INFECTION AND IMMUNITY, Nov. 2006, p. 6163–6170 0019-9567/06/$08.00⫹0 doi:10.1128/IAI.00857-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 11
Novel Surface Structures Are Associated with the Adhesion of Actinobacillus actinomycetemcomitans to Collagen Teresa Ruiz,1 Christopher Lenox,2 Michael Radermacher,1 and Keith P. Mintz1* Department of Molecular Physiology and Biophysics1 and Department of Microbiology and Molecular Genetics,2 University of Vermont, Burlington, Vermont Received 30 May 2006/Returned for modification 23 July 2006/Accepted 3 August 2006
Actinobacillus actinomycetemcomitans is a gram-negative, facultative, anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis and adult periodontitis and is the causative agent for other serious systemic infections. Recently, we have identified a protein, EmaA (extracellular matrix protein adhesin A), that mediates the adhesion of A. actinomycetemcomitans to collagen. The conserved sequence and predicted secondary structure suggest that EmaA is an orthologue of the Yersinia enterocolitica adhesin YadA. Electron microscopy examinations of A. actinomycetemcomitans have identified antenna-like protrusions associated with the surface of the bacterium. These structures are absent on emaA mutant strains and can be restored by transformation of the mutant strain with emaA in trans. The loss of these structures is associated with a decrease in the binding of this bacterium to collagen. The antenna-like structures are composed of a long rod that terminates in an ellipsoidal head region. The analysis of these structures using image processing techniques has provided an initial estimate of the overall dimensions, which suggests that the appendages are oligomeric structures formed by either three or four subunits. Together, the data suggest that emaA is required for the expression of novel appendages on the surface of A. actinomycetemcomitans that mediate the adhesion of the bacterium to collagen. assays (7, 23). In the nonfimbriated variants, the specific binding is mediated by nonfimbrial adhesins (1, 7, 28). Three nonfimbrial adhesins have been identified to date: Aae (28), Omp100 (1), and EmaA (21). Aae is a 130-kDa surface protein that is homologous to an epithelial cell adhesin (Hap) produced by Haemophilus influenzae (12). Aae is a member of the autotransporter family of bacterial proteins, which are characterized by a C-terminal domain that becomes integrated into the bacterial outer membrane and an N-terminal domain (the passenger domain) that is exposed on the cell surface (7, 28). Omp100 is an outer membrane protein with a predicted molecular mass of 32 kDa that migrates anomalously in sodium dodecyl sulfate-polyacrylamide gels as a 100-kDa protein (17). This protein is associated with adhesion to and invasion into epithelial cells as well as serum resistance. EmaA is an outer membrane protein with a molecular mass of ⬃201 kDa and is associated with the binding of A. actinomycetemcomitans to collagen (21). Preliminary sequence analysis of EmaA indicates that this protein belongs to a novel family of adhesins that is represented by the multifunctional adhesin of Yersinia enterocolitica, YadA (27, 34), collectively known as Oca (oligomeric coiled-coil adhesins). The Oca family of proteins includes YadA, UspA1, and UspA2 of Moraxella catarrhalis; Hia of Haemophilus influenzae; and Vomps (variably expressed outer membrane proteins) of Bartonella quintana (39). The Yersinia and Moraxella proteins oligomerize to form surface structures associated with the outer membranes of the bacteria (13). The dimensions of these structures vary depending on the mass of the individual proteins: 41 to 44 kDa for YadA (13) and 83 and 67 kDa for UspA1 and UspA2, respectively (13). Biochemical and structural studies have shown that three molecules of YadA form the structures found on the surface of Y. enterocolitica (25).
Surface proteinaceous structures, which act as adhesins, are assembled on bacterial membranes for colonization and persistence within host tissues. These structures include fimbriae (pili) and nonfimbrial adhesins. Fimbriae are long filamentous structures composed of multiple copies of either a single subunit or different subunits (32). The nonfimbrial adhesins form shorter structures using a single subunit or small oligomers of identical subunits that fold to expose the receptor binding domain at the distal end (3, 13, 15, 25, 26, 37). These structures are crucial for the adhesion of the bacterium to the requisite ligand within the host tissue including cells and extracellular matrix proteins (26). Actinobacillus actinomycetemcomitans, a gram-negative, facultative, anaerobic bacterium, is included in a clinically relevant group of microbes that includes Haemophilus, Actinobacillus, Cardiobacter, Eikenella, and Kingella. This pathogen is implicated as the etiological agent of several forms of periodontal disease and other serious systemic diseases including endocarditis (16, 38). A. actinomycetemcomitans isolates that are typically isolated from the oral cavity exhibit filamentous fimbriae, encoded by the flp gene within the tad loci, as visualized by electron microscopy (14). The fimbriated, or rough, phenotype can be converted, under laboratory conditions, to a nonfimbriated, or smooth, phenotype. The fimbriated strains autoaggregate and therefore do not display saturation kinetics in binding assays. This form was suggested to display nonspecific adhesion (6). The loss of fimbriae reduces autoaggregation, and saturation kinetics have been observed in binding
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Room 118, Stafford Hall, University of Vermont, Burlington, VT 05405. Phone: (802) 656-0712. Fax: (802) 656-8749. E-mail:
[email protected]. 6163
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RUIZ ET AL. TABLE 1. Bacterial strains and plasmids Strain or plasmid
Reference or source
Strains E. coli JM109
recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi-1 mcrA ⌬(lac-proAB) 关F⬘ traD36 proAB⫹ lacIq lacZ⌬M15兴
A. actinomycetemcomitans SUNY465 VT1169 Plasmids pDMG4 pVT1423
Genotype
Description or relative phenotype
Host for routine genetic constructions and cloned PCR products
22 22
Wild-type strain Wild-type strain; Nalr Rifr
10 22
pSU20 derivative Kanamycin gene isolated from pUC-4K in SmaI site of pBS SK(⫺) pDGM4 containing kan Low-copy-no. plasmid pQE-30 containing 1-5,910 bp of emaA pQE-30 containing entire emaA plus 500 bp upstream of start codon pKM1 containing entire emaA plus 500 bp upstream of start codon
pKM1 pMW119 pKM7 pKM8
This study 9 This study This study
pKM9
This study
In this study, we have used electron microscopy to analyze the cell surface of a smooth variant of A. actinomycetemcomitans as well as isogenic mutants with abrogated EmaA production. Investigation of the cell surface demonstrated that emaA mediates the formation of antenna-like surface protrusions that have not been described previously for this organism. The presence of EmaA surface structures is correlated with the binding of this pathogen to collagen. MATERIALS AND METHODS Bacterial strains. The A. actinomycetemcomitans strains used in this study are smooth variants derived from the clinical strain SUNY465 (Table 1). A spontaneous rifampin- and nalidixic acid-resistant strain of SUNY465, VT1169 (22), was utilized as the recipient strain in conjugation experiments. All A. actinomycetemcomitans strains were grown statically in trypticase soy broth supplemented with 0.6% yeast extract (TSBYE) in a humidified, 10% CO2 atmosphere at 37°C. The emaA mutant strain was generated by the insertion of an antibiotic cassette coding for resistance to spectinomycin into a unique HindIII site within the emaA sequence (21). The wild-type gene was replaced by the disrupted gene by allelic replacement via conjugation. Mutant strains were grown as described above and in the presence of 50 g/ml spectinomycin. The wild-type collagen binding phenotype was restored by electroporation of the mutant using a plasmid (pKM9) (see below) containing the complete emaA sequence including the endogenous promoter (GenBank accession number AY344064). This strain was grown in the presence of 50 g/ml kanamycin. Plasmid development. We restored the collagen binding phenotype of the null mutant by transformation with a replicating shuttle plasmid containing fulllength emaA and the upstream sequence. A derivative of the A. actinomycetemcomitans replicating plasmid pDMG4 (10), pKM1, was used to restore the collagen binding activity in the emaA mutant. The spectinomycin adenyltransferase (aad9) of pDMG4 was deleted by inverse PCR using primers engineered with StuI sites (5⬘ primer, 5⬘-CTAGATTATATATCATATGATC-3⬘; 3⬘ primer, 5⬘-GGGAAATATCATTCTAATTG-3⬘). The aminoglycoside phosphotransferase gene (kan) of pUC-4K (Amersham Pharmacia Biotech, Piscataway, NJ) was isolated from pVT1423 (22) by digestion with HincII and blunt-end ligated with the inverse PCR product restricted with StuI to generate pKM1. Electrocompetent Escherichia coli JM109 cells were transformed with the ligation mix and selected on LB agar plates containing 50 g/ml kanamycin. The emaA sequence including 500 bp upstream of the start methionine was cloned into pKM1 using a two-step cloning strategy. The open reading frame was amplified by PCR from chromosomal DNA using Pfu polymerase (Stratagene, La Jolla, Calif.). The primers were engineered with an SphI restriction enzyme site in the 5⬘ primer beginning at the start methionine (5⬘-ATGAATAAAGTC TTTAAACTAATC-3⬘) and an XmaI restriction enzyme site in the 3⬘ primer
initiated at the stop codon 5,910 bp downstream of the start methionine (5⬘-G GAATAAGCGCATTTTACCA-3⬘). The PCR product was isolated, restricted with the appropriate restriction enzymes, and ligated with the low-copy-number plasmid pMW119 (9) digested with the same enzymes. Following transformation into E. coli JM109 cells and recovery of the plasmid, the 6-kbp insert was excised with SphI/XmaI and cloned into the expression plasmid pQE-30 (QIAGEN Sciences, Valencia, Calif.), generating pKM7. The emaA sequence contains a unique KpnI site 1,140 bp downstream of the start methionine. This site was incorporated into a 2,525-bp PCR product corresponding to 500 bp upstream and 2,025 bp downstream of the start methionine of emaA. An SphI restriction enzyme site was engineered into the 5⬘ primer (5⬘-AACAAATCGCCGTCCAT CGCC-3⬘), and the reverse primer (5⬘-GACTGCTAAATTCTTTCCTGCC-3⬘) was used with chromosomal DNA in the presence of Pfu polymerase to amplify the DNA sequence. The PCR product was restricted with SphI/KpnI, and a 1,600-bp product was isolated and ligated with pKM7 restricted with the same enzymes, generating pKM8. The construct containing the putative promoter and emaA was digested with SphI and SmaI to release the 6.5-kbp product and ligated with the shuttle plasmid pKM1, restricted with SphI and HincII, to generate pKM9. A. actinomycetemcomitans was transformed with the purified plasmid by electroporation (32a), and transformants were selected on TSBYE agar plates containing 100 g/ml kanamycin. Collagen binding assay. Quantitation of the interaction of A. actinomycetemcomitans with collagen was determined by an enzyme-linked immunosorbent assay described previously (21). Briefly, type V collagen (Sigma Chemical Co., St. Louis, Mo.) dissolved in 0.5 N acetic acid was diluted in carbonate coating buffer (pH 9.6), and 1 g of protein was adsorbed to the bottom of individual wells of a 96-well microtiter plate by incubation at 4°C overnight. The protein solution was removed, and bovine serum albumin (BSA) (10 mg/ml in 10 mM sodium phosphate, 0.15 M NaCl, pH 7.4) was added to block any uncoated plastic. Log-phase bacteria (2 ⫻ 107 cells) in growth medium were added to the proteincoated wells and incubated at 37°C for 1 h. The bacteria were removed, the wells were washed with phosphate buffer, and anti-A. actinomycetemcomitans antibodies (purified immunoglobulin fraction) diluted in 1% BSA–phosphate buffer were applied for 1 h at room temperature. Following washing with buffer, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Jackson Laboratory, Bar Harbor, Maine) diluted in 1% BSA–phosphate buffer was added and incubated for 1 h at room temperature. The immunoglobulin complexes were detected by the addition of hydrogen peroxide in the presence of o-phenylenediamine, and color development was determined by the absorbance at 490 nm. Electron microscopy. A small aliquot (5 l) of the bacterial suspension was diluted in phosphate-buffered saline (PBS) (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) to a concentration that provided good coverage on the grid and was applied to 400-mesh copper grids coated with a thin carbon film. The grids were first washed by gently streaming several drops of PBS over the grids. They were subsequently negatively stained by running a few drops of a stain (either 1%
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FIG. 1. Sequence comparison of the putative membrane anchor domain of EmaA. The 61 carboxyl-terminal amino acids of EmaA were aligned with the corresponding regions of YadA (31) and HiaA (2). The boldface lines indicate identical amino acids, and dashed lines represent conserved amino acids.
uranyl acetate [UA; Ted Pella, Redding, Calif.], 2% phosphotungstic acid, pH 7, with NaOH [PTA; Ted Pella], 2% ammonium molybdate, pH 7, with NH4 [Ted Pella], 2% methyl amine tungstate [MAT; Nanoprobes, Yaphank, NY], or Nanovan [Nanoprobes]) over them. The last drop was left on the grid for 30 s. Finally, the excess liquid was wicked off, and the grids were quickly air dried. The grids were observed using a Tecnai12 Philips electron microscope (FEI Company, Hillsboro, Oreg.) equipped with a LaB6 cathode operated in point mode (Kimball International, Jasper, Ind.) and a 14-m 2,048- by 2,048-pixel charge-coupled-device (CCD) camera (TVIPS, Gauting, Germany). The microscope was run under conditions identical to those that have been used in the past to obtain images that show Thon rings beyond 0.9-nm resolution in vitreous ice preparations (30). Images were recorded at an accelerating voltage of 100 kV and nominal magnifications in the range of ⫻40,000 to ⫻70,000 under low-dose conditions on either film (S0-163; Kodak) or the CCD camera. Image processing. Negatives were scanned with a 7-m raster size on a Zeiss flatbed scanner (Intergraph, Madison, Ala). The images were converted to SPIDER format and reduced by binning two times to a final pixel size on the image of 0.27 nm (nominal magnification, ⫻52,000). The appendages were boxed from the images using the command “helixboxer” in EMAN (18). The stalk regions were extracted from these boxes and processed to determine their diameters. In a first analysis, the straightest stalks were selected and subjected to high-pass and low-pass filtrations before the density profile was calculated (the density across the filament axis of the appendage was projected onto the short axis) using different commands in SPIDER (8). For a more accurate analysis of the diameter, stalk images were cut into smaller segments (64 by 64 pixels) using “boxer” in EMAN. The new images were aligned to the projection of a model cylinder. Rotational alignment was carried out using self-correlation functions (35), followed by translational alignment perpendicular to the cylinder axis only. The aligned images were analyzed by a self-organizing map algorithm (19) to check for consistency. All nodes in the resulting output map of the neural network showed centered rods with similar diameters. The only major differences were the surrounding stain distributions. A rotationally symmetrized three-dimensional reconstruction was calculated from the average of the aligned projections.
Sequence analysis of EmaA. The gene coding for EmaA predicts a 1,965-amino-acid protein. Results of a BLAST search of the complete protein did not reveal any significant sequence identity to non-A. actinomycetemcomitans proteins. However, the C-terminal sequence of EmaA (positions 1906 to 1965) contains a protein domain (E value of 6e⫺06) (20) conserved among the Yersinia enterocolitica adhesin YadA (GenBank accession number Q56930) (31) and other members of this family of adhesins, e.g., the Haemophilus influenzae adhesin Hia (2) (Fig. 1). The C-terminal 9 amino acids consist of alternating hydrophobic residues ending in F or W, which comprise a targeting motif for the outer envelope of gramnegative bacteria (27, 33). Scrutiny of the EmaA and YadA sequences revealed the presence of additional similarities between these two proteins. A schematic representation of the cognate sequences (Fig. 2A and B) indicates that these two proteins share common motifs or protein domains, although the proteins vary greatly in mass (⬃201 kDa for EmaA versus ⬃44 kDa for YadA). A signal sequence is present in both proteins, as predicted by the SignalP program (24). The signal peptidase cleavage site of EmaA is predicted to be between amino acids 56 and 57. The pentameric collagen binding consensus sequence, SVAIG, present in YadA is also present in the EmaA sequence. Twelve derivatives of this sequence are clustered in the amino terminus of the EmaA protein. Adjacent to the collagen binding domain of YadA is a unique 10-amino-acid sequence, TDAVNVGQLD, or “neck” sequence that is highly conserved in the Oca family of adhesins (13). Three of these “neck” sequences are present in the EmaA protein. The first sequence (TDAVNVAQLK) is adjacent to the first 10 collagen binding sequences (Fig. 2). Proximal to the first neck sequence are two additional collagen binding sequences followed by a second neck sequence (TDA VNGSQLF). The third sequence is located close to the carboxyl terminus of the protein (TDAVNMSQLQ). The remainder of the protein is divided into the stalk region and the membrane anchor domain. The stalk region of EmaA is approximately 1,400 amino acids long. A sequence within the stalk region, predicted to form a coiled-coil structure, is
FIG. 2. Schematic representation of the protein sequences of YadA and EmaA. The domains are delineated based on the information described previously by Hoiczyk et al. (13). SP, signal peptide; Head, collagen binding domain; NS, neck sequence; Stalk, coiled-coil region; AD, membrane anchor domain.
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FIG. 3. Micrographs of A. actinomycetemcomitans wild-type strain. Left panel, micrograph stained with uranyl acetate; center panel, micrograph stained with phosphotungstic acid; right panel, micrograph stained with methyl amine tungstate. Scale bar, 100 nm.
present in the molecule. An additional predicted coiled-coil structure sequence is found adjacent to the membrane anchor domain of the protein, which is 61 amino acids long. Detection of surface structures on A. actinomycetemcomitans. We prepared negatively stained samples of the wild-type (VT1169) strain of A. actinomycetemcomitans using five different staining solutions to visualize the bacterial surface: UA, PTA, 2% ammonium molybdate, pH 7, with NH4, MAT, and Nanovan. Although we obtained good results with preparations in UA, PTA, and MAT (Fig. 3), PTA provided the best combination of contrast and stain depth (Fig. 4, top) to visualize the bacterial surface. All stain preparations showed highly complex bacterial surfaces, and in some instances, membranous extensions on the surface and small vesicles in close proximity to the bacterium were observed. In addition, the bacteria presented antenna-like appendages protruding from the cell surface; however, these appendages do not seem to achieve a high degree of surface coverage. Electron microscopy preparations of isogenic mutants (emaA mutant strain) also showed similarly convoluted surfaces. However, this mutant lacks the antenna-like protrusions that are clearly visible on the surface of the wild-type strain (Fig. 4). Partial surface regions were recorded on the CCD camera, and whole bacteria were imaged on film, which shows larger sample areas at the same magnification. After inspecting a large number of bacteria, all the data confirmed that there are no protrusions on the surface of the emaA mutant. The antenna-like appendages are composed of a long rod or stalk, which terminates in an ellipsoidal head region (Fig. 5). A bend is also usually present within the stalk domain, which suggests that these structures are not rigid but show a certain degree of flexibility. We have characterized the dimensions of the different regions of the structures. The ellipsoidal head domain can be defined by three different diameters (d): d1 was 2.8 ⫾ 0.3 nm, d2 was 3.1 ⫾ 0.3 nm, and d3 was 4.6 ⫾ 0.3 nm. In some cases, the domain looks more globular, depending on the orientation of the structures on the carbon surface. The distance from the end of the head domain to the bend in the stalk was very conserved in all our images: l1 was 24.6 ⫾ 0.9 nm. The total length (l) of the stalk is difficult to measure in electron microscope images, as these are projections, and it can
only be assumed that the longest appendage observed is parallel to the imaging plane. The longest stalk region had a length, lT, of 150 nm. The initial analysis of the stalk density along the filament axis projected onto the short axis of the straightest stalks showed that the filaments are not hollow. An initial estimate of the stalk diameter, ds, of 3.8 ⫾ 0.3 nm (Fig. 6) was obtained
FIG. 4. Micrographs of A. actinomycetemcomitans stained with phosphotungstic acid (pH 7). Top, wild-type strain; bottom, EmaA mutant. Scale bar, 100 nm.
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FIG. 5. Left, antenna-like structures extracted from micrographs of the wild-type strain stained with phosphotungstic acid (pH. 7). Scale bar, 10 nm. Right, model structure of the EmaA appendages showing the dimensions obtained from electron micrographs. Model dimensions are not drawn to scale.
from the density profile. This value was calculated by assuming that the stalk ends had a density equal to the average density value of the background of the image. As mentioned above, these structures are flexible, and it was difficult to obtain a large data set for straight, long stretches of stalks to calculate profiles. To obtain a more accurate value for the diameter and use a larger number of stalk images, we cut the stalks into smaller segments (17.3 nm in length) (Fig. 7). Smaller segments have a higher probability of being straight for filaments of the same persistence length. All these segments (185 seg-
FIG. 6. Top, boxed stalk regions of the antenna-like structures extracted from micrographs of the wild-type strain stained with phosphotungstic acid (pH. 7). Scale bar, 10 nm. Bottom, the density profile across the filament axis of the appendage was projected onto the short axis.
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FIG. 7. (A) Short boxed stalk regions of the antenna-like structures extracted from micrographs of the wild-type strain stained with phosphotungstic acid (pH. 7). Scale bar, 10 nm. (B) Average after translational and rotational alignment of the short stalk regions. Scale bar, 4 nm. (C) Density profile across the filament axis of the average was projected onto the short axis. (D) Volume calculated using the aligned short stalk regions. Scale bar, 4 nm. (E) Stalk models for four ␣-helix coiled coils and three ␣-helix coiled coils.
ments) were aligned translationally and rotationally to a reference cylinder centered in the image box. The segments that did not align well were discarded before an average was calculated to increase the signal-to-noise ratio of the data (Fig. 7). The diameter of the average stalk segment was 4.1 ⫾ 0.3 nm. Additionally, we calculated a three-dimensional volume by assigning angles randomly to the aligned stalk segments. This volume also had a ds of 4.1 ⫾ 0.3 nm. Transformation of the isogenic mutant, lacking the surface structures, with emaA on a replicating plasmid restored the antenna-like appendages onto the surface of the bacteria. Our observations indicate that while in some cases the surface coverage in the complemented mutant was similar to that in the wild type, in other cases, the structures were more abundant than in the wild type strain (Fig. 8). The increase in the number and density of structures seen on the surface of this complemented strain is most likely due to the number of copies of the replicating plasmid inside the bacterium. Collagen binding is associated with surface protrusions. The collagen binding activities of the wild-type and isogenic mutant strains were assessed to determine the correlation between the presence of surface structures and collagen binding. Consistent with the hypothesis that the protrusions are associated with collagen binding, inactivation of emaA (loss of structures) resulted in a 50% reduction in bacteria binding to collagen compared with the wild-type strain (Fig. 9). Transformants of the isogenic mutant strain with the empty plasmid displayed the same reduction in binding to collagen as the mutant strain. However, transformation of the emaA mutant strain with the plasmid containing emaA restored and exceeded the binding activity compared with the parent strain. The collagen binding activity of the complemented emaA mutant exceeded the binding by the wild type strain by ⱖ40%. The increase in collagen binding correlates with the increase in the number of structures on the surface of the complemented bacterium (Fig. 8).
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FIG. 9. Collagen binding activity of wild-type and isogenic mutants: the isogenic emaA mutant containing the empty plasmid (emaA mutant/pKM1) and the mutant transformed with the plasmid containing emaA (emaA mutant/pKM9). The data are expressed as percentages of wild-type binding.
FIG. 8. Micrographs of A. actinomycetemcomitans emaA mutant strain complemented with the emaA gene. Micrographs were stained with phosphotungstic acid. Scale bar, 100 nm.
DISCUSSION We have visualized novel structures on the surface of the oral pathogen A. actinomycetemcomitans. These structures are associated with the presence of emaA and mediate the interaction of A. actinomycetemcomitans with extracellular matrix components (e.g., collagen). However, our results do not preclude the existence of additional collagen adhesins on the surface of A. actinomycetemcomitans. The emaA predicted protein sequence shares little sequence identity with any known adhesins of A. actinomycetemcomitans (Aae and Omp100) or proteins present in the NCBI protein database (21). The homology search revealed that EmaA shares a high level of structural similarity with extracellular matrix adhesins, which are members of the Oca family (e.g., YadA, UspA1, UspA2, HiA, and Vomps). These adhesins represent a novel class of autotransporter proteins that adopt a trimeric protein conformation and remain anchored to the surface of the bacterium after secretion (24). EmaA contains all the conserved features and structural elements present on the Oca family of adhesins: (i) an N-terminal Sec-dependent secretion signal, (ii) a head domain consisting of 14-amino-acid degenerate repeats, (iii) highly conserved neck regions, (iv) a stalk domain with regions of high probability of coiled-coil formation, and (v) a C-termi-
nal membrane anchor domain (13). EmaA can thus be considered a novel adhesin expressed by A. actinomycetemcomitans. Synthesized proteins are targeted to the inner membrane for translocation into the periplasmic space via a signal peptide that is typically composed of 15 to 20 amino acids (36). The putative signal sequence of EmaA is unusually long, containing 56 amino acids. This is twice the size of the homologue sequence in YadA (25 to 26 amino acids). Extended signal sequences have been observed only in the TpsA and Oca family members of the type V secretion system (4). In addition, these extended sequences appear to be associated exclusively with autotransporter proteins larger than 100 kDa (11). The biological significance of these highly conserved, atypical sequences is still unknown. Adhesins of the Oca family that mediate the specific interaction with epithelial cells and extracellular matrix proteins oligomerize to form surface structures anchored with the outer bacterial membrane. These structures are seen as proteinaceous protrusions in electron microscope images. We have visualized antenna-like structures on the surface of A. actinomycetemcomitans that are dependent on emaA (Fig. 4). These structures could not been seen on the emaA mutant but could be restored by transformation of emaA in trans. The EmaA structures are sparsely distributed over the surface of the wildtype strain compared with the densely covered surface protrusions seen on Yersinia and to a lesser extent in Moraxella (13). The number of structures and their distribution may be dependent on bacterial growth conditions. In addition, we have observed that the membrane surfaces are not smooth as in other gram-negative bacteria (e.g., Escherichia coli, Salmonella enterica serovar Typhimurium, and Y. enterocolitica) but present many folds, similar to those observed in M. catarrhalis. It remains to be confirmed if the presence of these rough, complex membrane surfaces is related to the ability of the bacteria to form and extrude vesicles into the media. We carried out all our studies using at least three different negative stains (UA, PTA, and MAT) to avoid any experimental artifacts caused by the staining procedure. Uranyl ions carry a positive charge at pH 4.0, phosphotungstic ions are negative at physiological pH, and methylamine tungstate is a very weakly ionic stain that has almost no buffering capability. This
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combination of stains was selected to discriminate and avoid any possible effects that might arise from ionic interactions between the stains and the samples. A similar combination was successfully used to characterize the central channel of the bacterial flagella of S. enterica serovar Typhimurium (29). In addition, most bacterial samples are prepared as suspensions in PBS. This buffer has a tendency to react with heavy metals in the staining solution, rendering the electron microscopy samples useless for structural determinations. A few laboratories wash their bacterial preparations on the electron microscope grid with a drop of double-distilled water prior to negative staining to avoid precipitation. It is possible that the osmotic shock and the drop in pH to which the samples are exposed during this procedure might disturb the stability of the preparations and might give rise to structural artifacts. We achieved successful results that can be obtained even with the highly reactive uranyl acetate stain if several stain drops (6 to 8 drops) are allowed to run freely on the surface of the grid before blotting. This technique removes any buffer/stain precipitates that might have developed and results in high-quality images that can be used for structural analysis. The three-dimensional structure of YadA suggests that the protein sequence may be divided into three domains, head, stalk, and membrane anchor domain, which are reflected in the secondary structure of the protein. The whole EmaA structure is at least 155 nm long, while YadA protrudes from the membrane surface by 23 nm and UspA1/UspA2 protrudes by at least 65 nm. The size of the head domain (approximately 5.5 nm) and the size of the membrane anchor domain (approximately 60 amino acids) are very similar for all three adhesins. Thus, the length of the stalk or rod-like segment of the EmaA protrusions is much longer. The differences in the length of the YadA and UspA1/UspA2 structures are attributed to the differences in the coiled-coil or stalk region (13). Hence, the difference in the length of the EmaA structures is most likely accounted for by the extended stalk region of EmaA compared with YadA or the UspA1/UspA2 proteins. The stalk region of the EmaA structure is comprised of approximately 1,400 amino acids, while those of UspA and YadA are approximately 350 and 120 amino acids long, respectively. Presently, we can confidently say only that the stalk region of EmaA is at least 150 nm long, because electron microscope images are projections. The exact length will become available only as a result of the electron tomography experiments that are currently in progress. The head domain represents the N-terminal portion of the molecule and contains the biologically active moiety of the protein, which mediates the adherence to eukaryotic cells or extracellular matrix proteins (27). The collagen binding consensus sequence, NSVAIGXXS, or derivatives are repeated multiple times in both YadA and EmaA. These sequences are required for the binding of YadA to collagen (34). Initial experiments indicate that the homologous EmaA sequences are important for the interaction with collagen (unpublished data). Twelve derivatives of this sequence are clustered in the amino terminus of the EmaA protein, in comparison with eight derivatives located within the first half of the YadA molecule. The volume occupied by the head domain was calculated to be 138 nm3 using the averaged dimensions obtained from analyzing the EmaA images from different stains. This volume is very
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close to the 130 nm3 calculated for the YadA structures (13). Thus, it is possible that not all of the 12 putative collagen binding domains are contained in the head region. Moreover, the first neck region sequence (TDAVNVAQLK) is located after the 10th collagen binding sequences in the same relative position as in the YadA sequence. The two additional collagen binding sequences are flanked by a second neck sequence. The dimensions extracted for the head and “bend” regions from the micrographs correlate well with a putative head region that contains the first 10 collagen binding motifs and a putative “bend” region located between the two first neck sequences. The role of these sequences in the formation of the EmaA structures on the surface of A. actinomycetemcomitans awaits high-resolution structural analysis. The members of Oca family form oligomers at the bacterial surface (13). We know that EmaA also forms oligomeric structures because it is not possible to visualize a single long ␣-helix attached to the bacteria by electron microscopy. However, the question that remains is how many EmaA subunits are forming the antenna-like appendages that we have observed in electron microscopy images. The volume deduced for the head domain could accommodate four subunits using a partial specific volume of 0.73 cm3/g. However, a similar composition was first inferred for YadA, and higher-resolution structural data has confirmed that the number of subunits in the oligomer is only three (25). We have additionally analyzed the diameter of the stalk region by projecting the density along the filament axis of the appendage onto the short axis using both long and short stalk regions. A comparison of the diameter of the molecule obtained by this method with the known diameters of three-, four-, and five-stranded coiled coils should allow us to deduce the number of EmaA subunits in the structures. The results obtained are not conclusive but have narrowed the number of subunits to either three or four. We had expected to obtain diameter values close to 3.0 nm or 4.6 nm, which would have defined the number of subunits to three or four, respectively. However, this was not the case. There are always inherent inaccuracies when the threshold density value for electron microscopy data is determined, even in cases where averages and three-dimensional reconstructions are calculated in the same fashion as the analysis presented here. The putative membrane anchor region of EmaA contains 61 residues, while the homologue regions of YadA and HiaA contain 70 and 76 residues, respectively. Therefore, it is safe to assume that each subunit can form four -strands. The carboxyl terminus of YadA is proposed to form a -barrel pore, which, after trimerization, consists of 12 -strands. EmaA probably also forms a -barrel pore consisting of 12 -strands if the active molecule is a trimer. Together, all of the data presented in this report are consistent with the hypothesis that the structures found on the A. actinomycetemcomitans cell surface are composed of oligomers of EmaA. Studies are in progress to confirm this hypothesis. The structures visualized in this study represent the first identification of a cell surface structure associated with a nonfimbrial adhesin of A. actinomycetemcomitans mediating collagen binding. YadA mediates activities ranging from host cell binding to the protection of the bacterium against complement (reviewed in reference 5). The structural and functional homology of EmaA to the members of the Oca family indicates
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