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1 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT, USA
2 Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT, USA
Correspondence
Keith P. Mintz
Keith.Mintz{at}uvm.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), is a non-motile, Gram-negative, capnophilic bacterium associated with periodontal disease. This bacterium is strongly implicated in localized aggressive periodontitis (LAP) (Haubek et al., 2002; Yang et al., 2004) that is characterized by unexpected, rapid periodontal attachment loss (Paju et al., 2000), bone destruction (Haubek et al., 2002) and neutrophil dysfunction. LAP occurs mainly in young individuals under 35 years of age, and is particularly common in ethnic Africans (Albandar et al., 2002; Haubek et al., 2002; Paju et al., 2000). In addition to oral infections, A. actinomycetemcomitans is an opportunistic pathogen that causes serious systemic infections, such as endocarditis (Paju et al., 2000; Paturel et al., 2004), pulmonary infections and osteitis (Paju et al., 2000). Recent studies also link this micro-organism with coronary heart disease (Pussinen et al., 2005; Spahr et al., 2006).
The pathogenesis of A. actinomycetemcomitans involves multiple virulence determinants, which contribute to the development of disease. These virulence molecules include LPS (Dixon & Darveau, 2005), leukotoxin (Kolodrubetz et al., 1989; Lally et al., 1994), bundle fimbriae (Kachlany et al., 2001), as well as afimbrial adhesins. These adhesins include an epithelial cell adhesin, Aae (Rose et al., 2003), a multifunctional protein involved in cell adhesion and invasion, Omp100 (Asakawa et al., 2003), and the extracellular matrix protein adhesin A (EmaA) which mediates the interaction of A. actinomycetemcomitans with collagen (Mintz, 2004; Ruiz et al., 2006).
EmaA was described initially as an outer-membrane protein (202 kDa), encoded by a 5898 bp ORF (Mintz, 2004). Recently, we have demonstrated that emaA is essential for the formation of antennae-like surface structures, composed of multimeric EmaA molecules, which are required for collagen binding (Ruiz et al., 2006). EmaA is an orthologue of the well-characterized, trimeric autotransporter and collagen-binding adhesin YadA of Yersinia enterocolitica (Hoiczyk et al., 2000; Skurnik & Wolf-Watz, 1989). EmaA may be grouped with the oligomeric coiled-coil adhesin (Oca) family. The Oca family of adhesins shares common structural characteristics, including an N-terminal globular head domain that contains active motifs, a coiled-coil pillar-like intermediate stalk, and a C-terminal transmembrane anchor domain (or pore-forming translocator domain) that inserts into the outer membrane and facilitates the localization of the adhesin on the bacterial surface (Cotter et al., 2006; Hoiczyk et al., 2000; Roggenkamp et al., 2003).
LPS is an important outer-membrane molecule of Gram-negative bacteria, and covers 75 % of the cell surface (Caroff & Karibian, 2003). The O polysaccharide (O-PS) component of LPS, which extends from the outer membrane, is the major antigen that stimulates the host immune response. According to distinct O-PSs, A. actinomycetemcomitans is categorized into six serotypes: a, b, c, d, e and f (Kaplan et al., 2001; Nakano et al., 2000, 1998; Suzuki et al., 2000; Yoshida et al., 1999, 1998). Serotype b is strongly implicated in aggressive periodontitis (Dogan et al., 1999; Haubek et al., 2002; Yang et al., 2004). Serotype c is predominant in most populations, including ethnic Asians (Yoshida et al., 2003), Caucasians (Dogan et al., 1999; Yang et al., 2004) and Hispanics (Teixeira et al., 2006). Both b and c serotypes are found in extra-oral infections more frequently than other serotypes (Paju et al., 2000).
In this study, emaA was found to be present in all 27 strains of A. actinomycetemcomitans investigated, covering six serotypes. Genetic variations appear to be serotype-related, and translate into three different forms of the protein (full-length, intermediate and truncated), which results in both structural and biological diversity. Only the full-length and intermediate proteins form antennae-like structures associated with the outer membrane. The strong linkage between EmaA and serotype suggests an association between this autotransporter and OP-S that determines the serotype of this organism.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These strains included three American Type Culture Collection (ATCC) reference strains, four laboratory strains and 20 clinical strains isolated from subgingival plaque of a Caucasian cohort, except for two strains (BL0293 and CU1000N). BL0293 was isolated from the blood sample of a Caucasian male with endocarditis; CU1000N was originally isolated from a 13-year-old female of African descent with LAP (Kaplan et al., 2001). The serotype of all strains examined was determined by serological tests (Zambon et al., 1983), as well as analysis of partial serotype-specific sequences (Kaplan et al., 2001). All A. actinomycetemcomitans strains were stored at –80 °C, and recovered on TSBYE agar plates (3 % trypticase soy broth, 0.6 % yeast extract, 1.5 % agar) (Becton Dickinson) at 37 °C with 10 % humidified CO2. The same growth conditions were used in all the assays mentioned in this study. The original rough-form colonies (1 mm diameter, translucent, with internal star shape) from clinical samples were used for purification of genomic DNA and sequencing of the emaA gene. The smooth-colony variants (round and opaque) of clinical strains without fimbriae were obtained after five to eight subcultures in TSBYE broth (Inouye et al., 1990). In order to clearly expose these fine structures and test the collagen-binding activity without the influence of fimbriae, the smooth phenotype was used for transmission electron microscopy (TEM) and collagen-binding assays.
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). The PCR reactions were performed using an Expand High-Fidelity PCR system (Roche). The amplicons were purified using a QIAquick gel extraction kit (Qiagen). All DNA sequence analyses were performed using an ABI 3130xl genetic analyser (Applied Biosystems) in the DNA Analysis Core Facility, Vermont Cancer Center, University of Vermont.
Prediction of protein sequences.
Protein sequences were deduced based on the nucleotide sequences using the Expert Protein Analysis System (ExPASy) of the Swiss Institute of Bioinformatics (SIB) (http://www.expasy.org). If the deduced protein sequences were truncated due to substitution, deletion or insertion mutation, the longest ORF was chosen for phylogenetic analyses. The nucleotide sequences and the deduced protein sequences were aligned using Needleman–Wunsch global alignment through the European Molecular Biology Laboratory, the European Bioinformatics Institute (EMBL-EBI) (http://www.ebi.ac.uk/emboss/align/).
Subtyping of emaA DNA sequences and corresponding EmaA proteins.
The emaA sequences were subgrouped into genotypes based on the aligned DNA sequences. In addition, different forms of EmaA were predicted at the protein level, according to the deduced amino acid sequences and homology with the prototypic structural domains of EmaA reported in our previous work (Mintz, 2004).
Development of mAbs against the stalk domain of EmaA.
mAbs targeting the stalk domain of the prototypic full-length EmaA were developed. A glutathione-S-transferase (GST) fusion protein was synthesized by amplification of a 2172 bp fragment from the prototype emaA sequence (emaA-L1, Fig. 1) using primers engineered with SmaI and SalI sites: emaA1892SmaI (5'-CCCCCGGGCAGTCGTGTAGAACA-3') and emaA4144SalI (5'-ATGTCGACTATCTGCACCACCCACAA-3'). The amplicon was cloned into the pGEX-6P1 vector (Amersham). The generated GST fusion protein included a fragment of EmaA between amino acids 631 and 1354 (Fig. 3). The fusion protein was expressed in Escherichia coli (strain BL21 pLysS), and purified using glutathione Sepharose according to the manufacturer's instructions. The GST tag was cleaved by incubation with Prescission Protease (Amersham) before injection into mice for generation of the anti-EmaA mAb.
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80 % confluency, the supernatant was removed and screened against the purified target protein using an ELISA. The culture supernatants were screened for antibody activity by ELISA using the antigen purified as a maltose-conjugated fusion protein. The fusion protein was expressed in the pMal system (New England) by using the primers StalkSalIF (5'-GTCGACAGTCGTGTAGAACAAGG-3') and StalkHindIIIR (5'-AAGCTTTATCTGCACCACCCACAA-3'). Proteins were purified following the manufacturer's instructions. Positive-reacted hybridomas were subcloned by limiting dilution in 96-well plates in 100 µl RPMI S10 HT with 1 % HCF. After two rounds of subcloning and screening by ELISA, positive hybridomas were expanded and injected into mice for ascites production. Antibody was purified from ascites fluid using protein affinity chromatography (Green Mountain Antibodies).
Preparation of membrane proteins.
The total membrane protein content of A. actinomycetemcomitans was prepared as described previously (Mintz, 2004). Briefly, 200 ml stationary-phase cells was harvested and resuspended in 3.0 ml 10 mM HEPES, pH 7.4, with 1 mM PMSF and 1x Complete Protease Inhibitor Cocktail (Roche). The cells were lysed using a French press at 9000 Pa three times. The lysate was centrifuged at 100 000 g for 40 min, and the pellet was resuspended in HEPES with 2 % (w/v) SDS as the membrane fraction.
Analysis using SDS-PAGE.
Membrane protein (250 µg) from each sample was prepared in a loading buffer containing 10 mM HEPES, 2 % SDS, 5 % (v/v) β-mercaptoethanol, 2 % (v/v) glycerol and 0.05 % (w/v) bromophenyl blue, boiled for 5 min, and loaded in a 4–15 % gradient polyacrylamide Tris/HCl ready gel (Bio-Rad) and run in Laemmli buffer (Laemmli, 1970). The separated proteins were transferred to a nitrocellulose membrane, probed with the anti-EmaA mAb, and detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich). Signals were detected using the SuperSignal West Pico chemiluminescent substrate (Pierce), and visualized by exposure to film (Kodak).
TEM.
The EmaA structures were visualized using TEM of whole-cell mount preparations, as described by Ruiz et al. (2006).
Collagen-binding activity of A. actinomycetemcomitans expressing different forms of EmaA.
Collagen-binding activity was evaluated using Bornstein & Traub (1979) type V collagen from human placenta (Sigma type IX, C3657, Sigma-Aldrich) incorporated into a 3D gel matrix (Matrigel, BD Biosciences). A final volume of 50 µl, including 25 µl Matrigel, 40 µg type V collagen and TSBYE, was added to individual wells of sterile 96-well tissue-culture plates. Following polymerization at 37 °C for 2.5 h, 200 µl TSBYE was added to each well and incubated at 37 °C overnight. Mid-exponential-phase cells (5x107) were added to each well and incubated at 37 °C with 10 % CO2 for 3 h. The matrix was washed with PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.4), immersed in 200 µl TSBYE, and incubated at 4 °C overnight, which resulted in the depolymerization of the gel. A pilot study proved that all the A. actinomycetemcomitans strains used in this study survived during the overnight depolymerization at 4 °C without significant changes in cell numbers. The bacterial suspension was removed from each well, diluted and enumerated on TSBYE agar plates. Quadruplicate individual experiments were performed, with duplicate assays for each strain examined in each individual experiment. An unpaired t test was performed (GraphPad InStat version 3.05), and P<0.05 was considered statistically significant.
Phylogenetic analysis.
The emaA nucleotide sequences from different strains, as well as the translated protein sequences, were aligned using both T-Coffee (Notredame et al., 2000) and MUSCLE (Edgar, 2004) programs, with default parameters. Phylogenetic analyses were performed using three different approaches: Mr Bayes 3.1 for the Bayesian method (Huelsenbeck & Ronquist, 2001), and PROTPARS/DNAPARS and PROTDIST/DNADIST-NEIGHBOR from the Phylogeny Inference Package (PHYLIP, version 3.6) for maximum-parsimony and neighbour-joining methods, respectively (Felsenstein, 2005). The SEQBOOT and CONSENSE programs of the PHYLIP package were used for the generation of bootstrapped datasets and consensus tree reconstructions, respectively. Phylogenetic analyses were performed using both nucleotide sequences and the deduced protein sequences. YadA of Y. enterocolitica (GenBank accession no. X13882), an orthologue of EmaA, was included as an outgroup for localization of the root of emaA.
Genotyping of aae.
Aae, another A. actinomycetemcomitans outer-membrane protein, was chosen as a control to determine if the serotype-related variations found in emaA also exist in other outer-membrane autotransporters. The aae amplicon was generated using primers AAE5 and AAE3 under identical conditions, as described by Rose et al. (2003). The genotyping of the aae gene was based on the sizes of generated amplicons.
GenBank accession numbers.
The GenBank accession numbers for emaA of the strains used in this study are as follows. Serotype a: ATCC 29523 (DQ991438), IDH1062 (DQ991439), IDH2303 (DQ991440), IDH3812 (DQ991441), IDH1445 (DQ991442), IDH4139a (DQ991443); serotype b: VT1169 (AY344064), Y4 (DQ991444), JP2 (DQ991445), ATCC 29522 (DQ991446); serotype c: ATCC 33384 (DQ991447), PM73 (DQ991448), IDH2681 (DQ991449), IDH84Aa (DQ991450), BL0293 (DQ991451); serotype d: IDH1344 (DQ991452), IDH3863 (DQ991453), IDH269 (DQ991454), IDH3196 (DQ991455); serotype e: IDHd-85 (DQ991456), IDHD13Ba (DQ991457), IDH2149 (DQ991458), IDH1147 (DQ991459), IDH3095 (DQ991460); serotype f: IDHp474a (DQ991461), CU1000N (DQ991462). The emaA nucleotide sequence of HK1651 was provided by the Los Alamos Oral Pathogen Sequence Database (http://www.oralgen.lanl.gov/).
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RESULTS |
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). The genotyping was based on the aligned nucleotide sequences and the homology to the prototypic emaA reported in our previous work (Mintz, 2004). emaA was arbitrarily divided into two groups based on the sizes of the nucleotide sequences: long emaA (>5890 bp; 17 strains) and short emaA (
5040 bp; 10 strains). The major difference between these two groups was an 837 bp in-frame deletion, corresponding to the region between nucleotides 1540 and 2378 of the prototypic sequence, which was downstream of the head domain.
The long emaA group was subdivided into six genotypes, emaA-L1 to emaA-L6, based on nucleotide sequence (Fig. 1). emaA-L1, the prototype (5898 bp) of emaA, corresponded to the intact ORF that encodes the full-length protein (1965 aa, 202 kDa). emaA-L1 was found in all serotype b strains examined, which shared 99 % sequence identity. Two serotype c strains were also included within this genotype. The remaining three serotype c strains were grouped as emaA-L2, which was the result of a 4 bp insert (5'-TTAA-3') at nucleotide 154. This frame-shift mutation generated a premature stop codon within the signal sequence (Fig. 2). Excluding the insertion mutation, emaA-L1 and emaA-L2 had an almost identical sequence.
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). emaA-L3, -L4 and -L6 genotypes were associated with serotype e only. emaA-L3 (5907 bp), found in one serotype e strain, was a single ORF encoding a protein (1968 aa, 202 kDa) with a size similar to that of the prototypic EmaA. emaA-L4, found in a single serotype e strain, contained an 888 bp insertion element (IS888) within the stalk domain. This insertion element disrupted the ORF of emaA (Fig. 2). Excluding the insertion IS888, emaA-L4 was identical to emaA-L3. emaA-L6 comprised the remaining three serotype e strains. The 8 and 6 % sequence variation in the stalk and transmembrane anchor domains differentiated emaA-L6 from emaA-L3, as well as emaA-L5. However, all of them shared the identical non-prototypic head sequence (Fig. 1). In addition, a point mutation at nucleotide 389 (C
A) of emaA-L6 resulted in a premature stop codon close to the N terminus (Fig. 2).
The emaA-L5 genotype was exclusively found in serotype f, and shared 99.8 % similarity with emaA-L3. Unlike emaA-L3, which encodes an intact full-length EmaA, a single base deletion at nucleotide 479 of emaA-L5 resulted in a premature stop codon within the head domain (Fig. 2). These two serotype f strains were isolated from distinct sources (a 13-year-old African-American female and a Scandinavian Caucasian), but the emaA sequences were 99.9 % identical.
The short emaA genotypes (emaA-S1, -S2, -S3 and -S4), containing the non-prototypic head sequence, were associated with serotypes a and d. Only emaA-S1, found in all serotype d and 33 % of serotype a strains, encoded an intact ORF (Fig. 2). emaA-S2, present in 33 % of serotype a strains, contained point mutations that created a premature stop codon close to the C terminus (Fig. 2), otherwise emaA-S2 was identical to emaA-S1. emaA-S3 was identical to emaA-S1 and -S2, except for an 8 bp deletion within the signal sequence, which generated a premature stop codon within the signal sequence (Figs 1 and 2). emaA-S4 was different from the other genotypes. The signal peptide, the head domain and the first half of the stalk region were completely missing in this genotype. However, the C terminus was highly conserved throughout all genotypes (Fig. 1).
Subtyping of EmaA proteins
Three forms of the EmaA protein were deduced: full-length, intermediate and truncated (Fig. 2). The full-length EmaA was predicted to be a 202 kDa protein, which was associated with 100 % of serotype b strains, 40 % of serotype c strains, and 20 % of serotype e strains investigated. The full-length EmaA of serotype e was homologous to the prototypic EmaA of serotypes b and c in the stalk and anchor domains, but differed in the head sequence. However, the putative collagen-binding motifs were invariant within the head domain sequence (data not shown).
The intermediate EmaA contained a 279 aa deletion between the head domain and the stalk region, and was predicted to be a 173 kDa (1679 aa) protein. The predicted protein was identical to the full-length EmaA found in serotype e, excluding the deletion. One hundred per cent of serotype d and 33 % of serotype a strains were associated with the intermediate EmaA. The truncated EmaA was found in 100 % of serotype f strains, 80 % of e strains, 67 % of a strains and 60 % of c strains. The deduced protein sequences indicated premature termination of translation in the signal sequence, the head or the stalk region.
Detection of the full-length and intermediate forms of EmaA
The mAb used in this study interacted with the denatured form of the protein only, and was targeted to an epitope within the stalk region of EmaA. Two individual forms of EmaA molecules were detected from the membrane fraction isolated from the different genotypes (Fig. 3). The full-length EmaA was detected in the emaA-L1 genotype (VT1169 and ATCC 33384), while the intermediate EmaA was found in the emaA-S1 genotype (IDH3863). However, no EmaA products were detected in the membrane protein of strains with the truncated EmaA using this mAb (Fig. 3). The four strains with the truncated form of EmaA included three strains with stop codons close to the N terminus: IDH2681 (emaA-L2), CU1000N (emaA-L5) and IDHd-85 (emaA-L4); and one close to the C terminus: ATCC 29523 (emaA-S1) (Fig. 3).
Visualization of EmaA structures
Antennae-like appendages were present on the surface of strains containing the full-length and intermediate EmaA only (Fig. 4). The full-length prototypic EmaA structures expressed a rod-like stalk and an ellipsoidal head domain at the end of each stalk. In comparison, the intermediate EmaA structure appeared to be shorter than the prototypic structures. The shorter appearance of the intermediate form was associated with the 279 aa deletion downstream of the head domain. In addition, the ellipsoidal head present on the prototypic structures was not visible on the intermediate EmaA structures containing the non-prototypic head sequence.
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). The strain containing the truncated form of EmaA demonstrated little binding activity to either incorporated collagen or Matrigel alone, and in this respect was identical to the emaA– mutant.
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). The data indicated that the emaA genotypes could be grouped into two major clusters: one included emaA-L1 (serotypes b and c) and emaA-L2 (serotype c); the other included emaA-L3 (serotype e), emaA-L4 (serotype e), emaA-L5 (serotype f), emaA-L6 (serotype e), emaA-S1 (serotypes a and d), emaA-S2 (serotype a) and emaA-S3 (serotype a). However, the unique emaA-S4 (serotype a) appeared to be distinct from the above two clusters (Fig. 6). The main difference between these two lineages was the functional head domain, which contains multiple collagen-binding motifs. emaA-S4, however, was divergent from these two lineages. Further analysis, based on the predicted protein sequences of EmaA and YadA, suggested that emaA-S4 was either an allele generated by multiple mutations or a member of another evolutionary clade.
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). To determine whether the number of repeats is linked to a specific serotype, the 3 kb amplicon of the aae gene from different serotypes was determined. Four different genotypes of aae, based on difference sizes of the amplicons, were found among 14 strains covering the six serotypes. Multiple aae alleles were found associated with the serotype b strains, which contrasted with the findings for emaA. Variation in the number of repeats within aae was also found in the other five serotypes of A. actinomycetemcomitans. However, the number of repeats was not linked to a specific serotype. Therefore, the conserved emaA genotypes found associated with specific serotypes did not apply to another outer-membrane protein, Aae. |
DISCUSSION |
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; Ruiz et al., 2006). Type V collagen is ubiquitously distributed in human interstitial tissues, including periodontal ligaments (Becker et al., 1991) and the extracellular matrix of aortic media (Dingemans et al., 2000). This matrix protein is exposed in inflamed or degenerated tissue (Hillmann et al., 1998), and may become a substrate for the colonization of bacteria, consequently causing both oral and extra-oral infections.
Population genetic analyses based on emaA suggest that serotypes b and c are closely related, when compared with the remaining four serotypes, which is consistent with previous data (Haubek et al., 1995; Kaplan et al., 2002; Poulsen et al., 1994). The previous work based on DNA fingerprinting, serotypes, 16S rRNA, lkt (leukotoxin), flp-1 (major fimbriae subunit) and cdt (cytolethal distending toxin) suggests that serotypes b and c are genetically related, as are a, d, e and f. Among the latter four serotypes, a and d are phylogenetically related, and e and f are closer to each other (Kaplan et al., 2002). The phylogenetic trees based on emaA suggest that two lineages diverged during evolution, representing the two different head domains of emaA. The serotype-specific genetic heterogeneity found in emaA appears to be additional evidence for the hypothesis of the clonal population structure in the evolutionary biology of A. actinomycetemcomitans (Poulsen et al., 1994).
However, the serotype-related genetic diversity associated with EmaA is not applicable to another autotransporter adhesin, Aae (Rose et al., 2003). Aae is orthologous to the epithelial adhesin Hap of Haemophilus influenzae (St Geme & Cutter, 2000), and mediates the interaction of A. actinomycetemcomitans with epithelial cells derived from humans and old-world primates (Fine et al., 2005; Rose et al., 2003). The allele of the aae gene varies due to different numbers of a 135 bp imperfect repeat (Rose et al., 2003). Examination of this gene among the strains investigated in this study indicates that the variation in the number of repeats in aae does not segregate with bacterial serotypes, which may be a result of spontaneous mutations stimulated by periodic selection (Spratt et al., 1995). In contrast with aae, the conserved emaA genotype within each subpopulation of this organism suggests a stable clonal linkage between emaA and other virulence determinants within the same descent (Poulsen et al., 1994; Spratt et al., 1995).
Serotyping of A. actinomycetemcomitans is based on the O-PS, and these groupings suggest a correlation between the emaA and the type of LPS present on the surface of this bacterium. Interestingly, Jain et al. (2006) have observed that O-PS stabilizes the membrane structure and is involved in the transmembrane secretion machinery of some large autotransporters in Gram-negative bacilli. Therefore, the O-PS of A. actinomycetemcomitans may regulate EmaA assembly or determine the type of EmaA assembled on the surface of the bacterium.
Heterogeneity in the chromosome of emaA is recapitulated at the protein level. Three different forms of EmaA are predicted, but only two forms of the protein are associated with the membrane. The diversity in the sequence of these proteins does not interfere with the formation of surface structures or collagen-binding activity. The collagen-binding motifs in the head domain are fully conserved in the full-length and intermediate forms, in spite of the sequence variation in this region. The conservation of these motifs lends support to the hypothesis that these motifs are important for collagen-binding activity. In addition, the higher collagen-binding activity in the strain with the non-prototypic head domain suggests that the amino acid variations may alter the structure of the molecule and consequently affect collagen-binding activity. Studies are under way to determine which sequences are important for the interaction between the structure and collagen.
A sequence of about 20 aa has been identified in YadA that shows high similarity to conserved sequences in other members of the Oca protein family. This region is located between the head and stalk domains and is termed the neck sequence (Roggenkamp et al., 2003). These sequences are important to assemble and maintain the structure of this class of proteins. Three conserved neck sequences are present in the EmaA sequence (Mintz, 2004). The first two are located between the head and the stalk regions, while the third is located close to the anchor domain. In YadA, deletion of this sequence abolishes collagen-binding activity (Roggenkamp et al., 2003). However, the intermediate EmaA protein lacks the second neck sequence and still maintains binding activity. Together, the data suggest that the second neck sequence is not essential for binding activity.
The deletion and the sequence variation within the head domain most likely contribute to the appearance of the shorter EmaA structures and the apparent absence of the ellipsoidal head (Fig. 4). These structural changes may explain the higher collagen-binding activity of the intermediate EmaA strain. The differences in the proteins lend additional support to the proposal that EmaA is the structural subunit of these surface appendages.
The positive correlation between the presence of EmaA surface structures and collagen-binding activity is also supported by the comparison of strains ATCC 29523 (truncated EmaA) and IDH1062 (intermediate EmaA) (Fig. 5). These two serotype a strains share an identical emaA sequence, except for a single thymidine deletion at position 4380 bp in ATCC 29523. The deletion results in a truncated EmaA protein lacking the pore-forming transmembrane domain. The C terminus is essential for translocation, stabilization and full-level function of the trimeric adhesins, such as Hia and YadA (Cotter et al., 2006). ATCC 29523 does not express surface structures and displayed minimal collagen-binding activity. This contrasts sharply with the presence of surface appendages and the 120-fold increase in the number of cells bound to collagen demonstrated with the clinical strain IDH1062. The data imply that the C terminus of the protein is required for the assembly of EmaA structures, as proposed for the orthologous region of YadA (Roggenkamp et al., 2003; Cotter et al., 2006).
In conclusion, two forms of intact EmaA molecules are found in A. actinomycetemcomitans, and may mediate the colonization of host tissues by this organism. Incomplete translation or assembly of this oligomeric protein adhesin, as suggested by the truncated forms of EmaA, results in the loss of adhesion activity. However, we cannot exclude the possibility that these forms of the protein have alternative functions. The conservation throughout evolution of such a large protein in the most common serotypes found in human periodontitis and extra-oral infections suggests an important role for this protein or structure in the persistence of this pathogen within the human oral cavity.
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ACKNOWLEDGEMENTS |
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Edited by: M. A. Curtis
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REFERENCES |
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Asakawa, R., Komatsuzawa, H., Kawai, T., Yamada, S., Goncalves, R. B., Izumi, S., Fujiwara, T., Nakano, Y., Suzuki, N. & other authors (2003). Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans. Mol Microbiol 50, 1125–1139.[CrossRef][Medline]
Becker, J., Schuppan, D., Rabanus, J., Rauch, R., Niechoy, U. & Gelderblom, H. (1991). Immunoelectron microscopic localization of collagens type I, V, VI and of procollagen type III in human periodontal ligament and cementum. J Histochem Cytochem 39, 103–110.[Abstract]
Bornstein, P. & Traub, W. (1979). The chemistry and biology of collagen. In The Proteins, 3rd edn, vol. 4, pp. 411–632. Edited by H. Neurath & R. L. Orlando, FL: Hill Academic Press.
Caroff, M. & Karibian, D. (2003). Structure of bacterial lipopolysaccharides. Carbohydr Res 338, 2431–2447.[CrossRef][Medline]
Cotter, S. E., Surana, N. K., Grass, S. & St. Geme, J. W. (2006). Trimeric autotransporters require trimerization of the passenger domain for stability and adhesive activity. J Bacteriol 188, 5400–5407.
Dingemans, K. P., Teeling, P., Lagendijk, J. H. & Becker, A. E. (2000). Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media. Anat Rec 258, 1–14.[CrossRef][Medline]
Dixon, D. R. & Darveau, R. P. (2005). Lipopolysaccharide heterogeneity: innate host responses to bacterial modification of lipid A structure. J Dent Res 84, 584–595.
Dogan, B., Saarela, M., Jousimies-Somer, H., Alaluusua, S. & Asikainen, S. (1999). Actinobacillus actinomycetemcomitans serotype e – biotypes, genetic diversity and distribution in relation to periodontal status. Oral Microbiol Immunol 14, 98–103.[CrossRef][Medline]
Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32, 1792–1797.
Felsenstein, J. (2005). PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle.
Fine, D. H., Velliyagounder, K., Furgang, D. & Kaplan, J. (2005). The Actinobacillus actinomycetemcomitans autotransporter adhesin Aae exhibits specificity for buccal epithelial cells from humans and old world primates. Infect Immun 73, 1947–1953.
Haubek, D., Poulsen, K., Asikainen, S. & Kilian, M. (1995). Evidence for absence in northern Europe of especially virulent clonal types of Actinobacillus actinomycetemcomitans. J Clin Microbiol 33, 395–401.[Abstract]
Haubek, D., Ennibi, O., Abdellaoui, L., Benzarti, N. & Poulsen, S. (2002). Attachment loss in Moroccan early onset periodontitis patients and infection with the JP2-type of Actinobacillus actinomycetemcomitans. J Clin Periodontol 29, 657–660.[CrossRef][Medline]
Hillmann, G., Dogan, S. & Geurtsen, W. (1998). Histopathological investigation of gingival tissue from patients with rapidly progressive periodontitis. J Periodontol 69, 195–208.[Medline]
Hoiczyk, E., Roggenkamp, A., Reichenbecher, M., Lupas, A. & Heesemann, J. (2000). Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins. EMBO J 19, 5989–5999.[CrossRef][Medline]
Huelsenbeck, J. P. & Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755.
Inouye, T., Ohta, H., Kokeguchi, S., Fukui, K. & Kato, K. (1990). Colonial variation and fimbriation of Actinobacillus actinomycetemcomitans. FEMS Microbiol Lett 57, 13–17.[CrossRef][Medline]
Jain, S., van Ulsen, P., Benz, I., Schmidt, M. A., Fernadez, R., Tommassen, J. & Goldberg, M. B. (2006). Polar localization of the autotransporter family of large bacterial virulence proteins. J Bacteriol 188, 4841–4850.
Kachlany, S. C., Planet, P. J., DeSalle, R., Fine, D. H. & Figurski, D. H. (2001). Genes for tight adherence of Actinobacillus actinomycetemcomitans: from plaque to plague to pond scum. Trends Microbiol 9, 429–437.[CrossRef][Medline]
Kaplan, J. B., Perry, M. B., MacLean, L. L., Furgang, D., Wilson, M. E. & Fine, D. H. (2001). Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f. Infect Immun 69, 5375–5384.
Kaplan, J. B., Schreiner, H. C., Furgang, D. & Fine, D. H. (2002). Population structure and genetic diversity of Actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients. J Clin Microbiol 40, 1181–1187.
Kolodrubetz, D., Dailey, T., Ebersole, J. & Kraig, E. (1989). Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans. Infect Immun 57, 1465–1469.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lally, E. T., Golub, E. E. & Kieba, I. R. (1994). Identification and immunological characterization of the domain of Actinobacillus actinomycetemcomitans leukotoxin that determines its specificity for human target cells. J Biol Chem 269, 31289–31295.
Mintz, K. P. (2004). Identification of an extracellular matrix protein adhesin, EmaA, which mediates the adhesion of Actinobacillus actinomycetemcomitans to collagen. Microbiology 150, 2677–2688.
Nakano, Y., Yoshida, Y., Yamashita, Y. & Koga, T. (1998). A gene cluster for 6-deoxy-L-talan synthesis in Actinobacillus actinomycetemcomitans. Biochim Biophys Acta 1442, 409–414.[Medline]
Nakano, Y., Yoshida, Y., Suzuki, N., Yamashita, Y. & Koga, T. (2000). A gene cluster for the synthesis of serotype d-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans. Biochim Biophys Acta 1493, 259–263.[Medline]
Nørskov-Lauritsen, N. & Kilian, M. (2006). Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. Int J Syst Evol Microbiol 56, 2135–2146.
Notredame, C., Higgins, D. G. & Heringa, J. (2000). T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302, 205–217.[CrossRef][Medline]
Paju, S., Carlson, P., Jousimies-Somer, H. & Asikainen, S. (2000). Heterogeneity of Actinobacillus actinomycetemcomitans strains in various human infections and relationships between serotype, genotype, and antimicrobial susceptibility. J Clin Microbiol 38, 79–84.
Paturel, L., Casalta, J. P., Habib, G., Nezri, M. & Raoult, D. (2004). Actinobacillus actinomycetemcomitans endocarditis. Clin Microbiol Infect 10, 98–118.[CrossRef][Medline]
Poulsen, K., Theilade, E., Lally, E. T., Demuth, D. R. & Kilian, M. (1994). Population structure of Actinobacillus actinomycetemcomitans: a framework for studies of disease-associated properties. Microbiology 140, 2049–2060.[Abstract]
Pussinen, P. J., Nyyssonen, K., Alfthan, G., Salonen, R., Laukkanen, J. A. & Salonen, J. T. (2005). Serum antibody levels to Actinobacillus actinomycetemcomitans predict the risk for coronary heart disease. Arterioscler Thromb Vasc Biol 25, 833–838.
Roggenkamp, A., Ackermann, N., Jacobi, C., Truelzsch, K., Hoffmann, H. & Heesemann, J. (2003). Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA. J Bacteriol 185, 3735–3744.
Rose, J. E., Meyer, D. H. & Fives-Taylor, P. M. (2003). Aae, an autotransporter involved in adhesion of Actinobacillus actinomycetemcomitans to epithelial cells. Infect Immun 71, 2384–2393.
Ruiz, T., Lenox, C., Radermacher, M. & Mintz, K. P. (2006). Novel surface structures are associated with the adhesion of Actinobacillus actinomycetemcomitans to collagen. Infect Immun 74, 6163–6170.
Skurnik, M. & Wolf-Watz, H. (1989). Analysis of the yopA gene encoding the Yop1 virulence determinants of Yersinia spp. Mol Microbiol 3, 517–529.[Medline]
Spahr, A., Klein, E., Khuseyinova, N., Boeckh, C., Muche, R., Kunze, M., Rothenbacher, D., Pezeshki, G., Hoffmeister, A. & Koenig, W. (2006). Periodontal infections and coronary heart disease: role of periodontal bacteria and importance of total pathogen burden in the Coronary Event and Periodontal Disease (CORODONT) study. Arch Intern Med 166, 554–559.
Spratt, B. G., Smith, N. H., Zhou, J., O'Rourke, M. & Feil, E. (1995). The population genetics of the pathogenic Neisseria. In Population Genetics of Bacteria, pp. 143–160. Edited by S. Baumberg, J. P. W. Young, E. M. H. Wellington & J. R. Saunders. Cambridge, UK: Press Syndicate of University of Cambridge.
St Geme, J. W., III & Cutter, D. (2000). The Haemophilus influenzae Hia adhesin is an autotransporter protein that remains uncleaved at the C terminus and fully cell associated. J Bacteriol 182, 6005–6013.
Suzuki, N., Nakano, Y., Yoshida, Y., Nakao, H. & Yamashita, Y. (2000). Genetic analysis of the gene cluster for the synthesis of serotype a-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans. Biochim Biophys Acta 1517, 135–138.[Medline]
Teixeira, R. E., Mendes, E. N., Rogue de Carvalho, M. A., Nicoli, J. R., Farias Lde, M. & Magalhaes, P. P. (2006). Actinobacillus actinomycetemcomitans serotype-specific genotypes and periodontal status in Brazilian subjects. Can J Microbiol 52, 182–188.[CrossRef][Medline]
Yang, H. W., Asikainen, S., Dogan, B., Suda, R. & Lai, C. H. (2004). Relationship of Actinobacillus actinomycetemcomitans serotype b to aggressive periodontitis: frequency in pure cultured isolates. J Periodontol 75, 592–599.[CrossRef][Medline]
Yoshida, Y., Nakano, Y., Yamashita, Y. & Koga, T. (1998). Identification of a genetic locus essential for serotype b-specific antigen synthesis in Actinobacillus actinomycetemcomitans. Infect Immun 66, 107–114.
Yoshida, Y., Nakano, Y., Suzuki, N., Nakao, H., Yamashita, Y. & Koga, T. (1999). Genetic analysis of the gene cluster responsible for synthesis of serotype e-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans. Biochim Biophys Acta 1489, 457–461.[Medline]
Yoshida, Y., Suzuki, N., Nakano, Y., Shibuya, K., Ogawa, Y. & Koga, T. (2003). Distribution of Actinobacillus actinomycetemcomitans serotypes and Porphyromonas gingivalis in Japanese adults. Oral Microbiol Immunol 18, 135–139.[CrossRef][Medline]
Zambon, J. J., Slots, J. & Genco, R. J. (1983). Serology of oral Actinobacillus actinomycetemcomitans and serotype distribution in human periodontal disease. Infect Immun 41, 19–27.
Received 9 January 2007;
revised 9 March 2007;
accepted 1 May 2007.
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