pleiotrophic effects of 2 enterococcus faecalis saga– like genes

E. faecalis salB, Biofilm, and ECM Binding • JID 2006:193 (15 January) • 231

M A J O R A R T I C L E

Pleiotrophic Effects of 2 Enterococcus faecalis sagA–Like Genes, salA and salB, Which Encode ProteinsThat Are Antigenic during Human Infection,on Biofilm Formation and Binding to CollagenType I and Fibronectin

Jamal A. Mohamed,1,2 Fang Teng,1,2 Sreedhar R. Nallapareddy,1,2 and Barbara E. Murray1,2,3

1Division of Infectious Disease, Department of Internal Medicine, 2Center for the Study of Emerging and Reemerging Pathogens,and 3Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston

Background. We have shown previously that Enterococcus faecium SagA has broad-spectrum binding to extra-cellular matrix (ECM) proteins. In the present study, 2 sagA-like genes, salA and salB, were identified in Enterococcusfaecalis.

Methods. We compared the salA and salB mutants; their parental strain, OG1RF; and the salB-complementedstrain for binding to ECM proteins and biofilm formation.

Results. The salB mutant (TX5123) grew more slowly but showed greater binding (∼10%–20% of cells bound)to fibronectin (FN) and collagen type I (CI) than did OG1RF (∼1% of cells bound) ( ). Although TX5123P ! .001showed decreased biofilm formation in tryptic soy broth plus 0.25% glucose (TSBG) ( vs. OG1RF), aP ! .001marked increase in biofilm formation was shown by TX5123 but not by OG1RF when they were grown in TSBGplus horse serum (HS) or TSBG plus FN, and the increase was coincident with increased attachment and hydro-phobicity of TX5123. Complementation of the salB mutant restored the wild-type phenotypes.

Conclusions. Whether SalB expression is ever sufficiently low in vivo to enhance adherence to ECM proteinsor the serum-elicited increase in biofilm formation seen with the salB mutant in vitro is not currently known,but it is a potential way in which this organism could increase its adherence and biofilm formation during infection.

Enterococci are common causes of endocarditis, with

Enterococcus faecalis causing the majority of cases [1,

2]. Because of their intrinsic and acquired resistance to

multiple antibiotics, the treatment of enterococcal en-

docarditis can be very difficult; in some cases, this re-

sults in treatment failure and the death of the patient.

The extracellular matrix (ECM) is a macromolecular

structure in eukaryotic tissues that is composed of gly-

Received 15 March 2005; accepted 2 August 2005; electronically published 12December 2005.

Potential conflicts of interest: none reported.Financial support: Division of Microbiology and Infectious Diseases, National

Institute of Allergy and Infectious Diseases, National Institutes of Health (grantR37 AI47923 to B.E.M.).

Reprints or correspondence: Dr. Fang Teng, Div. of Infectious Disease, Centerfor the Study of Emerging and Reemerging Pathogens, University of Texas MedicalSchool, MSB 2.112, 6431 Fannin St., Houston, TX 77030 ([email protected]).

The Journal of Infectious Diseases 2006; 193:231–40� 2005 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2006/19302-0009$15.00

coproteins and proteoglycans such as fibronectin (FN),

laminin (LN), and collagens. Microbial adherence to

various components of the ECM has been shown to be

important for colonization and infection of the host.

Our earlier studies showed that most E. faecalis clini-

cal isolates were able to bind to collagen type I (CI),

collagen type IV (CIV), and LN but could do so only

conditionally (e.g., when grown at 46�C), whereas only

1 (MD-8) of 44 strains tested showed conditional bind-

ing to FN (∼9% of cells bound, where 5% was consid-

ered to be significant) [3]. When grown in brain-heart

infusion (BHI) at 37�C, most isolates (including MD-

8 and a reference strain, OG1RF) did not show signif-

icant binding in our assay to any of the ECM proteins

tested—including fibrinogen (FG), FN, CI, CIV, and

LN [3]—although other assays may have shown low-

level binding [4]. Our subsequent studies of the con-

ditional adherence of enterococci to ECM identified a

collagen-binding protein, Ace, in E. faecalis and a spe-

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232 • JID 2006:193 (15 January) • Mohamed et al.

Table 1. Strains used in the study.

Strain (alternative name) CharacteristicsReference or

source

E. coliDH5a Host strain StratageneTX10107 (FT1) DH5a expressing 321 aa of the N-terminal part of SalA; Spcr Present studyTX10108 (YX36) DH5a expressing 349 aa of the C-terminal part of SalB; Kanr [10]

E. faecalisOG1RF (TX4002) Well-characterized strain; Rifr, FAr [16]TX10106 salA disruption mutant; Spcr Present studyTX5123 salB (p54) disruption mutant; Kanr [11]TX10109 OG1RF with pAT18; Eryr Present studyTX10110 TX5123 with pAT18; Kanr, Eryr Present studyTX10111 TX5123 with pAT18 containing salB; Kanr, Eryr Present study

NOTE. Ery, erythromycin; FA, fusidic acid; Kan, kanamycin; Rif, rifampin; Spc, spectinomycin.

Table 2. Oligonucleotides used in the study.

Oligonucleotide Sequence (5′r3′) Usage

6259-2 CATTAACAAGCGTAGCGTTG salA disruption/expression6259-3 GCCTTTTTCAGGAGTCGTTG salA disruption/expressionsalBupper CACGTGAAAACTAGTCGTAA salB complementationsalBlower TCATTGCTGATTAGGCTGAG salB complementationsalA1 GTATTAACTTCGGTTATGGT RT-PCRsalA2 CTGGAAAACGCCATACAACA RT-PCRsalA-down1 TGTTGTATGGCGTTTTCCAG RT-PCRsalA-down2 TCTATTAAAGCTGAAGCGAT RT-PCRsalB1 CAACTGAAACAACTACACCA RT-PCRsalB2 TTAGGCTGAGTGTCCTACGAT RT-PCRsalB-down1 ATCGTAGGACACTCAGCCTAA RT-PCRsalB-down2 GTAAAAGGAATCTGACTACT RT-PCR

NOTE. RT-PCR, reverse-transcription polymerase chain reaction.

cific collagen–binding adhesin, Acm, in E. faecium. Recombi-

nant Ace was found to adhere to CI, CIV, and LN, whereas

Acm specifically bound to collagens but not to other ECM

proteins [5, 6]. More recently, we identified a gene encoding a

major secreted antigen, SagA, by screening an E. faecium ge-

nomic-expression library with serum from patients with E. fae-

cium endocarditis. The E. faecium sagA gene is located down-

stream of mreC/D genes, which encode cell shape determinants,

and has been shown to be essential for E. faecium growth [7].

The SagA protein consists of 3 domains and has shown broad-

spectrum binding to ECM proteins, including FG, CI, CIV, FN,

and LN [7]. In the present study, the SagA sequence was used

to search for homologues in E. faecalis, and the sagA-like genes

of E. faecalis (salA and salB—EF3060 and EF0394, respective-

ly—from strain V583 [8]) were identified. Sequence analysis

of these genes revealed that salB had been identified previously

in our laboratory as p54 (named for the calculated molecular

mass of a previously identified protein [9]) and that it encodes

an antigen expressed during infection in humans [10], although

disruption of the gene did not have an effect in the mouse

peritonitis model [11]. Recently, the salB gene was studied by

Breton et al. [12] (who called it “sagA” because of its similarity

to the E. faecium sagA gene) and was shown to be important

for resistance to various stress conditions, including bile salts,

NaCl, SDS, ethanol, H2O2, heat shock, and alkaline and acid

pH. In that study, electron microscopy showed that disruption

of this gene resulted in an abnormal shape and cell surface

[12]. Although those researchers did not examine binding to

ECM proteins, our previous observation with E. faecium SagA

suggested that this would be a logical next step.

Another phenotype that reflects the ability of bacteria to

adhere and that may be important for pathogenesis is biofilm

formation. Biofilm has been shown in many other pathogens

to be important for virulence, and the biofilms that form on

heart valves are termed “vegetations” [13, 14]. In our previous

work, the first systematic study of biofilm in E. faecalis en-

docarditis isolates, we examined our large collection and found

that endocarditis isolates produced biofilm significantly more

often and in significantly greater amounts than did nonendo-

carditis isolates [15]. Some E. faecalis mutants were also eval-



Figure 1. Analysis of the sal genes and their products. A, Reverse-transcription polymerase chain reaction (RT-PCR) and PCR of sal genes.Lane 1, intragenic fragment of salA (with primers salA1 and salA2); lane2, fragment spanning salA and its downstream gene (with primers salA-down1 and salA-down2); lane 3, intragenic fragment of salB (with primerssalB1 and salB2); lane 4, fragment spanning salB and its downstreamgene (with primers salB-down1 and salB-down2). RT-PCR and PCR wereperformed with RNA and genomic DNA of OG1RF. B, Western blots. Lane1, protein extracts from YX36; lane 2, protein extracts from FT1. Antibodieseluted from YX36 (SalB peptide) were used in the experiment shown inthe left panel and antibodies eluted from FT1 (SalA peptide) were usedin the experiment shown in the right panel.

uated in that study, and the disruption of the epa, atn, gelE,

and fsr genes resulted in significantly less primary attachment

and less biofilm formation on a polystyrene surface [15]. In

the present study, the effect of the disruption of E. faecalis

OG1RF salA and salB genes on ECM protein binding was stud-

ied, as was the effect of serum and ECM proteins on biofilm

formation.

MATERIALS AND METHODS

Bacterial strains and media. Bacterial strains used in the

study are listed in table 1. Escherichia coli strains were grown

in Luria-Bertani medium (Difco Laboratories) with appropriate

antibiotics, and BHI (Difco) or tryptic soy broth (TSB; Becton

Dickinson) medium with different supplements, as described

below, were used for E. faecalis.

Nucleic-acid and protein techniques. Primers used in the

study are listed in table 2. Polymerase chain reaction (PCR)

amplification, DNA cloning, and DNA sequencing were per-

formed in accordance with standard methods [17]. RNA ex-

traction from OG1RF (grown in BHI at 37�C for 24 h) and

reverse-transcription (RT)–PCR were performed as described

elsewhere [18]. The 963-bp region near the 5′ end of the salA

gene was amplified from OG1RF with primers 6259-2 and

6259-3 (table 2), cloned into pTEX5235 [19] (which resulted

in pTEX5235-2), and used to construct the salA disruption

mutant of OG1RF, as described elsewhere [19]. pTEX5235-2

was also used in E. coli (as FT1 [TX10107]) to express the N-

terminal part of SalA. Complementation of the salB mutant

was performed by cloning the whole length of the salB gene

with the ∼200-bp upstream region into the shuttle vector pAT18

[20]; the construct was introduced into the salB mutant by

conjugation, using S17-1 as the donor [19]. The pAT18 vector

was also introduced into OG1RF and the salB mutant by the

same method [19]. E. coli clones FT1 and the previously made

YX36 (TX10108) (expressing partial SalB [10]) were used for

the elution of anti-SalA and anti-SalB antibodies, respectively,

from serum from patients with E. faecalis endocarditis, using

methods described elsewhere [21] for protein extraction and

Western blotting [21].

Adherence assay. E. faecalis adherence to ECM proteins FG,

FN (Enzyme Research Laboratories), CI, CIV (Sigma Chemi-

cal), and LN (Invitrogen) was performed as described elsewhere

[3, 5], except that, for labeling, ∼107 cfu of bacteria were in-

oculated into 5 mL of BHI broth with 10 mCi of Tran35S-label/

mL, and the cultures were grown at 37�C to an OD600 of ∼0.9

(for OG1RF and the salA mutant) or ∼0.8 (for the salB mutant

TX5123), at which point they entered a stationary phase. 35S-

labeled TX5123 cells (OD600 adjusted to 0.2 in PBS) were in-

cubated with various concentrations of either CI or FN (0, 1,

50, or 100 mg/mL) for 1 h at 37�C and then centrifuged at 3400

g, followed by resuspension in PBS with 0.1% Tween 80 and

0.1% bovine serum albumin to remove excess unbound ECM

proteins, before the addition of labeled cells to the ECM-coat-

ed wells in adherence assay [3, 5].

Biofilm formation assay. Biofilm formation by E. faecalis

was determined quantitatively as described elsewhere [15], with

minor modifications. Briefly, E. faecalis strains were grown

overnight in TSB plus 0.25% glucose (TSBG) with appropriate

antibiotics at 37�C, and the OD600 was adjusted to 1.0 for each

inoculum. Cultures were diluted 1:100 in TSBG, TSBG plus

10% horse serum (HS; Invitrogen), TSBG plus FN (50 mg/mL;

Enzyme Research Laboratories or Sigma [the latter was used

in the biofilm formation experiment]), or TSBG plus CI (50

mg/mL), and 200 mL of this bacterial suspension was inoculated

into sterile 96-well polystyrene microtiter plates, followed by

incubation at 37�C. Bacterial growth in the microtiter plates

was measured by reading the optical density at 600 nm, and

biofilm formation was assessed by crystal violet staining and

phase-contrast microscopy, as described elsewhere [15]. To test

the stability of the salB-complemented strain in TSBG plus HS,

bacteria were taken from the wells at 6 and 24 h, serially diluted,

and plated on plain BHI, BHI-kanamycin (Kan; 2000 mg/mL),

and BHI-erythromycin (Ery; 10 mg/mL) plates.

Primary adherence assay. The initial adherence of OG1RF

and its isogenic mutants to a polystyrene surface was deter-

mined as described elsewhere [15], except that bacteria grown

in TSBG were adjusted to an OD600 of 0.1 with TSBG, TSBG



Figure 2. Growth curves of OG1RF, the sal mutants, and the salB-complemented strain. Bacteria were grown in brain-heart infusion (BHI) broth ina test tube, and growth in tryptic soy broth plus 0.25% glucose (TSBG), TSBG plus horse serum (HS), TSBG plus fibronectin (FN), and TSBG pluscollagen type I (CI) was assessed in microtiter plates as growth controls for biofilm formation. TX10109 is OG1RF with pAT18, TX10110 is the salBmutant with pAT18, and TX10111 is the salB mutant with pAT18 that contains salB (the salB-complemented strain).

plus HS, TSBG plus FN, or TSBG plus CI (as described above)

and then processed as described elsewhere [15], with the num-

bers of bacteria in 5 different microscopic fields counted in at

least 2 independent determinations.

Hydrophobicity assay. Cell surface hydrophobicities of E.

faecalis OG1RF and its mutants were tested as described else-

where [22], with some modifications. Briefly, bacterial strains

were grown overnight in 3 mL of TSBG and TSBG plus HS at

37�C. After the cells were washed with PUM buffer (22.2 g of

K2HPO4, 7.26 g of KH2PO4, 1.8 g of urea, 0.2 g of MgSO4, and

distilled water added to reach 1 L [pH 7.1]), cells were resus-

pended in 3 mL of PUM buffer, and the initial optical density

at 470 nm (ODI) was measured. Then, 300 mL of n-hexadecane

(Sigma Chemical) was added to the 3 mL of bacterial suspen-

sion; this was incubated at 37�C for 15 min. The mixture was

vortexed vigorously for 90 s and then allowed to stand for 10

min at room temperature. The aqueous phase was carefully

removed, and the final optical density at 470 nm (ODF) was

measured. The percentage of cell hydrophobicity was calculated

as follows: .[1 � (OD /OD )] � 100F I



Figure 3. Adherence to extracellular matrix (ECM) proteins. A, Adher-ence to fibronectin (FN) and collagen type I (CI) by OG1RF, the sal mutants,the salB-complemented strain, and plasmid controls. For the salA mutant(TX10106), the experiment was performed 2 times, with duplicates ineach experiment; for the other strains, the experiment was performed 6times, with duplicates in each experiment. TX10109 is OG1RF with pAT18,TX10110 is the salB mutant with pAT18, and TX10111 is the salB mutantwith pAT18 that contains salB (the salB-complemented strain). B, Ad-herence to FN and CI by the salB mutant (TX5123) after preincubationwith FN or CI. The experiment was performed 6 times, with duplicatesin each experiment. The mean � SD percentages of radiolabeled cellsbound to the ECM proteins are shown.

Figure 4. Biofilm formation by the sal mutants and OG1RF at 24 h.The experiment was performed 3 times, with quadruplicates in each ex-periment, and the mean � SD optical densities at 570 nm are shown.CI, collagen type I; FN, fibronectin; HS, horse serum; TSBG, tryptic soybroth plus 0.25% glucose.

Statistical analysis. Student’s t test was used to compare

OG1RF and its isogenic mutants.

RESULTS AND DISCUSSION

Identification of sal genes in E. faecalis and characterization

of the Sal proteins. In our search for SagA homologues in E.

faecalis, we identified 2 sagA-like open-reading frames in the

V583 genome database, which we named salA (EF3060) and

salB (EF0394); we had previously identified the latter as an

antigen-encoding gene [10]. Analysis of the sequences revealed

that salA, like sagA of E. faecium, has the mreC/D genes in its

immediate upstream region, whereas genes encoding hypo-

thetical proteins were found upstream of salB. Downstream of

salA and salB is a putative transcriptional regulator (EF3059)

and a putative methionine synthase (EF0395), respectively. Se-

quence analysis revealed transcriptional terminators after salA

and salB genes and separate promoters for EF3059 and EF0395;

RT-PCR with primers crossing salA (salB) and their down-

stream genes did not produce products (figure 1A), which sup-

ports the prediction that salA and salB are not cotranscribed

with their downstream genes. The salB gene has also recently

been identified by another group and named sagA on the basis

of its sequence similarity to the sagA gene of E. faecium [12].

However, because it shares similarity to SagA only in the N-

terminal part (as described below), we feel that the use of a

different name is more appropriate.

Comparison of predicted polypeptide sequences of SalA,

SalB, and SagA revealed a similar N-terminal domain (44% and

50% identity and 52% and 62% similarity for SalA and SalB,

respectively, vs. SagA of E. faecium), which were predicted by

Coilscan (Wisconsin Package version 10.0; Genetics Computer

Group) to form a coiled-coil structure but a different C-ter-

minal domain from each other. The repeat domain present in

the middle of SagA was not found in SalA and SalB. Similar

to SagA, both SalA and SalB were predicted by PSORT (avail-

able at: http://psort.nibb.ac.jp/) to be secreted proteins with a

cleavable 27-aa signal peptide. The N-terminal part of SalA

expressed in E. coli was found to react with E. faecalis patient

serum, which suggests that SalA, like SagA [7] and SalB [10],

is also an antigen expressed in vivo. Eluted antibodies from

Western blots that contained the N-terminal part of SalA (FT1)

did not react with protein extracts of YX36 (which contains

partial SalB) or vice versa (multiple bands were shown on the

Western blots, probably because of degradation of the proteins;

figure 1B), which indicates a specific reaction between these

proteins and their antibodies.



Figure 5. Attachment and hydrophobicity. A, Initial attachment of OG1RF, the sal mutants, and the salB-complemented strain to a polystyrenesurface. The experiment was performed 2 times, and 5 fields were counted in each experiment. The mean � SD no. of bacteria per field are shown.B, Hydrophobicity of wild-type OG1RF, the sal mutants and the salB-complemented strain. The experiment was performed 2 times, with 1 duplicatein each experiment, and the mean � SD percentage of hydrophobicity are shown. TX10106 is the salA mutant, TX5123 is the salB mutant, TX10109is OG1RF with pAT18, TX10110 is the salB mutant with pAT18, and TX10111 is the salB mutant with pAT18 that contains salB (the salB-complementedstrain). CI, collagen type I; FN, fibronectin; HS, horse serum; TSBG, tryptic soy broth plus 0.25% glucose.

Effect of the disruption of salA and salB on E. faecalis

growth in vitro. Unlike SagA in E. faecium, which could not

be disrupted without prior complementation, disruptions in

salA and salB were successfully constructed. In BHI broth, the

salA disruption mutant (TX10106) showed a growth curve sim-

ilar to that of OG1RF; in contrast, the salB mutant (TX5123)

generated a slightly lower optical density at 600 nm than did

OG1RF starting from 1 h, and the difference was obvious after

2 h and at entry into the stationary phase (figure 2). The growth

of TX10106, TX5123, and OG1RF were also compared in TSBG,

TSBG plus HS, TSBG plus FN, and TSBG plus CI in a 96-well

microtiter plate—the same conditions used for determining bio-

film formation. The optical-density change for TX5123 lagged

behind that of OG1RF and TX10106, but their optical densities

beyond 18 h were approximately the same. The growth of TX10106

and OG1RF was similar under all these conditions (figure 2). The

growth of the salB-complemented strain (TX10111) in BHI was

similar to that of OG1RF that contained pAT18 (TX10109),

whereas the salB mutant that contained only pAT18 (TX10110)

showed lower optical densities at 600 nm (figure 2). The growth

of TX10109 and TX10111 in TSBG, TSBG plus HS, TSBG plus

FN, and TSBG plus CI in a microtiter plate was similar to that of

OG1RF, whereas the growth of TX10110 was similar to that of

TX5123 under these conditions (data not shown).

Effect of the disruption of salA and salB on E. faecalis

binding to ECM proteins. The salB mutant TX5123, after

growth in BHI broth at 37�C, showed significant binding to CI

and FN (∼10% or ∼20% of salB mutant cells bound to CI or

FN), whereas OG1RF and the salA mutant TX10106 were non-

binders (∼1% of cells bound, with !5% considered to indicate

no binding) under these conditions ( for the salB mu-P ! .001

tant vs. OG1RF) (figure 3A). The salB-complemented strain,

like OG1RF, did not bind to CI or FN (figure 3A). Binding to

other ECM proteins—including FG, CIV, and LN—was not

detected for the sal mutants or for OG1RF under this condition.

After preincubation with 50 or 100 mg of CI or FN, binding



Figure 6. Biofilm development by the sal mutants and OG1RF. The experiment was performed 2 times, with quadruplicates in each experiment,and the mean � SD optical density at 570 nm at each time point is shown. CI, collagen type I; FN, fibronectin; HS, horse serum; TSBG, tryptic soybroth plus 0.25% glucose.

of the salB mutant to both CI and FN was significantly reduced

( ) (figure 3B). Preincubation with 1 mg of CI or FNP ! .001

did not affect binding (figure 3B).

Effect of the disruption of salA and salB on biofilm for-

mation of E. faecalis. Testing of the sal mutants and OG1RF

after 24 h of growth in TSBG in microtiter plates showed a

small (∼8%) but statistically significant reduction in biofilm

formation for the salA mutant TX10106 ( vs. OG1RF),P p .03

whereas the disruption of salB markedly impaired biofilm pro-

duction (∼54% reduction; vs. OG1RF) (figure 4). TheP ! .001

sal mutants and OG1RF were next grown in TSBG plus HS;

subsequently, FN and CI and were tested for biofilm formation.

OG1RF and TX10106 formed less biofilm in TSBG plus HS

and TSBG plus FN than in TSBG alone. However, the salB

mutant TX5123 formed very strong biofilm in TSBG plus HS

and TSBG plus FN ( vs. in TSBG and vs. OG1RF andP ! .001

TX10106 in TSBG, TSBG plus HS, and TSBG plus FN); in-

creased biofilm was not seen in TSBG plus CI (figure 4).

Effect of the disruption of salA and salB on initial attach-

ment to a polystyrene surface and hydrophobicity. To de-

termine whether salA and salB affect biofilm formation at the

primary adherence stage, the sal mutants and OG1RF were com-

pared for initial adherence to a polystyrene surface under dif-

ferent conditions. There was a significant reduction in initial

adherence by the salB mutant, compared with that of OG1RF

and the salA mutant, in TSBG ( ) (figure 5A), whichP p .002

suggests that SalB is important for initial attachment on the

surface; this is a prerequisite for biofilm formation. In contrast,

in TSBG plus HS and TSBG plus FN, the initial attachment of

the salB mutant was greater than that of OG1RF and the salA

mutant ( ) (figure 5A), which is consistent with theP ! .001

increased biofilm formed by the salB mutant under these con-

ditions. The salB mutant also showed higher initial adherence

in TSBG plus CI (figure 5A).

Because initial bacterial adhesion may be related to physi-

ochemical properties of the bacterial and biomaterial surfaces,

such as hydrophobicity or electrostatic charge [23], the hydro-

phobic properties of OG1RF and the mutants were compared

after growth in TSBG and TSBG plus HS. Results showed that

the salB mutant grown in TSBG plus HS had an increase in

hydrophobicity, compared with OG1RF and the salA mutant,

but that the hydrophobicity of the mutants and parental OG1RF



Figure 7. Biofilm formation by the salB-complemented strain. The experiment was performed 2 times, with quadruplicates in each experiment, andthe mean � SD optical density at 570 nm at each time point is shown. TX10109 is wild-type OG1RF with pAT18, TX10110 is the salB mutant withpAT18, and TX10111 is the salB mutant with pAT18 that contains salB (the salB-complemented strain). CI, collagen type I; FN, fibronectin; HS, horseserum; TSBG, tryptic soy broth plus 0.25% glucose.

were almost the same when grown in TSBG (figure 5B). The

salB-complemented strain TX10111 showed adherence and hy-

drophobicity similar to that of TX10109 (figure 5).

Time course of biofilm development. A time-course ex-

periment over 24 h was performed to study biofilm develop-

ment in the sal mutants and OG1RF under different conditions.

OG1RF and the salA mutant grown in TSBG displayed a steady

increase in biofilm formation up to the final time point, whereas

biofilm formation by the salB mutant was severely hindered at

all time points in TSBG (figure 6). In TSBG plus HS or FN,

salB mutant biofilm formation increased steadily up to an OD570

of 3–4 at 24 h, whereas the salA mutant and wild-type (wt)

OG1RF showed a slight decrease in biofilm formation under

these conditions (figure 6). Biofilm formation by the salB mu-

tant in TSBG plus CI reached a maximum (OD570, ≈1.3) at 6

h, after which the optical density decreased, whereas the for-

mation of biofilm by OG1RF and the salA mutant in TSBG

plus CI approximated that in TSBG (figure 6). The biofilm

formation of the salB-complemented strain TX10111 was sim-

ilar to that of TX10109 (OG1RF with the cloning vector) under

these conditions, except that, in TSBG plus HS and after 6 h,

TX10111 showed increased biofilm formation versus TX10109

to a level approximately one-half that of TX10110 at 24 h (fig-

ure 7). To rule out the possibility that, in TSBG plus HS, the

salB-complemented strain may lose the shuttle vector, we tested

the stability of the plasmid and found comparable colony-form-

ing units on BHI, BHI-Kan, and BHI-Ery plates. When Ery

was added to TSBG plus HS, biofilm formation by TX10109,

TX10110, and TX10111 was similar to that formed in TSBG

plus HS without Ery; these results indicate that the salB-com-

plemented strain was stable under this condition.

Phase-contrast microscopic analysis of biofilm. Microscopic

images taken 3 and 24 h after inoculation are shown in figure

8. After 3 h of incubation, the salB mutant started forming

clusters of cells in TSBG plus HS but not in TSBG. After 6 h

in TSBG, OG1RF and the salA mutant covered the surface,

forming microcolonies, whereas the salB mutant did not form

microcolonies; there were few attached bacteria, leaving broad

empty areas of plastic surface. In contrast, the salB mutant in

TSBG plus HS produced thick and mature biofilm after 6 h of

inoculation. The salB-complemented strain TX10111 showed

biofilm formation in TSBG and TSBG plus HS (figure 8). These



Figure 8. Phase-contrast microscopic analysis of biofilm structure. A, Bacteria grown in tryptic soy broth plus 0.25% glucose (TSBG). B, Bacteriagrown in TSBG plus horse serum. TX10106 is the salA mutant, TX5123 is the salB mutant, and TX10111 is the salB mutant with pAT18 that containssalB (the salB-complemented strain).

data are consistent with the indirect determination of biofilm

formation by the crystal violet staining method.

In summary, our results indicate that the absence of SalB

allows E. faecalis OG1RF to adhere to FN and CI and to form

a biofilm in the presence of serum. This is the first time that

we have been able to show, using our adherence assay, high-

level E. faecalis binding to FN or that an OG1RF derivative can

adhere to collagen when it is grown in BHI at 37�C. The salB-

complemented strain, like OG1RF, did not bind to FN or CN,

which confirms the negative effect of salB in this process. Fur-

thermore, our results show that serum and FN each elicited

strong biofilm formation by the salB mutant, whereas these

components had no effect on the salA mutant and OG1RF, and

that the increased initial attachment to the polystyrene surface

by the salB mutant grown in HS could be due, at least in part,

to the increased hydrophobic nature of the cell surface of the

salB mutant. Because both FN and CI increased the initial

attachment of the salB mutant to the polystyrene surface, it

may be that the salB mutant binds first to FN and CN, which

then attach to the plates; the subsequent increase in optical

density with HS or FN but not with CI suggests several pos-

sibilities, including that CI may be destroyed in the process,

that HS and FN may promote intercellular adherence, and/or

that HS and FN cause the activation or induction of additional

factors that further increase biofilm formation. Complemen-

tation fully restored wt phenotypes in biofilm formation (e.g.,

in TSBG and TSBG plus FN) and partially restored them in

TSBG plus HS, which suggests that the increased number of

copies of salB creates a disequilibrium and/or that FN (or some

other factor) in serum is not present in sufficient quantities to

lead to the same effect when salB is present in multiple copies.

The abnormal shape and cell surface of the salB mutant dem-

onstrated elsewhere [12] also suggested that the salB mutant

has an altered cell surface that may unmask factors involved

in FN/CI binding and biofilm formation. Reduced binding of

the salB mutant to both FN and CI after preincubation with



either FN or CI suggests that some specific binding factor(s)

on the salB mutant may be involved in binding to both FN

and CI. Recently, we identified a family of putative microbi-

al surface component–recognizing adhesive matrix molecules

(MSCRAMMs) from E. faecalis [24], which includes the pre-

viously identified collagen/laminin-binding MSCRAMM Ace

[5]. Future studies with these putative MSCRAMMs will ad-

dress whether any of these proteins are expressed and/or ex-

posed on the surface of the salB mutant. Although these in

vitro observations with a constructed mutant may not have

clinical relevance, it is tempting to speculate that, under certain

in vivo conditions—perhaps on an intravenous catheter or in

a cardiac vegetation—salB expression might be sufficiently

down-regulated to allow in vivo FN/CI adherence and a serum-

elicited increase in biofilm formation, which might increase the

capability of this organism to cause infection.

References

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pleiotrophic effects of 2 enterococcus faecalis saga– like genes

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