JOURNAL OF BACTERIOLOGY, Aug. 2005, p. 5330–5340
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 187, No. 15
Identification and Characterization of a Novel Adhesin Unique to
Yiping W. Han,1,2* Akihiko Ikegami,1Chythanya Rajanna,1Hameem I. Kawsar,1
Yun Zhou,4Mei Li,1Hakimuddin T. Sojar,3Robert J. Genco,3
Howard K. Kuramitsu,3and Cheri X. Deng4
Departments of Biological Sciences,1Pathology,2and Biomedical Engineering,4Case Western Reserve
University, Cleveland, Ohio, and Department of Oral Biology, School of Dental Medicine,
State University of New York at Buffalo, Buffalo, New York3
Received 10 January 2005/Accepted 27 April 2005
Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections
of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging
from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is im-
portant for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacte-
rium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is
composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses
of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum,
Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent
in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium navi-
forme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and
ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A
double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonopo-
ration procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster
ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an
important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobac-
terium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from
other parts of the body may originate from the oral cavity.
Fusobacterium nucleatum is a long filamentous, gram-nega-
tive anaerobe associated with various human diseases, includ-
ing periodontal, peritonsillar, orofacial, brain, chest, lung, ab-
dominal, blood, and obstetrical and gynecological abscesses
and infections, existing either as a mixed infection or as the
sole infecting agent (3, 6, 8–10, 12, 13, 16, 28, 39, 41, 45, 48, 52).
The primary colonization site of F. nucleatum in humans is the
oral cavity. It is one of the most abundant gram-negative
anaerobes in subgingival plaque and can be isolated in healthy
periodontal sites. During periodontal infection, its cell mass
increases as much as 10,000-fold, making it one of the most
abundant anaerobic species in the diseased sites (48). F. nu-
cleatum is also one of the predominant anaerobes associated
with preterm birth, having been frequently isolated from am-
niotic fluids and placentas of women delivering prematurely
(27). F. nucleatum may originate in the oral cavity and be
transmitted to the uterus via a hemotogenous route. It has
been shown in mice that, once in the bloodstream, F. nuclea-
tum colonizes specifically in the placenta, causing preterm and
term stillbirths (25).
Several virulence phenotypes of F. nucleatum have been
identified. F. nucleatum is recognized as an “adhesive” organ-
ism because it binds to a variety of host mammalian cells,
including epithelial and endothelial cells, polymorphonuclear
leukocytes, monocytes, erythrocytes, fibroblasts, and HeLa
cells, as well as salivary macromolecules, extracellular matrix
proteins, and human immunoglobulin G (IgG) (2, 25, 26, 50,
59, 62, 63). It also coaggregates with a wide array of microor-
ganisms in the oral cavity and plays an important role in plaque
formation (1, 7, 20, 22, 29, 36–38, 46, 53). Identification of the
adhesin molecules on F. nucleatum is thus essential for under-
standing its pathogenesis. It has been suggested that F. nuclea-
tum possesses both lectin-like and non-lectin-like adhesins (44,
49, 54, 58, 60, 61). Three components, a 40- to 42-kDa major
outer membrane porin protein (FomA) and 39.5-kDa and 30-
kDa polypeptides, have been suggested as possible adhesins
from F. nucleatum that are involved in interbacterial coaggre-
gation (33, 34, 55). FomA was also found to bind to the human
IgG Fc fragment (23). A high-molecular-mass component,
ranging from 300 to 330 kDa, has been suggested as a galac-
tose-binding agglutinin (49). However, it is unclear if any of
these components are involved in F. nucleatum binding to the
F. nucleatum invades epithelial and endothelial cells in vitro,
a mechanism presumably employed for its spreading into
deeper tissues (25, 26). Invasion of F. nucleatum into endothe-
lial cells was observed in vivo in the mouse placenta (25). A
spontaneous mutant defective in tissue cell attachment and
invasion, F. nucleatum 12230 lam, has been isolated, but the
nature of its mutational change is unknown (26). The lam
* Corresponding author. Mailing address: Department of Biological
Sciences, School of Dental Medicine, Case Western Reserve Univer-
sity, 10900 Euclid Avenue, Cleveland, OH 44106-4905. Phone: (216)
368-1995. Fax: (216) 368-0145. E-mail: email@example.com.
mutant exhibited virulence similar to that of the wild type in
causing fetal death in the mice (25).
F. nucleatum also induces an array of host cell responses. It
is a strong stimulator of the production of interleukin-8 from
epithelial cells, indicating its ability to induce inflammation
(15, 26). It stimulates apoptosis of human peripheral white
blood cells and suppresses T-cell responses (30, 56). The or-
ganism also induces production of innate antimicrobial pep-
tides, human ?-defensins, in gingival epithelial cells (40). This
is presumably a mechanism to suppress the growth of compet-
In this study, we report the identification of a novel 13.6-kDa
adhesin peptide from F. nucleatum involved in attachment to
mammalian cells and the construction of its deletion mutant by
a novel sonoporation method.
MATERIALS AND METHODS
Bacterial strains, culture conditions, plasmids, and enzymes. Bacterial strains
and plasmids used in this study are listed in Table 1. All fusobacterial strains were
maintained on either Trypticase soy or Columbia agar (BBL) supplemented with
5% defibrinated sheep blood (Cleveland Scientific, OH) or in Trypticase soy or
Columbia broth (BBL) and incubated as previously described (25). The Esche-
richia coli strains were maintained in LB broth (Difco) or on LB agar (Difco) and
incubated at 37°C in air. Restriction endonucleases and ligase were purchased
from New England BioLabs (Beverly, MA), and PfuUltra high-fidelity DNA
polymerase was from Stratagene (La Jolla, CA).
To construct plasmids pYH1378 and pYH1426, a 526-bp fragment, “upfadA,”
and a 510-bp fragment, “downfadA,” corresponding to the upstream and down-
stream regions flanking the fadA gene, respectively, were amplified using primer
sets fadA1f (5?AGGTCAAGAAGCAAAAGG3?)-fadA1r (5?TTTTTGGTACCC
TTGCTGCATCAGTTGC3?) and fadA2f (5?TTTTTGGATCCTCAAGCTTTA
AGAGCTGG3?)-fadA2r (5?AGGGTTACTTGATTCAGG3?), generating a
KpnI site in “upfadA” and a BamHI site in “downfadA” at ends adjacent to fadA.
A KpnI-BamHI fragment containing the ermF-ermAM cassette from pVA2198
(18) was then ligated with the “upfadA” and the “downfadA” fragments, followed
by cloning into pCR2.1 (Invitrogen, Carlsbad, CA). The resulting plasmid,
pYH1378, was digested with EcoRV, and a sacB gene from pRL250 (11) was
inserted into the EcoRV site to generate pYH1426.
Preparation of biotinylated and nonlabeled KB surface proteins. The human
oral mucosal epithelial cell line KB (ATCC CCL-17; American Type Culture
Collection, Manassas, VA) was maintained in MEM medium (GibcoBRL, Rock-
ville, MD) supplemented with 10% fetal bovine serum (Mediatech, Herndon,
VA). The cultures were grown in four 75-cm2tissue culture flasks (Fisher Sci-
entific, Pittsburgh, PA) under 5% CO2at 37°C to near confluence. The cells were
detached from the flasks by using enzyme-free cell dissociation buffer (Gibco-
BRL). Following washes with sterile phosphate-buffered saline (PBS) (Sigma, St.
Louis, MO), the cells were incubated in 2 ml of 1 mM sulfo-NHS-LC-biotin
(Pierce Chemical Co., Rockford, IL) at 4°C for 2 h. The outer membrane
components were extracted with 1% Triton X-100 (Sigma) at room temperature
for 1 h, followed by centrifugation. The supernatant was transferred to a Cen-
tricon YM-3 column (Millipore, Bedford, MA) and centrifuged at 7,500 ? g. The
centrifugation was repeated twice, adding 2 ml sterile 10 mM Tris, pH 7.5, each
time to the sample reservoir to change the buffer. At the end of centrifugation,
a total of approximately 50 ?l of concentrated sample was recovered and stored
at 4°C. The protein concentration was determined with bicinchoninic acid (BCA)
(Pierce, Rockford, IL). Nonlabeled KB surface proteins were prepared following
the same procedures except that the KB cells were not incubated with sulfo-
Far-Western analysis. A total of approximately 1 ? 108to 5 ? 108CFU of F.
nucleatum 12230 or 10 ?g of fractionated F. nucleatum components, unless
TABLE 1. Bacterial strains and plasmids
Strain or plasmid Source of isolation or relevant characteristicSource or reference
F. nucleatum 12230
F. nucleatum 12230-US1
F. nucleatum ATCC 10953
F. nucleatum ATCC 23726
F. nucleatum ATCC 25586
F. nucleatum ATCC 49256
F. nucleatum ATCC 51190
F. nucleatum PK 1594
F. nucleatum DUMC1356
F. nucleatum DUMC2079
F. nucleatum DUMC2929
F. nucleatum DUMC3156
F. nucleatum DUMC3349
F. gonidiaformans DUMC CF65-1
F. gonidiaformans DUMC CF63-1
F. naviforme DUMC CF108-1
F. mortiferum ATCC 25557
F. periodonticum ATCC 33693
F. russii ATCC 25533
F. simiae ATCC 33568
F. ulcerans ATCC 49185
Transtracheal isolate, working strain in the lab
F. nucleatum 12230 ?fadA::ermF-ermAM
Inflamed gingiva; F. nucleatum subsp. polymorphum
F. nucleatum subsp. nucleatum
Cervicofacial lesion; F. nucleatum subsp. nucleatum
Periodontal pocket; F. nucleatum subsp. vincentii
Sinusitis in upper jaw; F. nucleatum subsp. fusiform
Amniotic fluid; preterm birth
Placenta; preterm birth
Amniotic fluid; preterm birth
Placenta; preterm birth
Placenta; preterm birth
Vaginal tract; bacterial vaginosis
Vaginal tract; bacterial vaginosis
Vaginal tract; bacterial vaginosis
Infection in a cat
Monkey dental plaque
P. E. Kolenbrander
P. E. Kolenbrander
P. E. Kolenbrander
P. E. Kolenbrander
P. E. Kolenbrander
P. E. Kolenbrander
G. B. Hill
G. B. Hill
P. E. Kolenbrander
P. E. Kolenbrander
P. E. Kolenbrander
P. E. Kolenbrander
P. E. Kolenbrander
Cosmid vector used for library construction (21.6 kb)
pLAFR2 clone containing fadA (48.2 kb)
Cloning vector (3.9 kb)
pCR2.1 carrying 2.4-kb fragment containing fadA (6.3 kb)
Source of the ermF-ermAM cassette (9.2 kb)
pCR2.1 carrying ?fadA::ermF-ermAM and flanking regions of fadA
Source of sacB (14.3 kb)
pYH1378 containing sacB (9.2 kb)
VOL. 187, 2005FUSOBACTERIUM ADHESIN 5331
otherwise indicated, were subjected to 12% sodium dodecyl sulfate-polyacrylam-
ide gel electrophoresis (SDS-PAGE) and then transferred to Immobilon-P poly-
vinylidene difluoride (PVDF) membranes (0.45-?m pore size; Millipore). The
membranes were blocked with 1% bovine serum albumin (Sigma), followed by
incubation with biotinylated KB surface proteins at a 1:500 dilution in TBST (50
mM Tris, pH 7.5, 0.5 M NaCl, 0.1% Tween 20) at room temperature for 1 h. The
membranes were washed with TBST and incubated with avidin-horseradish per-
oxidase (HRP) conjugate (Bio-Rad, Hercules, CA) at a 1:1,000 dilution. The
membranes were developed using 4-chloro-1-naphthol (Bio-Rad) and hydrogen
peroxide (Sigma). For controls, the membranes were incubated directly with
avidin-HRP conjugate without incubation with biotinylated KB surface proteins.
For competitive far-Western analysis, the membrane was preincubated with
nonbiotinylated KB surface proteins in 20-fold excess at room temperature for
1 h prior to incubation with biotinylated KB surface proteins.
Preparation of “40P.” One liter of freshly grown F. nucleatum 12230 culture
was centrifuged, and the cell pellet was resuspended in 10 ml sterile PBS,
followed by 10 min of ultrasonication in an ice-water bath with a 3-mm microtip
at 20-W output pulse setting at a 50% duty cycle (Vibra Cell, model VC250;
Sonic and Materials Inc., Danbury, CT). The suspension was then centrifuged at
3000 ? g, and the supernatant was centrifuged again at 100,000 ? g. The
twice-centrifuged supernatant was designated the cell extract, to which ammo-
nium sulfate was added to a final concentration of 40% (wt/vol) and incubated at
4°C with agitation for ?4 h. The suspension was centrifuged at 100,000 ? g for
2 h, and the pellet was dissolved in 10 mM Tris, pH 7.5, followed by dialysis
against 10 mM Tris, pH 7.5, at 4°C. The resulting solution was designated “40P,”
and its protein concentration was determined by BCA.
Construction and screening of F. nucleatum 12230 cosmid library. Chromo-
somal DNA of F. nucleatum 12230 was purified, partially digested with Tsp509I,
and cloned into the EcoRI site of cosmid pLAFR2 (19). The ligation mixture was
incubated with Gigapack III XL packaging extract (Stratagene), and the cosmid
phage lysate was prepared according to the manufacturer’s instructions. The
phage lysate was used to transfect JM109, and the cosmid clones were selected
on LB plates containing 20 ?g/ml tetracycline. The clones were saved in 96-well
plates and stored at ?80°C. Four degenerate oligonucleotide pools were de-
signed based on the protein N-terminal sequence and the Codon Usage Data-
base (Table 2). The probes were labeled with digoxigenin (DIG) by using the
DIG DNA labeling and detection kit (Roche, Indianapolis, IN). They were then
used to screen the F. nucleatum 12230 cosmid library by colony hybridization as
described previously (21). Putative positive clones were examined by Southern
blotting analysis using pool 4 probes.
fadA sequence analysis. The DNA sequence of the 2.4-kb fragment from
pYWH401 was determined (CAMBI Nucleic Acid Facility, Buffalo, NY), first
using the M13 reverse primer and the T7 primer and then using synthetic
oligonucleotide primers M13ext1 (5?GCTTCCATTTGTTAAAACCACC3?),
M13ext2 (5?GCAATTAAACTTACAATCTGAAAGCC3?), M13ext3 (5?CAGT
TAGACCAAAGGGTCCTG3?), T7ext1 (5?TCCATAACCAAATAACTTATA
C3?), and T7ext2 (5?CTAGCAGCGTCAGCTTGTGCTC3?), derived from the
sequence of the fragment. The open reading frames (ORFs) were identified
using the National Center for Biotechnology Information ORF Finder (http:
//www.ncbi.nlm.nih.gov/gorf/gorf.html). The fadA genes from F. nucleatum
ATCC 49256, DUMC1356, and ATCC 33693 were amplified with oligonucleo-
tide primers fadA1Kf (5?CTTTTAAAACCTCTCCAAGC3?) and T7ext1. All
other fadA genes were amplified with primers M13ext2 and T7ext1. The PCR
conditions were as following: denaturing at 94°C for 30 s, annealing at 50°C for
30 s, and extension at 72°C for 5 min, with repeats of 30 cycles. The PCR
products were treated with Exo/SAP-IT (USB, Cleveland, OH) and their nucle-
otide sequences determined (Molecular Biotechnology Core, Lerner Research
Institute, Cleveland, OH) using primers fadAfor (5?TTAGCTGTTTCTGCTTC
AGC3?) and fadArev (5?TTACCAGCTCTTAAAGCTTG3?).
DNA dot blotting. Chromosomal DNA of Fusobacterium species was dena-
tured by heating at 95°C for 10 min. A total of 0.5 ?g denatured DNA was
spotted onto an Immobilon-NY? membrane (Millipore). A DIG-labeled 359-bp
fadA fragment was used as the probe. It was amplified by PCR using primers
fadAfor and fadArev with chromosomal DNA of F. nucleatum 12230 as the
Construction of fadA mutant of F. nucleatum 12230 via sonoporation. Log-
phase F. nucleatum 12230 cells were washed and resuspended to a final concen-
tration of 1 ? 1010CFU/ml in PBS supplemented with 0.1 mM CaCl2and 0.1
mM MgCl2. A total of 100 ?l of the bacterial suspension was mixed with 50 ?g
plasmid DNA and 50 ?l Optison (Perflutren protein-type A microspheres for
injection, USP; Amersham, Princeton, NJ) in a 96-well plate and subjected to
ultrasound (US) treatment. A custom-made regular planar piezoelectric lead-
zirconate-titanate US transducer of a circular aperture with a diameter of 5.1 cm
(center frequency of 0.96 MHz) was vertically directed upward to irradiate the
bacteria in the 96-well plate. A signal generator (33250A; Agilent Technologies,
Palo Alto, CA) controlled the duty cycle and initial amplitude of the input signal,
which was amplified using a 75-W power amplifier (75A250; Amplifier Research,
Souderton, PA). The amplified signal was connected to the US transducer to
generate the desired US field. Pulsed US exposures at a duty cycle of 50% and
a pulse repetition frequency of 1 Hz were used for a total duration of 90 s. The
US beam profile was measured using a calibrated hydrophone system (HPM04/1;
Precision Acoustics, United Kingdom), and the effective US output powers were
calibrated using a US power meter (UPM-DT-10; Ohmic Instrument Co, Easton,
MD). The acoustic pressure of US exposure was 0.5 MPa (corresponding to an
initial input signal at 130 mV). Following US treatment, the suspension was
plated onto Columbia blood agar plates and incubated under anaerobic condi-
tions at 37°C for 24 h. The bacteria were then replicated onto Columbia blood
agar plates containing 0.4 ?g/?l clindamycin and incubated for 3 additional days.
The clindamycin-resistant colonies were purified on plates before being inocu-
lated in Columbia broth containing 0.4 ?g/?l clindamycin. The genetic nature of
the mutants was verified by PCR, using primers fadAfor and fadArev, and by
Southern blot analysis, using the same 359-bp DIG-labeled fadA probe used for
DNA dot blotting.
Western blot analysis. Whole-cell F. nucleatum was boiled for 3 min in Lae-
mmli sample buffer, subjected to 15% SDS-PAGE, and blotted onto a PVDF
membrane. The membrane was incubated overnight with polyclonal anti-FadA
serum (unpublished results) at a 1:1,000 dilution at 4°C. After washing, the
membrane was incubated with goat anti-rabbit IgG–HRP at a 1:1,000 dilution at
room temperature for 1 h, followed by color development as described above.
Northern blot analysis. RNA was prepared from mid-log-phase F. nucleatum
by phenol extraction. A total of 10 ?g RNA/lane was loaded onto a 1.5%
agarose-formaldehyde gel, alongside a 0.16- to 1.77-kb RNA ladder (Invitrogen),
followed by electrophoresis at 50 V for 1.5 h. The RNA was then transferred
onto a Zeta-Probe GT blotting membrane (Bio-Rad) by alkaline blotting for 4 h.
The above-mentioned 359-bp fadA fragment was used as a probe, using the ECL
direct nucleic acid labeling and detection system (Amersham Biosciences) ac-
cording to the manufacturer’s instructions. The membrane was washed, exposed
on an X-ray film, and developed. The experiment was repeated at least twice.
RT-PCR. RNA was prepared from mid-log-phase F. nucleatum by using the
RNeasy minikit (QIAGEN, Valencia, CA), followed by treatment with RNase-
free DNase (QIAGEN). DNA contamination in the RNA samples was deter-
mined by PCR amplification of ORF2, fadA, and ORF3 with primers Orf2-F
(5?GGAGGGGAAGATGGAAGAAG3?) and Orf2-R (5?TCTTCTGCTATTG
CTGGATGAA3?), fadAfor and fadArev, and Orf3-F (5?AAGGGTTACTTGA
TTCAGGAATTG3?) and Orf3-R (5?CAATTCCTGAATCAAGTAACCCTT3?),
respectively. Samples with no detectable DNA contamination were used for
reverse transcription-PCR (RT-PCR). Reverse transcription was performed us-
ing SuperScript II (Invitrogen) with 1 ?g DNA-free RNA and 10 pmol of the
TABLE 2. Degenerate oligonuleotide probes used to identify the putative adhesin from an F. nucleatum genomic library
FadA N-terminal sequencea........................................................... A
aSequence identified by protein N-terminal microsequencing.
bX, A or T. The nucleotides that differ in pools 1, 2, and 3 are underlined.
5332HAN ET AL. J. BACTERIOL.
forward primer for each gene in a final volume of 50 ?l per reaction. An aliquot
of 2 ?l of the RT reaction mix was then used for PCR amplification of 25 cycles
(94°C for 45 s, 55°C for 30 s, and 72°C for 1 min, followed by a 7-min extension
at 72°C), using both the forward and reverse primers described above. The PCR
products were subjected to electrophoresis on a 1.0% agarose gel. Each exper-
iment was repeated at least twice.
Bacterial growth curve. Fresh broth cultures of F. nucleatum were transferred
into fresh medium at a 1:4 dilution. An aliquot was taken out every hour, and its
optical density at 600 nm was measured using a Genesys 5 UV-visible spectro-
photometer (Thermo Electron, Waltham, MA). The experiment was repeated
Tissue culture cell attachment assay. KB cells were cultured as described
above. Chinese hamster ovary (CHO) cells were maintained in F12K medium
(Mediatech) supplemented with 10% fetal bovine serum. The attachment assays
were carried out as previously described (26). Briefly, KB or CHO cells were
seeded into 24-well trays and allowed to grow to near confluence. Immediately
before the assay, the spent medium was replaced with fresh nonsupplemented
medium. F. nucleatum strains were harvested and resuspended in PBS to a
density of 5 ? 108cells/ml. Approximately 5 ? 106CFU was added into each well
and incubated at 37°C under 5% CO2for 1 h. The monolayers were then washed
four times with PBS and lysed with water. F. nucleatum attached to the cells was
enumerated on blood agar plates. Attachment values were expressed as the
percentage of bacteria associated with the host cells relative to the total number
of bacteria initially added.
Nucleotide sequence accession number. The nucleotide sequence of the 2.4-kb
fragment from F. nucleatum 12230 containing the fadA gene has been deposited
in the GenBank database with an assigned accession number AY850357. The
accession numbers for the FadA sequences from other fusobacterial strains
and species are as follows: DQ012969 for F. nucleatum ATCC 10953, DQ012970
for F. nucleatum ATCC 23726, DQ012971 for F. nucleatum ATCC 25586,
DQ012972 for F. nucleatum ATCC 49256, DQ012973 for F. nucleatum
ATCC 51190, DQ012974 for F. nucleatum DUMC1356, DQ012975 for F. nu-
cleatum DUMC2079, DQ012976 for F. nucleatum DUMC2929, DQ012977 for
F. nucleatum DUMC3156, DQ012978 for F. nucleatum DUMC3349, DQ012979
for F. nucleatum PK1594, DQ012980 for F. periodonticum ATCC 33693, and
DQ012981 for F. simiae ATCC 33568.
Identification of a putative adhesin molecule from F. nuclea-
tum 12230. It was shown previously that F. nucleatum binds to
both KB cells and normal human gingival epithelial cells (26).
Therefore, KB cells were used in this study for ease of manip-
ulation. Sulfo-NHS-LC-biotin is a nonspecific biotinylating
agent which does not penetrate mammalian cell membranes.
Thus, upon incubation with the KB cells, it nonspecifically
labeled proteins on the KB cell surface. Triton X-100 extrac-
tion of the KB cells produced a “cocktail” of biotinylated KB
surface proteins. When this “cocktail” was incubated with F.
nucleatum components immobilized on PVDF membranes,
binding between F. nucleatum adhesins and their receptors on
the KB cells could occur. When whole-cell F. nucleatum 12230
was tested, one dark and two light bands were identified (Fig.
1I, panel c). When the same components were incubated di-
rectly with avidin-HRP, the two light bands were not detected,
while the dark band remained strongly visible (Fig. 1I, panel
b). This observation indicates that the components identified
by the two light bands bound KB surface proteins, while the
dark band represents an F. nucleatum component(s) natu-
rally bound with biotin, such as a carboxylase. The two light
bands had apparent molecular masses of 40 kDa and 16
kDa. The 40-kDa component was detected only in the boiled
F. nucleatum and not in the nonboiled sample, indicating it
likely formed oligomers too large to migrate into the gel
(Fig. 1I, panel c). The size of the monomer and its oligomer-
ization suggest that this component could be the trimer-
forming major outer membrane porin protein FomA (35).
Since the 16-kDa component was more prominent, it was
Incubation of F. nucleatum 12230 cell extract with 40% (wt/
vol) (NH4)2SO4resulted in the precipitation of a limited num-
ber of F. nucleatum components, designated “40P” (Fig.1II,
panel a). Far-Western blot analysis of 40P using biotinylated
KB surface proteins identified one major band (Fig. 1II, panel
c), which was not detected if the membrane was incubated only
with avidin-HRP (Fig.1II, panel b). The component in Fig.1II,
panel a, corresponding to that in Fig.1II, panel c, was identified
by aligning the two PVDF membranes. This component was
designated “FadA,” for Fusobacterium adhesin A, and its N-
terminal amino acid sequence was determined by protein mi-
crosequencing (ProSeq, Boxford, MA) (Table 2). Binding be-
tween FadA and the biotinylated KB surface proteins
increased as the FadA quantity increased (Fig.1III, panel a).
Furthermore, the binding was inhibited by preincubation with
nonlabeled KB surface proteins prior to incubation with the
biotinylated proteins (Fig.1III, panel b). These results further
indicate that FadA bound specifically to a component(s) on the
KB cell surface.
Identification of the fadA gene. A genomic library of F.
nucleatum 12230 was constructed by cloning the bacterial chro-
mosomal DNA into the cosmid vector pLAFR2. A total of 576
cosmid clones were saved in six 96-well plates. AseI digestion
of 10 randomly picked clones showed that all were indepen-
dent clones (data not shown). With a mean insert size of
approximately 20 kb, and assuming that all clones were inde-
pendent, this library should have covered the entire F. nuclea-
tum 12230 genome four to five times. A total of four different
degenerate oligonucleotide pools were used to screen the li-
brary (Table 2). Pools 1 to 3 correspond to the first 10 amino
acids of the FadA N-terminal sequence. The only difference
between these three pools was the codon used for the serine
residue at position 6. Pool 4 corresponds to the amino acid
sequence from position 7 through 16. Since the F. nucleatum
genome consists of more than 70% AT, the pools were de-
signed such that only the third position in selected codons
carried a mixture of A and T. This design reduced the degen-
eracy of the oligonucleotide pools. Through repeated colony
hybridization and Southern blot analyses, one true positive
clone was identified and designated YWH1 (data not shown).
Cosmid pYWH1 was purified and digested with different re-
striction endonucleases. A 6.2-kb EcoRV fragment, a 1.3-kb
EcoRI fragment, and a 2.4-kb Sau3AI fragment were identified
through Southern blot analysis using pool 4 oligonucleotides as
probes (data not shown). The 2.4-kb Sau3AI fragment was
subcloned into the BamHI site of pCR2.1 to generate
pYWH401, and its DNA sequence was determined (data not
shown). A total of four ORFs were identified. The smallest
ORF encodes 129 amino acids, with the first eighteen residues
corresponding to a typical signal peptide, which should be
absent in the secreted form. The next 16 residues perfectly
matched the N-terminal peptide sequence of FadA (Table 2),
indicating that the component identified by far-Western anal-
ysis was the secreted form. FadA is alanine (20%) and leucine
(10%) rich. It shares no homology with any known adhesins.
Secondary structure analysis preformed by the Ph.D. method
at the European Molecular Biology server indicated that it was
VOL. 187, 2005FUSOBACTERIUM ADHESIN 5333
composed almost exclusively of ?-helix. The intact FadA had a
calculated molecular mass of 13.6 kDa, while the secreted form
was 12.6 kDa, smaller than the apparent molecular mass of 16
kDa identified by SDS-PAGE.
Conservation of FadA among fusobacteria. The presence of
fadA among other species and strains of fusobacteria was ex-
amined by DNA dot blotting with 12 strains of F. nucleatum, 2
strains of F. gonidiaformans, and one strain each of F. mor-
tiferum, F. naviforme, F. periodonticum, F. russii, F. simiae, and
F. ulcerans (Fig. 2). The fadA gene appeared to exist in the
three most closely related species, F. nucleatum, F. periodon-
ticum, and F. simiae, but was absent in the other species (Fig.
FIG. 1. I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with
Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes were incubated
with streptavidin-HRP conjugate, followed by chemiluminescence reaction. c. F. nucleatum 12230 (I) or 40P (II) components immobilized on
PVDF membranes were first incubated with biotinylated KB surface proteins, followed by incubation with streptavidin-HRP. “M,” protein size
marker, with sizes indicated on the left; “NB,” nonboiled whole-cell F. nucleatum 12230 (I) or 40P (II); “B,” boiled whole-cell F. nucleatum 12230
(I) or 40P (II). The arrows point to bands visible in c but not in b, indicating binding to biotinylated KB proteins. The bottom arrow in Ic and the
arrow in IIc indicate FadA. III. Competitive far-Western analysis. 40P or F. nucleatum 12230 whole-cell components immobilized on PVDF
membranes were incubated directly with biotinylated KB surface proteins (a) or preincubated with 20? nonlabeled KB surface proteins prior to
incubation with biotinylated KB surface proteins (b). “M,” protein size markers, as indicated on the left; lanes 1 to 5, 40P in increasing amounts
(0.375, 0.625, 1.25, 2.5, and 5 ?g, respectively); lanes 6, F. nucleatum 12230.
5334 HAN ET AL.J. BACTERIOL.
2). DNA sequence analysis showed that FadA was highly con-
served among the fadA-positive species (Fig. 3).
Following identification of the fadA gene, the genomic se-
quence of F. nucleatum ATCC 25586 became available (31). A
BLAST search showed that the 2.4-kb fragment containing the
fadA gene was highly conserved between F. nucleatum 12230
and ATCC 25586 (data not shown). The ORF corresponding
to fadA in F. nucleatum ATCC was FN0264. The three addi-
tional ORFs near fadA were identified as follows: ORF1
(FN0262), which transcribes in the opposite direction relative
to fadA, encodes a formate acetyltransferase; ORF2 (FN0263),
immediately upstream of fadA, encodes a peptidyl-prolyl cis-
trans-isomerase; and ORF3 (FN0265) is downstream of fadA
and encodes a cell division protein, FtsX, with 29% identity to
the ABC transporter permease cell division protein FtsX of
Listeria monocytogenes (data not shown). In addition, a paral-
ogue (i.e., homologue on the same chromosome), FN1529, is
present in F. nucleatum ATCC 25586, sharing 31% identity
with FadA (Fig. 3).
More recently, the genome sequence of another strain, F.
nucleatum subsp. vincentii ATCC 49256, became available (32).
DNA dot blot and PCR analyses indicate the presence of fadA
in this strain (Fig. 2 and 3). However, a BLAST search failed
to identify fadA in the gapped genome sequence (data not
shown). Instead, a FadA paralogue, FNV2159, which is 31%
identical to FadA and 98% identical to FN1529, was identified
Construction of a fadA deletion mutant of F. nucleatum
12230. To the best of our knowledge, currently no report is
available on the construction of genetic knockout mutants of F.
nucleatum. Since fadA is made up of fewer than 400 bases, it
would be difficult to construct a knockout mutant by integrat-
ing a suicide plasmid containing an internal fragment of fadA.
Following unsuccessful attempts to generate a correct fadA
deletion mutant of F. nucleatum 12230 by either electropora-
tion or conjugation, DNA delivery via sonoporation, i.e., tran-
sient membrane permeabilization by ultrasound, was tested.
Plasmid pYH1426, which contains a homologous fragment of
approximately 500 bp at either end of fadA and a 2.1-kb ermF-
ermAM cassette replacing fadA, was used (Fig. 4A). The ermF-
ermAM cassette confers erythromycin and clindamycin resis-
tance (24). The plasmid also carries a 2.1-kb fragment
containing a sacB gene, conferring sucrose sensitivity (11).
Intact pYH1426 was mixed with F. nucleatum 12230 and Op-
tison, followed by a 90-s (pulse repetition frequency, 1 Hz; duty
cycle, 50%) ultrasonic treatment. Optison is a Food and Drug
Administration-approved contrast agent consisting of albumin-
coated perfluoropropane (C3F8) gas bubbles and is routinely
used in ultrasound imaging for cardiac diagnosis. It has been
used to facilitate sonoporation in mammalian cells (47). Under
these experimental conditions, the viability of F. nucleatum
12230 was not affected; nor was there any detectable DNA
damage when examined by agarose gel electrophoresis (data
not shown). Ultrasonic delivery of pYH1426 into F. nucleatum
12230 produced more than 30 independent transformants, at
an efficiency of approximately 0.05 transformant/?g DNA. All
transformants were genetically identical double-crossover fadA
deletion mutants, as determined by PCR (data not shown) and
Southern blot analyses (Fig. 4B). Loss of FadA in these mu-
tants was verified by Western blotting using anti-FadA poly-
clonal antibodies (Fig. 4C). One of the mutants was designated
F. nucleatum 12230-US1 (Table 1).
In order to determine if insertional inactivation had any
polar effects on the downstream gene ORF3, Northern blotting
and RT-PCR were performed (Fig. 5). Northern blotting
using fadA as a probe revealed a transcript of approximately
400 bases, indicating that fadA was transcribed monocis-
tronically (Fig. 5A). DNA sequence analysis indicated the
likely existence of a rho-independent transcription termina-
tor immediately downstream of fadA from position 1893 to
1941 (data not shown). Transcription of both neighboring
genes of fadA, the upstream ORF2 and the downstream
ORF3, was unaffected in F. nucleatum 12230-US1 as indi-
cated by RT-PCR (Fig. 5C).
Characterization of the fadA deletion mutant. F. nucleatum
12230-US1 was characterized by its growth rate, aerotolerance,
and ability to bind to KB and CHO cells, each in comparison
with its parental strain F. nucleatum 12230. The mutant con-
sistently grew at a lower rate, with a doubling time in the
exponential phase of approximately 6 h, compared to 5 h for
the parental strain (Fig. 6). The mutant exhibited aerotoler-
ance similar to that of the wild type (data not shown). When
tested for binding to KB and CHO cells, F. nucleatum 12230-
US1 was found to be severely defective. Although the percent
attachment levels varied when KB or CHO cells were used, the
difference between the wild type and the mutant remained
consistent, with the mutant exhibiting a reduction of approxi-
mately 70 to 80% (Fig. 7). These observations indicate that
FadA is nonessential for bacterial integrity but is required for
its binding to host cells.
FIG. 2. DNA dot blot analysis of the fadA gene in different Fuso-
bacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidi-
aformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. navi-
forme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum
ATCC 25586; 7, F. nucleatum ATCC 23726; 8, F. nucleatum 12230; 9,
F. nucleatum ATCC 49256; 10, F. nucleatum ATCC 51190; 11, F.
nucleatum PK1594; 12, F. nucleatum DUMC2929; 13, F. nucleatum
DUMC3349; 14, F. nucleatum DUMC3156; 15, F. nucleatum
DUMC1356; 16, F. nucleatum DUMC2079; 17, F. periodonticum
ATCC 33693; 18, F. russii ATCC 25533; 19, F. simiae ATCC 33568; 20,
F. ulcerans ATCC 49185.
VOL. 187, 2005 FUSOBACTERIUM ADHESIN 5335
F. nucleatum binds to a wide variety of partners, including
both eukaryotic and prokaryotic cells. Although a few putative
adhesins have been suggested to be involved in interbacterial
coaggregation or agglutinination of red blood cells, it is unclear
if they are also required for F. nucleatum binding to other host
cells. Known for its wide-ranging adherence properties, F. nu-
cleatum may possess multiple adhesins, some of which may be
partner specific while others may have multiple binding sub-
strates. In this study, a novel peptide, FadA, was identified,
with the ?-helix predicted as the predominant secondary struc-
ture. The secreted form of FadA has a larger apparent molec-
ular mass than its calculated molecular mass (Fig. 1). As a
putative adhesin, FadA is likely associated with the outer mem-
brane. It is not unusual for a membrane protein to have aber-
rant migration on SDS-PAGE. For instance, the 40-kDa
FomA protein has been reported to migrate as 37-kDa, 40-
kDa, 42-kDa, and 62-kDa proteins (5, 34, 35).
Loss of FadA resulted in a 70 to 80% reduction of the
organism’s ability to bind to KB and CHO cells (Fig. 7). Sev-
eral possibilities exist: (i) the loss of attachment was due to the
lower growth rate of F. nucleatum 12230-US1, (ii) the defect
was due to a polar effect on the downstream gene(s), (iii) FadA
serves as an accessory protein for binding, or (iv) FadA is a
major adhesin directly involved in F. nucleatum binding to host
cells. The first three possibilities are unlikely for the following
reasons: (i) the incubation time during the attachment assay
was 1 hour, during which the bacterial growth was minimal; (ii)
Northern blot and RT-PCR analyses indicated that fadA was
transcribed monocistronically and that transcription of ORF3
was unaffected by the mutational change in fadA (Fig. 5) (these
observations were also supported by the detection of a putative
transcription terminator immediately downstream of fadA),
and (iii) FadA was identified by far-Western analysis as directly
and specifically bound by biotinylated KB surface proteins
(Fig. 1). Taken together, the most reasonable explanation
would be that FadA is directly involved in binding. Further
supporting this notion is that expression of FadA in E. coli
enhanced the ability of E. coli to bind to mammalian cells
(unpublished results). Since F. nucleatum 12230-US1 was de-
FIG. 3. Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins.
The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are listed below
FadA. The conserved and identical residues between FadA and the paralogues are indicated. Fn, F. nucleatum; Fp, F. periodonticum; Fs, F. simiae.
The numbers above the sequence indicate amino acid positions in the secreted form of FadA, and the numbers beside the sequence indicate
positions in the intact form.
5336HAN ET AL.J. BACTERIOL.
fective in binding to both KB and CHO cells, it is likely that
FadA binds to a receptor(s) common to both types of cells. It
should be pointed out that although FadA appears to be a
significant adhesin for F. nucleatum to bind to host cells, an
additional adhesin(s) exists, likely accounting for the remain-
ing binding activities observed in F. nucleatum 12230-US1.
Although BLAST searches failed to identify FadA in the
gapped genome of F. nucleatum ATCC 49256, DNA hybrid-
FIG. 4. Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the ?fadA::erm mutant by double-
crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the fadA gene. The shaded
boxes represent regions hybridizing with the probe during Southern blotting. The sizes of the fragments hybridized with the probe are indicated.
H, HindIII cleavage sites; E, EcoRI cleavage sites. B. Southern blot analysis of F. nucleatum 12230 and F. nucleatum 12230-US1, using a 359-bp
fadA fragment as a probe. Lanes: 1, F. nucleatum 12230 digested with EcoRI; 2, F. nucleatum 12230-US1 digested with EcoRI; 3, F. nucleatum
12230 digested with HindIII; 4, F. nucleatum 12230-US1 digested with HindIII. The DNA size markers are indicated on the left. C. Western blot
analysis of F. nucleatum 12230 and 12230-US1, using anti-FadA antibodies. Lanes: 1, protein size markers, with molecular masses shown on the
left; 2, F. nucleatum 12230; 3, F. nucleatum 12230-US1. The arrow indicates FadA.
VOL. 187, 2005 FUSOBACTERIUM ADHESIN 5337
ization, PCR, and sequence analyses indicated that FadA is
highly conserved among F. nucleatum, F. periodonticum, and F.
simiae yet is absent in F. mortiferum, F. gonidiaformans, F.
naviforme, F. mortiferum, F. russii, and F. ulcerans (Fig. 2 and
3). F. nucleatum, F. periodonticum, and F. simiae have been
reported as three closely related oral species, forming a distinct
group within the genus (42, 51). The presence of FadA in these
three species and its absence in others are consistent with the
previously described genetic relatedness within the group.
Therefore, fadA may be used as a marker for identification of
orally related fusobacteria. The conservation of fadA in F.
nucleatum isolated from intrauterine infections and its absence
in the vaginal species F. gonidiaformans and F. naviforme fur-
ther support the hypothesis that intrauterine F. nucleatum orig-
inates from the oral cavity rather than the vaginal tract (25).
BLAST searches also identified two paralogues of FadA,
FN1529 from F. nucleatum ATCC 25586 and FNV2159 from
F. nucleatum ATCC 49256, which share 31% identity with
FadA and 98% identity with each other. The conservation of
the FadA paralogue among fusobacteria and its role in adher-
ence are currently under investigation.
Genetic manipulation of F. nucleatum has been difficult,
presumably due, in part, to its diversified restriction endonu-
clease systems, which differ between strains and cleave DNA
irrespective of the extent of methylation (43). Attempts to
construct a fadA deletion mutant of F. nucleatum 12230 by
either electroporation or conjugation were unfruitful. This
could be attributed to one or more of the following: (i) inef-
ficient DNA delivery by electroporation or conjugation, (ii)
inefficient homologous recombination between the exogenous
plasmid and the bacterial chromosome, (iii) exogenous DNA
being digested by a restriction endonuclease(s) before recom-
bination could occur, or (iv) killing of the bacteria by electro-
poration. DNA delivery via ultrasound has been employed with
mammalian cells (4, 14, 47, 57, 64). It has been suggested that
ultrasonic treatment of mammalian cells induces transient
membrane permeability, allowing uptake of extracellular com-
pounds, such as chemotherapeutic agents, genetic materials,
and fluorescence markers, which normally do not permeate the
cell membrane (17). Although ultrasound treatment in the
presence of Optison enhances sonoporation, its mechanism is
not clearly understood (47). Our results demonstrate that the
same technology could also be applied to bacteria, even though
the bacterial cell envelope is quite different from that of mam-
malian cells. Unlike electroporation, which kills the majority of
the bacteria, ultrasonic treatment under the testing conditions
used did not affect the viability of F. nucleatum. By mixing F.
nucleatum 12230 with intact pYH1426, we intended to first
obtain a single-crossover merodiploid construct through sono-
poration and then utilize the sacB gene on pYH1426 to select
FIG. 5. RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of
primers used for RT-PCR. The 2.4-kb fadA-containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin indicates the
location of a putative transcription terminator. B. Northern blot analysis of F. nucleatum 12230 (lane 2) and F. nucleatum 12230-US1 (lane 3), using
the 359-bp fadA fragment as a probe. Lane 1, 359-bp fadA fragment (positive control). C. RT-PCR analysis of expression of ORF2 (lanes 1 and
6), fadA (lanes 2 and 7), and ORF3 (lanes 3 and 8) in F. nucleatum 12230 (lanes 1 to 3) and F. nucleatum 12230-US1 (lanes 6 to 8). Lane 4, negative
control without RNA; lane 5, 1.0-kb Plus DNA ladder.
5338HAN ET AL.J. BACTERIOL.
for a double-crossover mutant on sucrose medium (11). Sur-
prisingly, all transformants obtained were fadA deletion mu-
tants. The mechanism of this one-step double-crossover allelic
exchange is unclear and is currently under investigation. It
should be pointed out that, as a preliminary test, the concen-
trations and ratios of the bacteria, plasmid, and Optison were
empirically determined and thus may be far from optimal.
Additional work is needed to understand the sonoporation
mechanism and to optimize its conditions.
In summary, a novel adhesin, FadA, which is unique to oral
fusobacteria, was identified. It was required for F. nucleatum
attachment to epithelial cells and thus may play an important
role in Fusobacterium colonization in the host.
We are indebted to Gale B. Hill and Paul E. Kolenbrander for
generously providing fusobacterial strains.
This work was supported in part by NIH grants DE 14924 and DE
14447 and Philip Morris External Research grant to Y.W.H., NIH
grant DE 09821 to H.K.K., and start-up funds from the Department of
Biomedical Engineering, Case Western Reserve University, to C.X.D.
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FIG. 6. Growth of F. nucleatum 12230 (solid triangles and solid
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