Department of Pediatric Dentistry, The University of Texas
Health Science Center at San Antonio, San Antonio, Texas
782841 and
Oral Infection and
Immunity Branch, National Institute of Dental Research, National
Institutes of Health, Bethesda, Maryland 208922
Received 10 September 1997/Returned for modification 6 November
1997/Accepted 23 December 1997
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INTRODUCTION |
Two major fimbrial types have been
identified in strains of Actinomyces naeslundii that
colonize the oral cavity. Fimbriae designated type 1 mediate bacterial
adherence to salivary proline-rich proteins that coat the tooth enamel
(9, 18). In contrast, those designated type 2 exhibit a
lectin activity (3) that was initially detected by the
lactose-sensitive coaggregation of A. naeslundii strains
with several streptococcal strains, such as Streptococcus
oralis 34 (27), that also colonize teeth. Type 2 fimbriae also mediate bacterial adhesion to various host cells (3), including erythrocytes, epithelial cells, and
polymorphonuclear leukocytes. Activation of the latter cell type by
type 2 fimbriated Actinomyces strains results in
phagocytosis and bacterial killing (32) and the release of
mediators such as superoxide (33) that may contribute to the
initiation of gingival inflammation. Consequently, the identification
of the fimbrial lectin(s) would provide an improved understanding of
bacterium-host cell interactions. However, the nature of the type 2 fimbria-associated lectin activity, whether it is a part of the major
fimbrial subunit or a minor fimbrial component, remains unknown. A
major obstacle in distinguishing between these alternatives is the
inability to dissociate A. naeslundii fimbriae to monomer
subunits.
The lectin-like adhesins of several gram-negative bacteria have been
identified in studies of fimbria biogenesis at the genetic level
(22, 39). However, little is known concerning bacterial adhesins and assembly of fimbriae in gram-positive bacteria. The expression of both type 1 and type 2 fimbriae by A. naeslundii T14V (8) makes this strain a model system
for studies of biogenesis of fimbriae in gram-positive bacteria.
The genes that encode the structural subunits of A. naeslundii T14V type 1 and type 2 fimbriae and A. naeslundii WVU45 type 2 fimbriae have been cloned previously, and
results indicate that these genes encode proteins of approximately 54 to 59 kDa (13, 45-47). Nucleotide sequencing of the type 1 subunit of strain T14V and the type 2 subunit of strain WVU45
(47) revealed significant similarity between the encoded
proteins. These studies also showed the presence in each subunit of an
N-terminal leader and a C-terminal cell wall sorting signal, which is
common among gram-positive cell surface proteins (37). The
detection of a cell wall sorting signal in the fimbrial subunits is of
interest since individual subunits are not expected to become
covalently anchored to the cell wall peptidoglycan. The possible role
of this sorting signal in fimbrial processing and polymerization in
A. naeslundii has not been examined. Interestingly, results
from a recent study showed that mutant strains generated by insertional
inactivation of a fimbria-associated gene, orf4, 3' to the
A. naeslundii T14V type 1 fimbrial subunit gene expressed
subunits that were not assembled into functional type 1 fimbriae
(49). A comparison of unassembled to polymerized subunits
would provide insights into assembly of fimbriae.
The A. naeslundii T14V type 2 fimbrial subunit gene,
fimA, was cloned and expressed previously in
Escherichia coli from a recombinant cosmid, pAV1402
(13). This clone expressed a protein of approximately 59 kDa
that was detected with an antibody raised against type 2 fimbriae
(5). In this report, we present the nucleotide sequence of
fimA and an additional gene, designated orf365,
3' to fimA. Mutants generated by allelic replacement of either fimA or orf365 were examined for type 2 fimbria expression and fimbria-mediated adherence. The immunoreactions
of fimbrial antigens from wild-type and isogenic mutants were compared
with those of antibodies against either type 2 fimbriae from A. naeslundii T14V or a 20-amino-acid synthetic peptide prepared from
the predicted C-terminal sequence of the fimbrial subunit. The results
demonstrate clearly that expression of both fimA and
orf365 was required for the synthesis of type 2 fimbriae.
Moreover, the carboxyl-terminal peptide of the precursor fimbrial
subunit appeared to have been cleaved during assembly. To our
knowledge, the proposed posttranslational modification is a novel step
in biogenesis of fimbriae.
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MATERIALS AND METHODS |
Bacteria and plasmids.
Bacterial strains and plasmids used
in this study are described in Table 1. A
complex medium (7) or Lactobacillus-carrying medium (15) supplemented with 20 mM
D,L-threonine was used to prepare cultures of
Actinomyces strains, and Luria-Bertani (LB) (31)
was used for E. coli strains. The antibiotics (Sigma
Chemical Co., St. Louis, Mo.) used in this study were kanamycin
sulfate, streptomycin, and ampicillin, at 40, 50, and 100 µg/ml,
respectively.
Preparation of A. naeslundii T14V type 2 fimbrial
antigens.
Type 2 fimbriae were isolated from A. naeslundii 5951, a spontaneous mutant that expresses only type 2 fimbriae (Table 1). Bacteria from the stationary phase of growth were
washed with Tris HCl-buffered saline (TBS; 0.15 M NaCl, 0.02 M Tris-HCl
[pH 7.8], 0.1 mM CaCl2, 0.1 mM MgCl2, 0.02%
sodium azide) and subjected to sonication (4). Fimbriae
collected in fractions at or near the void volume of a Sephacryl S400
(Pharmacia Biotech, Inc., Piscataway, N.J.) column were purified by
fractional ammonium sulfate precipitation at 4°C as described
previously (5, 29). Edman degradation of purified type 2 fimbriae was performed as described previously (47).
The precursor subunit protein (FimA) from E. coli AV3502 was
purified by a procedure similar to that described previously (45). A sonicated extract was applied to a DEAE-Sephacel
(Pharmacia Biotech) column and eluted with a gradient of 0.05 to 0.2 M
NaCl in TBS. FimA was monitored by solid-phase immunoassay with
anti-A. naeslundii T14V type 2 fimbrial antibody and further
purified by Sephacryl S-300 (Pharmacia Biotech) gel filtration column
chromatography. Final purification of FimA was by immunoaffinity
chromatography with a column prepared with an anti-A.
naeslundii T14V type 2 fimbrial monoclonal antibody and by elution
with 3 M sodium thiocyanate. The concentration of antigens was
determined by a micro-bicinchoninic acid protein assay (Pierce,
Rockford, Il.), using bovine serum albumin as the standard.
A synthetic peptide consisting of an amino-terminal cysteine followed
by the carboxyl-terminal 20 amino acid residues
(VGSVLVARYRERKQNANLAL) of A. naeslundii T14V FimA
was synthesized on a 430A automated peptide synthesizer (Applied
Biosystems, Inc., Foster City, Calif.). The amino acid composition of
the peptide was determined by amino acid analysis as described
previously (47). For immunization, the peptide was
conjugated to keyhole limpet hemocyanin (KLH), using
sulfo-m-maleimidobenzoyl-N-hydroxysulfo-succinimide
ester (Pierce) as the cross-linker.
Antisera and immunological methods.
A rabbit was immunized
with peptide-KLH conjugate (1 mg) in Freund's complete adjuvant on day
1 and the same amount of conjugate in incomplete adjuvant on days 21, 42, and 63. The antipeptide antiserum (JC8) was obtained 1 week after
the last injection. The production of rabbit antiserum (R55) against
purified A. naeslundii T14V type 2 fimbriae and the
preparation of monospecific immunoglobulin G (IgG) fractions from these
antisera have been described previously (5, 9).
Enzyme-linked immunosorbent assay (ELISA) was performed with
flat-bottom wells of Immulon I plates (Dynatech Laboratories, Inc.,
Alexandria, Va.) that were coated overnight at 4°C with purified type
2 fimbriae (3 µg/ml), purified recombinant subunit (3 µg/ml), or
synthetic peptide (100 µg/ml). The amount of antibody bound to
adsorbed antigen was detected with a secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, Calif.). Western blotting was performed as described previously (44), using either rabbit immune IgG (1 µg/ml)
or antiserum diluted to at least 1:500. Horseradish
peroxidase-conjugated goat anti-rabbit IgG was the secondary antibody,
and blots were developed with reagents supplied in the immunoassay kit
(Bio-Rad Laboratories).
Molecular DNA manipulations.
Restriction endonuclease maps
of plasmid DNA were determined by standard methods (31).
Subcloning and construction of integration vectors were performed with
DNA fragments eluted from agarose gels with reagents from an Elu Quik
kit (Schleicher & Schuell, Keene, N.H.). Ligations and transformations
of E. coli with various constructs were performed by
procedures described previously (14, 31). The host strain
for plasmids pAV3022, pAV2621, and pAV2606 was E. coli
JM109. For plasmids pMY221, pMY2366, and pMY2201, E. coli
DH5
was the host strain, and E. coli TG1 carrying the resident plasmid pGP1-2 (Amersham Life Science Inc., Arlington Heights,
Ill.) (40) was the host strain for plasmid pAV3502. Transformants were selected on LB agar containing antibiotics, and
plasmid DNA was isolated by the alkaline lysis method and purified by
CsCl-ethidium bromide density gradient centrifugation (31).
The nucleotide sequence of A. naeslundii T14V chromosomal DNA in plasmids pAV3502 and pAV2621 was determined by the Sanger dideoxy-chain termination procedure (34), using a Sequenase kit (version 7.0; United States Biochemical Corp., Cleveland, Ohio) and
[35S]dATP (12.5 mCi/ml; DuPont England Nuclear, Boston,
Mass.). Primers for DNA sequencing and for amplification of DNA
fragments by PCR were prepared on an Applied Biosystems model 391 DNA
synthesizer. PCR was performed with either Pfu DNA
polymerase (Stratagene, La Jolla, Calif.) or Taq DNA
polymerase (Life Technologies, Inc.), using conditions similar to those
described previously (44). Nucleotide sequences were
analyzed by using the software package of the Genetics Computer Group
(version 9.0; University of Wisconsin Biotechnology Center)
(12). Sequence homology searches to other bacterial proteins
in public databases were performed with the program BLAST
(1).
Isolation and characterization of mutants.
Purified
integration plasmid DNA (100 ng) was used to transform A. naeslundii T14V by electroporation (48), and
transformants were selected on brain heart infusion agar containing
kanamycin and streptomycin. Chromosomal DNA from mutants was digested
with restriction endonucleases, separated by agarose gel
electrophoresis, and analyzed by Southern blot hybridization, under
conditions of high stringency, to various 32P-labeled DNA
probes (43). Briefly, DNA on filters was prehybridized at
42°C for 2 to 4 h in a solution containing 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 50% formamide, 10% dextran sulfate (Pharmacia Biotech), 1× Denhardt's solution, 1% sodium dodecyl sulfate (SDS), 1 M NaCl, 0.5% sodium pyrosphosphate, and 200 µg of denatured herring sperm DNA (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) per ml. Hybridizations were at the same temperature for 18 to 20 h. Posthybridization washes were with 0.1×
SSC-0.5% SDS at 65°C for 1 h with one change of buffer.
Sonicated bacterial cell extracts from A. naeslundii strains
were prepared as described previously (44). A cell extract
enriched for cytoplasmic proteins also was obtained by disruption of
washed bacteria which had been passed twice at 500 lb/in2
(equivalent to cell pressure of 8,000 lb/in2) through a
French pressure cell (American Instrument Co., Silver Spring, Md.).
Cell debris was removed by centrifugation at 16,000 rpm in a SW40Ti
rotor (Beckman Instruments, Inc., Fullerton, Calif.) for 1 h at
4°C, and the supernatant fluid was brought to 40% saturation with
solid ammonium sulfate at 4°C. The precipitated proteins were
dissolved in TBS and dialyzed extensively against TBS prior to
SDS-polyacrylamide gel electrophoresis (PAGE) (25) on 10% polyacrylamide gels and transfer to nitrocellulose.
The adherence properties of each mutant strain were assessed by the
coaggregation assay using S. oralis 34 as the partner strain
(26). Cell suspensions (5 × 108 in a final
volume of 50 µl) of A. naeslundii parent or mutant strains
and S. oralis 34 were mixed in wells of microtiter plates, and results were scored as described previously (7).
Reversibility of coaggregation was determined in the presence of 125 mM
lactose.
Nucleotide sequence accession number.
The nucleotide
sequence reported in this study was assigned GenBank accession no.
AF019629.
| |
RESULTS |
Restriction site mapping, subcloning, and expression.
A
restriction endonuclease map was determined for the recombinant cosmid
pAV1402 (13), and fimA was localized on the
inserted DNA fragment by immunological screening of various subclones
with anti-A. naeslundii T14V type 2 fimbrial antibody. The
59-kDa type 2 fimbrial subunit protein was detected by Western blot
analysis in a subclone carrying the plasmid pAV3022, which contained a 9.0-kb HindIII DNA fragment from pAV1402 (Fig.
1). Shotgun subcloning of pAV3022 after
SmaI digestion resulted in the isolation of plasmids pAV3502
(Fig. 1) and pMY221 (Table 1), which had an insertion of a 2.4-kb
SmaI DNA fragment that encoded the 59-kDa subunit protein.
Two additional derivatives, designated pAV2606 and pAV2621, which
contained BamHI DNA fragments from pAV3022 were obtained. These subclones encoded proteins of approximately 22 and 35 kDa, respectively, that were immunostained with anti-A.
naeslundii T14V type 2 fimbrial antibody. From the physical maps
of the plasmids, these proteins represented the truncated N- and
C-terminal portions, respectively, of the structural subunit (Fig. 1).
Expression of these truncated proteins was directed by the promoter of
the vector, and the A. naeslundii T14V DNA in pAV2621 was
fused in frame to the lacZ' sequence of pUC13.
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FIG. 1.
Restriction endonuclease maps of recombinant cosmid
pAV1402 and its derivatives. The size of the inserted A. naeslundii T14V DNA in each plasmid and the apparent molecular
weights of the plasmid-encoded proteins detected by immunostaining with
the anti-A. naeslundii T14V type 2 fimbriae antibody are
indicated. Selected restriction endonuclease recognition sites are
included for reference. Symbols:  , pHC79 DNA;
_____, pUC13 DNA;  , pGEM3Z DNA; and
 , A. naeslundii T14V DNA.
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Sequence analysis of fimA and a putative gene 3' to
fimA.
The nucleotide sequences of the 2.4-kb SmaI
and the 4.4-kb BamHI DNA fragments from pAV3502 and pAV2621,
respectively, were determined. The DNA sequence containing
fimA and an open reading frame, designated
orf365, is presented in Fig.
2.
Both fimA and orf365 started with an ATG
initiation codon that was preceded by a putative ribosomal binding site
(Fig. 2) (38). The fimA gene (nucleotides 482 to
2086) encoded a predicted protein of 535 amino acids, and the
amino-terminal 30+ residues of the deduced protein had properties
characteristic of a leader sequence (41). Results of Edman
degradation of type 2 fimbriae purified from A. naeslundii
T14V identified glutamate at position 34 of the predicted protein
sequence as the N-terminal amino acid. The next 29 amino acid residues
determined by amino acid sequencing were identical to the predicted
protein sequence (Fig. 2). Thus, the putative leader sequence cleavage
site of the precursor protein encoded by fimA is between
threonine and glutamate at positions 33 and 34, respectively (Fig. 2).
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FIG. 2.
Nucleotide sequence of a 3.67-kb A. naeslundii T14V chromosomal DNA region containing the type 2 fimbrial subunit gene, fimA, and a putative gene,
orf365, involved in fimbrial biogenesis. The presumptive
ribosomal binding site (rbs; underline), two inverted repeats (arrow
and dotted underline) downstream of the termination codon, TGA (*),
of fimA, the amino-terminal amino acid sequence of type 2 fimbriae (dotted underline) determined by Edman degradation, the leader
peptide processing site (upward arrow) of the subunit precursor, the
conserved cell wall anchoring motif (LPXTG; boxed), the 20-amino-acid
carboxyl-terminal sequence (thick underline) of FimA used to prepare a
rabbit antipeptide antibody, and the putative transmembrane segment in
ORF365 (open bar) are indicated. Selected restriction endonuclease
recognition sites are included for reference.
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The predicted type 2 fimbrial subunit of A. naeslundii T14V,
obtained following cleavage of the leader, consisted of 502 amino acid
residues and had a calculated molecular weight of 52,847. The
hydropathy of the predicted protein, plotted by the Kyte-Doolittle method (24), suggested a molecule that was predominately
hydrophilic except for the presence of a hydrophobic region at the
C-terminal end. The sequence of the C-terminal 43 amino acid residues
resembled that of a cell wall sorting signal (28, 36, 37)
which has been noted in many gram-positive cell surface proteins and
other Actinomyces fimbrial subunits (46, 47).
This signal comprises the consensus cell wall anchoring motif, LPXTG
(Fig. 2), followed, in sequence, by a hydrophobic domain and a
positively charged hydrophilic domain. To detect the C-terminal end of
the fimbrial subunit protein, rabbit antibody was prepared against a
synthetic peptide consisting of the C-terminal 20 amino acid residues
(Fig. 2). This antiserum reacted strongly in ELISA with the recombinant subunit protein (FimA) purified from E. coli AV3502 and with
the unconjugated synthetic peptide. Significantly, the antibody did not
react above the level of preimmune serum with type 2 fimbriae purified
from strain T14V (Table 2). In contrast,
rabbit antiserum against type 2 fimbriae from strain T14V did not react
above the level of preimmune serum with the unconjugated peptide but
reacted strongly both with the recombinant fimbrial subunit and with
type 2 fimbriae. Comparable results were obtained with purified type 2 fimbriae and recombinant subunit protein that were subjected to
SDS-PAGE and transferred to nitrocellulose. The patterns from Western
blotting with anti-A. naeslundii T14V type 2 fimbriae antibody were similar to those observed previously (13) and consisted of a characteristic ladder of high-molecular-weight proteins
in addition to a relatively weak band of monomeric subunit for type 2 fimbriae and a single band at 59 kDa for the recombinant protein. In
contrast, the antipeptide antibody did not detect bands from
transferred fimbriae but reacted strongly with the recombinant FimA
(profile not shown). Thus, epitopes associated with the C-terminal 20 amino acids predicted by the nucleotide sequence of fimA
were detected in the recombinant fimbrial subunit but not in fimbriae
isolated from A. naeslundii T14V cell surface.
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TABLE 2.
Reactions of rabbit antisera with A. naeslundii T14V type 2 fimbriae and related antigens measured
by ELISA
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Two inverted repeats were located immediately 3' of the termination
codon of fimA (nucleotides 2097 to 2134 and 2217 to 2762) (Fig. 2). The calculated free energies (17) of these
potential hairpin structures were 
35 and 49 kcal, respectively. A
predicted RNA secondary structure encompassing these repeat sequences
generated by the program FOLD (50) had an overall calculated
free energy of 137.7 kcal. This region of dyad symmetry was followed
by a putative gene, orf365 (nucleotides 2468 to 3562), that
encoded a predicted protein of 365 amino acids with a calculated
molecular weight of 39,425. Similar to the type 2 fimbrial structural
subunit, ORF365 also was predominately hydrophilic (24).
However, unlike the subunit protein, no detectable leader sequence or
cell wall sorting signal motifs were observed in ORF365. Further
analysis of ORF365 suggested that this protein contained one membrane
helix (between amino acids 238 and 255) (Fig. 2) (42).
Results of a topology prediction (with a reliability of 7 on a scale of
0 to 9, with 9 being most reliable) (30) indicated that the
N-terminal two-thirds and the C-terminal one-third of ORF365 were
located outside and inside the cytoplasmic membrane, respectively.
Sequence homology between FimA or ORF365 and other bacterial proteins
was noted only with fimbria-associated proteins from A. naeslundii T14V or WVU45 (46, 47, 49). Sequence
alignments by the program Bestfit (Genetics Computer Group) showed
significantly greater sequence similarity between FimA of strain T14V
and the type 2 fimbrial subunit of strain WVU45 (65% sequence identity and 77% similarity) (Fig. 3A)
(46) than between FimA and the type 1 fimbrial subunit
(FimP) of strain T14V (31% sequence identity and 38% similarity)
(Fig. 3B). Significant homology (40% identity and 47.5% similarity)
also was noted between the amino-terminal half of ORF365 and the
central region of the predicted protein encoded by orf4, a
type 1 fimbria-associated gene of A. naeslundii T14V
(49).
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FIG. 3.
Sequence homology between the deduced amino acid
sequences of type 2 fimbrial subunits of A. naeslundii T14V
and A. naeslundii WVU45 (A), type 2 (FimA) and type 1 (FimP)
fimbrial subunits of A. naeslundii T14V (B), and the
N-terminal portion of the protein encoded by orf365 flanking
fimA and the central portion of the protein encoded by
orf4 flanking fimP of the type 1 fimbrial gene
cluster (C). Identical (|) and conserved substituted
(:) amino acid residues are indicated. The N-terminal amino acid
(downward arrow) determined by Edman degradation of purified fimbriae
and the consensus cell wall anchoring motif (LPXTG; boxed) are
indicated.
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Construction of fimA and orf365
mutants.
Integration plasmids pMY2201 and pMY2366 were constructed
by substituting the kanamycin resistance (kan) gene from
pJRD215 (10) for the 975-bp KpnI and 600-bp
BstXI DNA fragments internal to fimA and
orf365, respectively (Table 2 and Fig. 2).
Kanamycin-resistant transformants were obtained by transformation of
A. naeslundii T14V with these plasmids. The physical maps of
representative mutants (Fig. 4) were
determined by Southern blot analysis of genomic DNA digested with
various restriction endonucleases and hybridized to various DNA probes,
including pUC13 DNA, kan, fimA, and
orf365. Results of these analyses showed that strains
MY2T-DC7 and MY2366-DC2 were generated by allelic replacement of
fimA and orf365, respectively, with the
kan gene. The lack of hybridization signal between strain
MYT2-DC7 and the 975-bp KpnI DNA internal to
fimA, and between strain MY2366-DC2 and the 600-bp
BstXI DNA internal to orf365, confirmed that each
specific DNA sequence was deleted from the respective mutant. As
expected from the insertion-and-duplication mechanism predicted by
Campbell (2), two types of single-crossover mutants were
obtained with pMY2201. Those like strains MYT2-SC8 and MYT2-SC3 each
contained a copy of the intact fimA sequence (Fig. 4). The
only single-crossover mutants obtained with pMY2366 were those like
MY2366-SC1 (Fig. 4); mutants with insertions of pMY2366 between the
BamHI site in fimA and the 5' BstXI
site of orf365 were not isolated.
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FIG. 4.
Restriction endonuclease maps of A. naeslundii wild-type strain T14V and isogenic mutants generated by
allelic replacement of fimA (strain MYT2-DC7) or
orf365 (strain MY2366-DC2) and those generated by single
crossover with the integration plasmid pMY2201 (strains MYT2-SC8 and
MYT2-SC3) or pMY2366 (strain MY2366-SC1). Only the chromosomal DNA
region flanking fimA and orf365 and selected
restriction endonucleases are included. Symbols: _____,
A. naeslundii T14V DNA; ----, pUC13
DNA; , kan gene;  , fimA;
 , orf365. The chromosomal region where plasmid
integration occurred as mediated by the Campbell insertion-duplication
mechanism is also indicated ( ).
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Roles of fimA and orf365 in biogenesis of
fimbriae.
Type 2 fimbria-mediated adherence, as determined by the
lactose-sensitive coaggregation of Actinomyces strains with
S. oralis 34, was completely abolished by allelic
replacement of fimA in strain MYT2-DC7 and by integration of
pMY2201 in fimA of strain MYT2-SC8 or MYT2-SC3. No type 2 fimbrial antigens were detected in strains MYT2-DC7 and MYT2-SC8 (Fig.
5A, lanes 2 and 4, respectively), as
shown by Western blot analysis of sonicated cell extracts with anti-type 2 fimbrial antibody. Thus, although strain MYT2-SC8 contained
a copy of fimA (Fig. 4), the lack of FimA production suggested that fimA might be part or an operon or that the
expression of genes 5' to fimA was required for
fimA expression. Some high-molecular-weight protein bands
along with the fimbrial subunit were present in strain MYT2-SC3 (Fig.
5A, lane 3). However, minor differences were noted between the
immunostained protein profile of this strain and that of the wild-type
strain (Fig. 5A, lane 1), suggesting the possibility of truncated type
2 fimbriae produced by this strain.
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FIG. 5.
(A) Composite of Western blots of sonicated cell
extracts of A. naeslundii T14V (wild type), mutant strains
MYT2-DC7, MYT2-SC3, MYT2-SC8, MY2366-DC2, and MY2366-SC1, strain 147, and strain 5951 with anti-A. naeslundii T14V type 2 fimbrial
antibody (lanes 1 through 8, respectively). Proteins were separated by
SDS-PAGE, and transferred proteins on nitrocellulose were immunostained
with anti-A. naeslundii T14V type 2 fimbrial antibody. The
apparent molecular sizes (in kilodaltons) are indicated on the left.
(B) Western blot of cell extracts of A. naeslundii mutant
strains MY2366-DC2, MYT2-SC3, 147, and 5951 and wild-type strain T14V
(lanes 1 through 5, respectively). Transferred proteins on
nitrocellulose were immunostained with rabbit anti-peptide antibody
prepared against the predicted C-terminal sequence of FimA. Arrows
indicate the 59-kDa type 2 fimbrial subunit.
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Lactose-sensitive coaggregation activity was also abolished by allelic
replacement of orf365 in mutant strain MY2366-DC2 but was
not affected by insertion of pMY2366 3' to orf365 in strain MY2366-SC1. Only the fimbrial subunit protein was detected in sonicated
extract of strain MY2366-DC2 along with immunoreactive bands of lower
molecular weight (Fig. 5A, lane 5). The latter bands were degradative
products of the subunit, as suggested previously (13). In
contrast, the immunoblot profile of sonicated extract of strain
MY2366-SC1 was similar to that observed in the wild-type strain T14V or
strain 5951, which expresses only type 2 fimbriae (Fig. 5A; compare
lane 6 to lanes 1 and 8). The subunit expressed by strains MY2366-DC2
and MYT2-SC3 was also detected by Western blotting with the antipeptide
antibody prepared against the C-terminal end of FimA. The subunit
protein appeared as a sharp narrow immunostained band with this
antibody but as a broadly stained band with the anti-type 2 fimbrial
antibody, even though equal amounts of sonicated extract were used in
both analyses. A difference in the amount of antigen detected by the
antipeptide antibody in strain MY2366-DC2 and MYT2-SC3 also was
indicated by the greater intensity of the subunit band observed with 35 and 70 µg, respectively, of sonicated extract from these strains
(Fig. 5B, lanes 1 and 2). Significantly, the antipeptide antibody did
not react with 150 µg of sonicated extracts or French press extracts
from strains 147 and 5951 and wild-type strain T14V (Fig. 5B, lanes 3, 4, and 5, respectively). However, the subunit and
higher-molecular-weight type 2 fimbrial antigens were readily detected
by Western blotting with anti-type 2 fimbrial antibody in the extracts
of strains T14V and 5951 (Fig. 5A, lanes 1 and 8, respectively). The
specificity of the anti-type 2 fimbrial antibody used in these
experiments was indicated by the absence of any reaction with strain
147, which lacks both type 1 and 2 fimbriae (Fig. 5A, lane 7; Fig. 5B,
lane 3). Thus, the unassembled subunit expressed in the mutant strain
MY2366-DC2 or MYT2-SC3 also contained the C-terminal peptide of the
precursor subunit FimA.
| |
DISCUSSION |
Results from this study demonstrate that at least two genes,
namely, the fimbrial subunit gene, fimA, and the 3' adjacent gene orf365, are necessary for the synthesis of functional
type 2 fimbriae in A. naeslundii T14V. The conclusion is
supported by the lack of expression of type 2 fimbrial antigen by the
fimA mutant MYT2-DC7 and of assembled fimbriae by the
orf365 mutant MY2366-DC2. The mutant strains, MYT2-DC7 and
MYT2-SC8, that lacked type 2 fimbriae were unable to coaggregate with
S. oralis 34, which has a receptor polysaccharide for the
type 2 fimbrial lectin (26). However, expression of
fimA alone was not sufficient for the adherence properties
observed in the wild-type strain, since mutant strains MYT2-SC3 and
MY2366-DC2, which synthesized only the fimbrial subunit, also failed to
coaggregate with S. oralis 34. In a previous study of strain
T14V type 1 fimbriae (44, 49), mutants that produced the
unassembled type 1 subunits but not type 1 fimbriae did not adhere to
proline-rich proteins that are specific receptors of these fimbriae
(18). Further studies to identify and characterize the genes
involved in biogenesis of type 1 and type 2 fimbriae should provide a
firm basis for associating the receptor binding sites of these
structures either with the structural subunits, FimP and FimA,
respectively, or with minor fimbrial proteins.
The hypothesis that orf365 is involved in the assembly of
type 2 fimbriae was supported by the following observations. First, mutants generated by allelic replacement of orf365 coding
sequence with the kan cassette expressed only monomeric
fimbrial subunit. Second, significant sequence similarity was observed
between the predicted protein encoded by orf365 and that
encoded by orf4, a gene which is located immediately 3' to
the A. naeslundii T14V type 1 fimbrial subunit gene,
fimP (49). Third, an isogenic mutant of
orf4 created by allelic replacement also expressed only unassembled fimbrial subunits (49). These similarities
suggested that orf365 and orf4 may play similar
roles in the synthesis of type 2 and type 1 fimbriae, respectively. It
is of interest that the mutant strain, MY2366-SC1, which contained
pMY2366 integrated beyond the 3' end of orf365 produced
functional type 2 fimbriae. Thus, orf365 may be the last
member of the type 2 fimbrial gene cluster(s). Indeed, analysis of the
nucleotide sequence of the chromosomal DNA region between the
SmaI site 3' to orf365 and the downstream
BamHI site (Fig. 5; note restriction endonuclease map of
strain T14V [Fig. 4]) revealed the presence of genes encoding ribosomal proteins (data not shown). Further analysis of the DNA sequence 5' of fimA may reveal additional fimbria-associated
genes, as is the case with the type 1 fimbrial gene cluster in A. naeslundii T14V (49), which consists of seven genes,
including fimP.
Striking similarities have been observed among different fimbrial types
from E. coli and related gram-negative bacteria (11, 16, 19, 23, 39). The similarities include significant homologies
between structural subunit and fimbria-associated proteins. In
addition, the organizations of genes that encode major and minor
fimbrial components, chaperone proteins, or other proteins involved in
subunit transport and control of fimbrial assembly are similar
(11, 16, 19, 23). Information gained from the sequence
analysis of fimA and its gene product in this study extends
previous observations that common characteristics also exist among
various A. naeslundii fimbrial genes. Thus, each of three
fimbrial subunit genes (type 1 of A. naeslundii T14V and type 2 of strains T14V and WVU45) encodes a precursor subunit that has
a typical leader sequence of approximately 30 amino acids. The fimbrial
subunits of A. naeslundii are generally more hydrophilic than the subunits of gram-negative bacterial fimbriae (21). At the protein level, significant sequence homology (33%) was observed
between the type 1 fimbrial subunit of A. naeslundii T14V
and the type 2 fimbrial subunit of strain WVU45 (47). A similar level of sequence homology also was observed between the fimP- and fimA-encoded proteins (Fig. 3B),
suggesting that an overall sequence identity of approximately 30% may
be expected between the structural subunits of A. naeslundii
type 1 and type 2 fimbriae. Greater homology would be anticipated
between the subunits of functionally similar fimbriae. Indeed, an
overall sequence similarity of 77% was found between the A. naeslundii T14V and WVU45 type 2 fimbrial subunits (Fig. 3A) even
though the type 2 fimbriae of these strains are only weakly
cross-reactive (6). Finally, the relative locations of
orf365 and orf4 with respect to the structural
subunit genes would favor the hypothesis that the type 1 and type 2 fimbrial gene clusters are organized similarly.
An epitope(s) associated with the predicted carboxyl terminus of the
type 2 fimbrial subunit was detected in recombinant FimA from E. coli and unassembled FimA synthesized by the orf365
knockout mutant but not in fimbriae from A. naeslundii T14V.
Thus, the C terminus of FimA must either become inaccessible to
antibody or, alternatively, be removed during assembly of fimbriae. The latter possibility is favored by the results of Western blotting of
cell extracts of strains 5951 and T14V in which FimA monomer was not
detected by antibody against the predicted C-terminal peptide of FimA
(Fig. 5B, lanes 4 and 5, respectively) but was readily detected by
antibody against type 2 fimbriae (Fig. 5A, lanes 8 and 1, respectively). In addition to these findings, the removal of the
peptide at the C-terminal end of FimA during assembly of fimbriae would
be consistent with the presence of a cell wall sorting signal in the
subunit (Fig. 2). Based on the general model for trafficking of surface
proteins in gram-positive bacteria (28, 35), it is likely
that the carboxyl terminus of the precursor fimbrial subunit may be
cleaved between threonine and glycine of the LPLTG sequence and that
the C-terminal threonine, instead of being anchored to cell wall
peptidoglycan, is linked to another subunit either directly or through
a peptidoglycan fragment that has yet to be detected in mature
fimbriae. Alternatively, the processed subunit may be transiently
associated with a cell wall protein that functions to initiate subunit
assembly. In this regard, a cell-bound nucleator protein that primes
the polymerization of E. coli curlins during pilus assembly
has been described (20). The possible existence of covalent
linkages between subunits of A. naeslundii fimbriae would
account for the inability of techniques such as SDS-PAGE to dissociate
these structures to subunits (3). Consistent with the
predicted primary sequence and experimental data, the A. naeslundii fimbrial subunit precursor would be expected to undergo
two posttranslational modifications: removal of the amino-terminal
leader sequence during export of the precursor through the cytoplasmic
membrane, and removal of the carboxyl-terminal peptide at the cell wall
anchoring sequence during subunit assembly. However, the possibility
that carboxyl-terminal peptide processing and fimbrial assembly are
independent events cannot be excluded. Clearly, further studies are
needed to define the mechanism(s) of fimbrial subunit polymerization.
The results of such studies should advance our knowledge of fimbrial
biosynthesis in this gram-positive species.
We thank Frank Robey (NIDR) for preparation of the synthetic
peptide and KLH conjugate, Bob Harr (NIDR) for technical
assistance, and Linda Lee (UTHSC at San Antonio) and Donald J. LeBlanc
(Indiana University School of Dentistry) for critical review of the
manuscript.
This study was supported by grant DE11102 awarded to M.K.Y. from the
National Institute of Dental Research.
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Abstract
Introduction
