical S. haemo-
lyticus isolates from humans, four (NRS50, NRS69, NRS115,
and NRS116) produced amplicons identical in size to those of
JCSC1435 (Table 1). PCR analysis indicated that three clinical
isolates (NRS9, NRS62, and M176) did not carry capsule genes
similar to those of JCSC1435, nor did they react with S. hae-
molyticus CP-specific antiserum by immunodiffusion or immu-
noblotting (Fig. 2). Likewise, six bovine isolates of S. haemo-
lyticus and deletion mutant 8HT4 failed to react by
immunoblotting or immunodiffusion with the JCSC1435 CP
antiserum (not shown). Strains NRS9 and NRS62, but not
M176, were positive by PCR for the phoB gene just down-
stream of the capShlocus (Fig. 1), whereas all three strains
were negative by PCR for four genes (SH0383 to SH0386)
upstream of the capShlocus. All of the S. haemolyticus iso-
lates, but not an S. aureus strain, yielded a positive amplicon
with primers specific for S. haemolyticus recA (not shown).
To determine whether the capsule genes were expressed by
the clinical S. haemolyticus isolates, we performed RT-PCR
experiments with primers that yielded products ?1.6 kb in size
and spanned the length of the capShlocus (Table 2). Our
results revealed that S. haemolyticus strains JCSC1435, NRS50,
NRS69, and NRS115 expressed the capShgenes since they
were positive by RT-PCR with primer pairs with homology to
capABCSh, capEFSh, capHISh, and capKLSh. Furthermore, the
clinical isolates NRS50, NRS69, and NRS115 each produced a
serologically detectable CP when tested with antiserum specific
for the JCSC1435 CP (Fig. 2). Although strain NRS116 was
positive for transcription of the upstream capShgenes, it was
negative for the expected bands corresponding to the down-
stream genes (capHIShand capKLSh), suggesting that strain
NRS116 may carry a defective promoter downstream of
capFSh. This observation is consistent with our inability to
detect CP production by this strain (Fig. 2).
Influence of culture medium and bacterial growth phase
on S. haemolyticus CP production. To investigate environ-
mental influences on CP production, we cultivated S. hae-
molyticus JCSC1435 on different growth media, including
TSB, TSB plus 1% glucose, brain heart infusion broth, Co-
lumbia broth, or Columbia broth or agar supplemented with
2% NaCl. CP, visualized by electron microscopy and immu-
nogold labeling, was abundant when strain JCSC1435 was
grown in brain heart infusion broth, TSB plus 1% glucose,
or Columbia broth plus 2% NaCl (data not shown). In
contrast to that observed with S. aureus (19, 20), cultivation
on Columbia salt agar plates was suboptimal for CP produc-
tion by S. haemolyticus.
Similar experiments were performed to determine the
influence of the bacterial growth phase on capsule expres-
sion by strain JCSC1435. Similar to CP expression by S.
aureus, little CP was detected on the surface of S. haemo-
lyticus cells until late in the exponential growth phase. Max-
imal CP was observed on the bacterial cells during the
postexponential and early-stationary phases of growth (Fig.
3 and 4). This finding was consistent with RT-PCR experi-
JCSC1435. S. haemolyticus capShtranscripts were detected
once the culture reached an optical density at 600 nm of 0.5
but were absent from cells harvested from 24-h cultures
(data not shown).
To better visualize the S. haemolyticus CP, we prepared
ultrathin sections of strain JCSC1435 harvested from overnight
TSB plus 1% glucose cultures. Electron micrographs of S.
haemolyticus JCSC1435 that were incubated with CP-specific
antibodies showed abundant surface-associated CP (Fig. 3A)
that was lacking on bacterial cells incubated with preimmune
serum (Fig. 3B).
FIG. 2. Colony immunoblotting of staphylococcal strains reacted
with S. haemolyticus CP-specific antiserum. The strains tested are de-
scribed in Table 1. A capsule serotype 5 S. aureus strain was used as a
1652 FLAHAUT ET AL.J. BACTERIOL.
Differentiation between PGA and CP production by S. hae-
molyticus. PGA is a surface-associated polymer produced by
Bacillus anthracis and members of the S. epidermidis group,
and PGA has been shown to play a role in staphylococcal
resistance to high osmolarity (13). Because the genes encoding
PGA biosynthesis are present within the S. haemolyticus ge-
nome (25), we wanted to ensure that the capsule-like material
visualized on the surface of JCSC1435 was CP and not PGA.
To accomplish this, we performed immunogold labeling exper-
iments of JCSC1435 with antibodies to either PGA or the S.
haemolyticus CP. Both S. haemolyticus JCSC1435 and acapsu-
lar mutant 8HT4 harvested from Columbia salt agar plates
showed immunogold labeling with PGA antibodies; the gold
particles were distributed in small clusters closely associated
with the bacterial cells (Fig. 4A and B). Similar findings were
observed on bacteria cultivated in TSB (data not shown). Our
data corroborate those of Kocianova et al., who reported that
PGA production by S. epidermidis is sparse, i.e., 1.2 ?g PGA/
liter (13). Our experimental results indicate that PGA synthe-
sis and CP production by S. haemolyticus are not linked, since
PGA was produced by wild-type strain JCSC1435 and the
acapsular mutant 8HT4 (Fig. 4A and B), whereas CP was
apparent on the wild-type strain (Fig. 4C) but not on mutant
8HT4 (Fig. 4D).
S. haemolyticus biofilm formation. Adherence of bacteria to
polystyrene microtiter plates is a convenient assay for biofilm
formation (2, 6). S. haemolyticus JCSC1435 is a poor biofilm
producer (Fig. 5), and it lacks the ica genes implicated in
biofilm formation (25). PCR analysis of the S. haemolyticus
clinical isolates revealed that they also lacked the ica genes
(not shown). However, other cell wall structures, such as wall
teichoic acid and protein, have been shown to mediate staph-
ylococcal biofilm formation in strains lacking or not expressing
the ica locus (8, 14). Several of the S. haemolyticus clinical
isolates produced biofilms in vitro that were comparable to
that of the control strain S. epidermidis RP62A (Fig. 5). Al-
though the acapsular mutant 8HT4 showed strongly enhanced
biofilm production (P ? 0.001) compared to the wild-type
strain (Fig. 5), there were no other obvious correlations be-
tween CP production and biofilm formation among the clinical
Effect of CP on in vitro opsonophagocytic killing of S. hae-
molyticus. To determine whether there were differences in re-
sistance to phagocytosis attributable to CP production by S.
haemolyticus, we measured the killing of strains JCSC1435 and
the acapsular mutant 8HT4 by human PMNs. As shown in Fig.
6, ?78% of the JCSC1435 and 8HT4 inocula were killed by
PMNs in the presence of both CP-specific antibodies and com-
FIG. 3. Transmission electron micrographs of S. haemolyticus
JCSC1435. To visualize the capsule, the bacteria were incubated with
S. haemolyticus CP-specific antiserum (A). Bacteria incubated with
preimmune serum showed no evidence of CP (B). Magnification,
?22,500. Bars, 0.2 ?m.
FIG. 4. Detection of S. haemolyticus PGA or CP by immunogold
staining and visualization by transmission electron microscopy. Sam-
ples (A and B) incubated with PGA antiserum were harvested from
Columbia salt agar plates, since salt enhances PGA expression (13).
Samples (C and D) incubated with S. haemolyticus CP-specific anti-
serum were harvested from postexponential-phase cultures in TSB. (A
and C) S. haemolyticus JCSC1435; (B and D) cap deletion mutant
8HT4. Magnification, ?45,000. Bars, 0.1 ?m.
FIG. 5. Biofilm production by S. haemolyticus strains. Each bar
represents the mean ? standard deviation of 12 separate determina-
tions. S. aureus Reynolds and S. epidermidis RP62A are control nega-
tive and positive biofilm-producing strains, respectively. The S. hae-
molyticus CP phenotype of each strain is indicated.
VOL. 190, 2008S. HAEMOLYTICUS CAPSULAR POLYSACCHARIDE1653
plement. Similarly, ?70% of the mutant 8HT4 (CP?) inocu-
lum was effectively opsonized for phagocytic killing by comple-
ment alone. The addition of CP antibodies to the mutant strain
did not improve the killing observed by PMNs and comple-
ment. In contrast, the JCSC1435 inoculum bacteria increased
in number when incubated with PMNs and complement but no
antibodies. In serum with no complement activity, rabbit CP-
specific antibodies were not opsonic for either strain. No killing
was observed in control samples lacking PMNs or in samples
with PMNs but no opsonin. These results confirm the pre-
dicted antiphagocytic activity of the capsule produced by S.
Purification of S. haemolyticus JCSC1435 CPs. Capsule was
extracted and purified from 20 liters of a JCSC1435 TSB cul-
ture. The extract (?1 g), released into the supernatant by
autoclaving the bacterial pellet, was clarified by DNase,
RNase, and protease treatments. The crude CP preparation
was then separated from contaminating teichoic acids by ion-
exchange chromatography. The CP (?10% of the crude ex-
tract) eluted from the DEAE column with 0.14 to 0.18 M NaCl
and was detected by immunodiffusion assays with rabbit S.
haemolyticus antiserum. The serologically active fractions were
pooled, dialyzed, concentrated, and further purified by size-
exclusion chromatography. The purified CP (13 mg) eluted
near the void volume of an S-300 Sephacryl column with a Kav
of 0.01. The final CP yield was ?0.65 mg of capsule per liter of
culture. The capsule preparations used for biochemical char-
acterization contained ?2% protein, 0.5% nucleic acid, and
Structural characterization of S. haemolyticus CP. A set of
the NMR spectra—correlation spectroscopy, total correlation
spectroscopy, NOESY, heteronuclear single quantum correla-
tion, and HMBC—was recorded for the S. haemolyticus CP.
Spectra contained signals of spin systems of three mono-
saccharides—?-fucosamine (?-FucN, unit A), ?-glucosamine
(?-GlcN, unit B), and 2,4-diamino-2,4,6-trideoxy-?-glucose
(bacillosamine or QuiN4N, unit C) in pyranose form and an
aspartic acid residue (Table 3). Relative configurations of the
constituent monosaccharides were identified on the basis of
vicinal proton coupling constants and
shifts. Anomeric configurations were deduced from the J1,2
coupling constants and chemical shifts of H-1, C-1, and C-5
signals and by observations of intraresidual NOE connectivi-
ties (H-1:H-3, H-1:H-5) characteristic of the ?-pyranosides for
?-bacillosamine unit C. The sequence of the monosaccharides
was determined from NOE and HMBC data, which showed
correlations from anomeric proton to transglycosidic proton:
A1:C3, B1:A3, and C1:B4, establishing the sequence [A-C-B]n
For determination of the position of the aspartic acid resi-
due, a NOESY spectrum of the polysaccharide was recorded in
10% D2O-90% H2O in order to reveal correlations for NH
protons. The1H NMR spectrum contained five amide signals
(Fig. 8A), corresponding to four amino groups of the amino
sugars (A2NH, B2NH, C2NH, and C4NH) and the amino
group of the aspartic acid (Asp2NH). They showed NOE cor-
13C NMR chemical
FIG. 6. Opsonophagocytic killing of S. haemolyticus JCSC1435 and
acapsular mutant 8HT4 by human PMNs. The percentage of bacteria
killed (mean ? standard error of the mean) was calculated as a re-
duction in CFU/ml after 2 h at 37°C compared with the CFU/ml in
each sample at time zero. The data were pooled from experiments
performed two to six times. Ab, S. haemolyticus CP-specific antibodies;
C’, guinea pig serum with complement activity.
TABLE 3. NMR data for the S. haemolyticus CP (?, ppm)
FIG. 7. Structure of the S. haemolyticus JCSC1435 CP.
1654FLAHAUT ET AL. J. BACTERIOL.
relations (Fig. 8B) with the protons at the site of the amide
location (A2NH:A2; B2NH:B2; C2NH:C2; C4NH:C4), to the
ones of the neighboring protons (B2NH:B3; C2NH:C3; C4NH:
C3, C4NH:C6), and to the CH3of the acetyl groups (A2NH:
Ac; B2NH:Ac; C2NH:Ac). The proton of the aspartic acid
amino group showed a NOE correlation with a CH3of an
acetyl group (Asp2NH:Ac) due to N acetylation of the aspartic
acid residue. Aspartate H-3 gave strong NOE correlation with
the C4NH (Asp3:C4NH, Fig. 8B), whereas Asp2 gave no cor-
relation with C4NH; this indicated the attachment of the as-
partate to the bacillosamine 4-amino group through the C-4
carboxyl of the aspartate. Thus, it could be determined that
amino groups at A2, B2, and C2 and the amino group of the
aspartic acid are acetylated and the amino group at position 4
of the bacillosamine residue is acylated with the 4-carboxyl
group of the N-acetyl aspartate (Fig. 8B). Similar structures
with N-acetyl-aspartate were described as constituents of the
O-antigen polysaccharides of enterobacteria of the genera
Providencia and Proteus (31).
The NMR data were confirmed by composition analyses that
showed the presence of GlcN, FucN, and Asp. BacN was not
detected after hydrolysis. The absolute configurations of GlcN
and FucN were determined by GC of (S)-2-butylglycoside ac-
etates. The absolute D-configuration of the BacN residue was
deduced from13C NMR chemical shift effects from L-FucN
and D-GlcN, as described by Lipkind et al. (18). The structure
of the CP purified from S. haemolyticus strain JCSC1435 was a
trisaccharide repeating unit (Fig. 7): ?3-?-L-FucNAc-3-(2-
CP production is an important determinant of bacterial vir-
ulence, and it is a trait common to many invasive bacterial
pathogens. Although clinical isolates of S. aureus were recog-
nized as encapsulated by the early 1980s (10), the CoNS were
considered nonencapsulated species. However, when the S.
haemolyticus JCSC1435 genome was sequenced by Takeuchi et
al. (25), they reported a capsule operon located within the
“oriC environ” of the chromosome. The oriC environ includes
genes specific to each staphylococcal species, and this region of
the S. haemolyticus genome is particularly subject to frequent
rearrangements and deletions (25, 28). We sought to deter-
mine whether a CP with antiphagocytic properties was actually
expressed by S. haemolyticus JCSC1435 and to elucidate the
structure of the CP encoded by the capShgenes.
Previous reports indicated that at least some bovine strains
of S. haemolyticus produced an uncharacterized surface anti-
gen that was serologically cross-reactive with S. aureus CP5 (21,
26). Poutrel et al. reported that 13 of 19 S. haemolyticus strains
from six bovine herds in France reacted with antibodies to CP5
(21), and Tollersrud et al. indicated that all seven Norwegian
bovine isolates of S. haemolyticus reacted with both monoclo-
nal and polyclonal antibodies to S. aureus CP5 (26). DNA
prepared from the encapsulated bovine isolates showed weak
hybridization with S. aureus cap5 DNA probes under low-
stringency conditions. Our studies indicate that the CP pro-
duced by bovine S. haemolyticus isolates is unrelated at the
genetic and serologic levels to that produced by the human
isolate JCSC1435. In contrast, four of seven S. haemolyticus
clinical isolates from humans carried the capAShto capMSh
genes, and three of the four produced a CP that reacted with
JCSC1435 CP antiserum. Whether the strains lacking the capSh
genes carry genes encoding a different capsule type has not yet
Biochemical characterization of the purified CP from S.
haemolyticus JCSC1435 revealed a new polysaccharide struc-
Glc)-4-?-GlcNAc-. This structure is consistent with the pre-
dicted functions of the genes within the capShlocus. The first
seven genes of the S. haemolyticus JCSC1435 cap operon
showed high amino acid identity to S. aureus cap5ABCDEFG
or cap8ABCDEFG. The capABCD genes are conserved among
the cap loci of S. haemolyticus, Staphylococcus saprophyticus,
and S. aureus (cap1, cap5, and cap8) (15, 17, 22, 25). The capA
gene product may be involved in chain length determination,
capB encodes a putative tyrosine protein kinase, and capC
encodes a putative phosphotyrosine-protein phosphatase.
Cap5D is a putative 4,6-dehydratase, converting UDP-GlcNAc
to UDP-2-acetamido-2,6 dideoxy-D-xylo-4-hexulose, a precur-
sor for S. aureus UDP-D-FucNAc or, in the case of S. haemo-
lyticus, 2-NAc-4-N-Asp-2,4,6-trideoxy-D-Glc. The presence of
L-FucNAc in the S. haemolyticus CP was predicted by the
presence of the capEFGShgenes, which show 89%, 71%, and
87% identity, respectively, to the cap5EFG or cap8EFG genes
responsible for L-FucNAc synthesis in S. aureus (11). The S.
haemolyticus CP is the first example of a staphylococcal poly-
saccharide that includes a trideoxy sugar residue, and it is also
unique in containing aspartic acid as an N-acyl substituent of
QuiNAc4N, which has been described only in the O polysac-
FIG. 8. Structural analysis of CP produced by S. haemolyticus
JCSC1435. (A)1H NMR spectrum of the S. haemolyticus CP in 10%
D2O with WET water suppression. (B) Fragment of the NOESY
spectrum of the polysaccharide showing correlations from amide pro-
VOL. 190, 2008S. HAEMOLYTICUS CAPSULAR POLYSACCHARIDE1655
charides of Proteus and Providencia spp. (12). The putative
aminotransferase capLShgene likely adds the amino group to
C-4 of UDP-QuiNAc, which is then acylated by the aspartic
acid residue, possibly involving capHSh.
Despite the similarity between the S. aureus and S. haemo-
lyticus CP biosynthetic genes, there is little nucleotide homol-
ogy upstream of the cap5A gene, suggesting that regulation of
CP biosynthesis may differ for the two species. Furthermore,
the putative transcriptional regulator at the 3? end of the capSh
locus is absent from the S. aureus cap5 or cap8 gene cluster.
The effects of other regulators of S. aureus CP expression, such
as agr, arlS, mgrA, and sigB, on S. haemolyticus CP production
are unknown. Although CP production by both S. aureus and S.
haemolyticus was maximal during postexponential growth, cul-
tivation of S. haemolyticus cells in a liquid medium resulted in
CP production greater than that by bacteria grown on solid
medium with added salt, a condition that promotes S. aureus
CP production (19, 20).
The S. haemolyticus CP demonstrated biologic properties
consistent with those produced by other encapsulated bacterial
pathogens. Strain JCSC1435 was resistant to complement-
mediated opsonophagocytic killing by human neutrophils,
whereas the acapsular mutant 8HT4 was susceptible. The ad-
dition of anticapsular antibodies neutralized the antiphago-
cytic effect of the S. haemolyticus capsule. In addition to CP, S.
haemolyticus produces PGA, a surface polymer that has been
shown to have antiphagocytic properties when expressed by S.
epidermidis (13). PGA is produced by many CoNS, including S.
haemolyticus ATCC 29970. We visualized PGA on the surface
of JCSC1435 and acapsular mutant 8HT4 by immunogold
labeling of bacteria examined by electron microscopy, indi-
cating that the biological differences that we observed be-
tween the two bacterial strains were due to production of CP
and not PGA.
Biofilm formation is an important characteristic of opportu-
nistic pathogens, such as CoNS. Some S. haemolyticus isolates
are associated with foreign body infections (7), but there is no
evidence that members of this species carry the ica genes that
are implicated in biofilm production (2, 6). The influence of CP
production on the development of staphylococcal biofilm pro-
duction has received little attention. We evaluated the biofilm-
forming ability of S. haemolyticus strains cultivated under
growth conditions favorable for capsule production. The ob-
servation that the capsule deletion mutant strain showed
greater biofilm formation than did the parental strain sug-
gested that CP might partially mask or inhibit surface factors
critical for biofilm production, as has been shown for other
encapsulated pathogens (5, 31). However, there was no corre-
lation among the clinical S. haemolyticus isolates between CP
production and biofilm production, suggesting that the biofilm
formation is strain dependent and mediated by factors that
remain poorly understood.
In conclusion, we have isolated, purified, and characterized
CP from the first CoNS shown to be encapsulated. Similar
studies characterizing the CP of an S. saprophyticus strain (15)
are in progress. CoNS are important agents of opportunistic
infections in compromised patients, and S. haemolyticus is no-
torious for its resistance to antibiotics. CP produced by strain
JCSC1435 cross-reacted serologically with CP detected on
other clinical isolates of S. haemolyticus. The capShlocus bears
some homology to the S. aureus cap5 or cap8 locus, but it is
unique since it carries genes that encode enzymes to synthesize
a trideoxy sugar residue that is N acylated by aspartic acid. We
have visualized the bacterial polysaccharide by electron mi-
croscopy and shown that it protects the bacterium from uptake
and killing by human neutrophils.
This work was supported by Public Health Service grant AI29040
(J.C.L.). S.F. was supported by a Fulbright-Nord-Pas de Calais
(France) grant from the Fulbright Franco-American commission.
We thank Irina Sadovskaya for her critical review of the manuscript
and Robert Solinga for technical assistance. Clinical isolates of S.
haemolyticus were obtained through the Network on Antimicrobial
Resistance in Staphylococcus aureus (NARSA) program supported
under NIAID, NIH contract no. N01-AI-95359. Tore Tollersrud kindly
provided the bovine S. haemolyticus isolates, and Julia Wang gener-
ously donated the antiserum to PGA.
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