Department of Microbiology and Molecular Genetics, Rm 110 Stafford Hall, University of Vermont, Burlington, VT 05405, USA
Correspondence
Keith P. Mintz
Keith.Mintz{at}uvm.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the complete A. actinomycetemcomitans emaA sequence reported in this paper is AY344064.
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INTRODUCTION |
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Actinobacillus actinomycetemcomitans is a Gram-negative facultative anaerobic bacterium that colonizes the human oral cavity and upper respiratory tract. This bacterium is strongly associated with periodontitis in young individuals and with cases of adult periodontitis (Slots et al., 1980; Slots, 1982
; Slots & Listgarten, 1988
; Zambon, 1985
). This pathogen has been associated with other serious human infections, including infectious endocarditis, soft-tissue abscesses and pneumonia, and it may contribute to cardiovascular disease (Beck et al., 1996
; Haraszthy et al., 2000
; Kaplan et al., 1989
). The periodontium is believed to be the source for these non-oral diseases, but little is known about the tropism used by A. actinomycetemcomitans to colonize the oral cavity and to infiltrate and disseminate in tissues.
Pathogens have evolved diverse strategies to be successful in colonization of host tissues. A common theme amongst these pathogens is the ability to initiate infection by adhesion to specific host macromolecules under stringent or hostile conditions (Finlay, 1990). These molecules include the proteins, secreted by host cells, that form the extracellular matrix (ECM). The ECM is a biologically active tissue composed of a complex mixture of macromolecules, including multiple collagen types, fibronectin, laminin and glycosaminoglycans. The ECM not only serves a structural function but also affects a number of cellular activities, including migration, proliferation and differentiation. ECM proteins that have been described to act as a substrate for bacterial adhesion include collagens, laminin, fibronectin, fibrinogen, vitronectin and heparan sulfate (Patti et al., 1994
). A large number of intracellular and extracellular human pathogens adhere to mammalian ECM proteins and have been shown to contribute to the virulence of the micro-organism (Patti et al., 1994
; Westerlund & Korhonen, 1993
).
A. actinomycetemcomitans invades and migrates through epithelial cells (Fives-Taylor et al., 1999), and is eventually found in contact with collagen fibres of connective tissue (Carranza et al., 1983
; Gillett & Johnson, 1982
). Previously, we have demonstrated the adhesion of A. actinomycetemcomitans to fibrillar collagens, with the greatest affinity for type V collagen and fibronectin. In the present study, we describe the generation and screening of a transposon mutant library to isolate variants of A. actinomycetemcomitans with altered binding to collagen and fibronectin. Mutants deficient for adhesion to each ECM protein were identified, showing that independent adhesins mediate the binding of this bacterium to collagen and fibronectin. A mediator required for collagen adhesion, EmaA (extracellular matrix protein adhesin), was identified and described. Based on the protein sequence, EmaA is predicted to be structurally related to the collagen-binding protein YadA of Yersinia enterocolitica (Roggenkamp et al., 2003
), and is likely to directly mediate adhesion to collagen.
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METHODS |
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The frequency of transposition was calculated by the number of spectinomycin-resistant cells divided by the total number of cells used in the conjugation mixture.
Detection of A. actinomycetemcomitans mutants altered in binding to ECM proteins.
The transposon library was replica plated to microtitre plates containing 250 µl TSBYE and incubated for 3 days. The bacteria were dispersed by pipetting and the bacterial growth determined by measurement of OD495. Bacterial suspension (100 µl) was transferred to microtitre plates previously coated with either human type V collagen (Sigma) or fibronectin (ICN Pharmaceuticals) and blocked with BSA (1 %) in Tris-buffered saline (TBS: 20 mM Tris, 150 mM NaCl, pH 7·4). Bacterial adhesion was detected by ELISA, as described previously (Mintz & Fives-Taylor, 1999). Following incubation of the bacteria for 2 h at 37 °C, the wells were washed with TBS, and purified polyclonal antisera, raised against the whole bacterium, in TBS containing 1 % BSA, were added to detect bound bacteria. The antibodies were incubated for 1 h at room temperature and the wells washed with TBS containing 0·1 % Tween 20 (TBST). The immune complexes were located by the addition of horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulins (Jackson Laboratories) in TBS containing 1 % BSA. Following incubation for 1 h, the wells were washed with TBST, and the immune complexes were detected by the addition of hydrogen peroxide and o-phenylenediamine in 80 mM citrate phosphate buffer, pH 5·0. The reaction was allowed to proceed and stopped by the addition of 4 M sulfuric acid. The absorbance values were recorded using an EL211s microplate reader (Bio-Tek). The ratio of the individual absorption reading divided by the mean absorption reading of all the wells was used to assess bacterial binding in the initial screens.
The strains from the initial screen that displayed differences in binding to human collagen and/or fibronectin were isolated, purified and screened again as follows. Mutant and parent strains were streaked for isolation and a single colony was inoculated into broth cultures and incubated overnight. The cultures were diluted 1 : 10 and allowed to grow for an additional 3 h under normal culture conditions. The bacterial cell density was calculated from an established standard curve of the parent strain, correlating OD495 with colony forming units. Equal numbers of bacteria from each strain were added to the wells in a final volume of 100 µl TSBYE. Assays were performed in triplicate, as described above, and repeated a minimum of three times.
Southern analysis.
Strains were grown in TSBYE containing 50 µg spectinomycin ml1, and DNA was isolated using the Puregene DNA extraction Kit (Gentra Systems). Chromosomal DNA was restricted with EcoRI and the fragments separated on a 0·7 % agarose gel in TAE buffer. The DNA fragments were transferred to Hybond nylon membranes (Amersham Life Sciences) and the membranes were treated following the method of Sambrook et al. (1989). The membranes were hybridized with DNA probes conjugated with horseradish peroxidase, using the conditions suggested by the manufacturer (Amersham Life Sciences). Hybridizing fragments were visualized using the ECL detection system (Amersham Life Sciences) and exposure to photographic film (XAR-5, Eastman Kodak).
Determination of transposon integration sites within the chromosome of A. actinomycetemcomitans.
The integration site of the transposon within the A. actinomycetemcomitans chromosome was determined by DNA sequencing of inverse PCR products. Total chromosomal DNA was extracted as described above and restricted with AluI or BanI. The enzymes were inactivated by heating, and the DNA fragments ligated by the addition of T4 DNA ligase. A portion of this material was used as the template for PCR. Oligonucleotide primers were synthesized (Sigma-Genosys) based on the published aad9 nucleotide sequence (complementary strand, bases 4867, 5'-CTCTTGCCAGTCACGTTACG-3') and a sequence adjacent to the unique AluI restriction site (bases 563578, 5'-GGAATCATCCTCCCAAACAAG-3') or the BanI restriction sequence within the spc gene (bases 420441, 5'-AGTCGTCGTATCTGAACCATTG-3'). The PCR products were analysed by agarose gel electrophoresis and the products were either gel purified (Qiagen) and subcloned into the T/A cloning vector pGEM T-Easy (Promega), or purified by QIAquik (Qiagen) and sequenced. DNA sequencing was performed at the University of Vermont Cancer Center DNA Analysis Facility. The generated sequences were used to search the A. actinomycetemcomitans strain HK1651 DNA database at the University of Oklahoma for homologous sequences.
Allelic replacement mutagenesis of emaA of A. actinomycetemcomitans.
Directed gene mutagenesis was achieved by conjugative transfer of a non-replicating broad-host-range plasmid modified for efficient use in A. actinomycetemcomitans (Mintz et al., 2002). An internal fragment of emaA was generated by PCR using the primers 5'-CCCTTTCTACCACTACAGATATACC-3' (corresponding to bases 80104) and 5'-CACTGCATCAGTATCATTACGACCAC-3' (corresponding to bases 12601285 of the complementary stand) and SUNY 465 DNA as the template. A unique HindIII restriction site present at base 609 was used to introduce aad9 by blunt-end ligation, resulting in the disruption of the emaA sequence. The PCR fragment was ligated with the T/A cloning vector pGEM T-Easy (Promega) and transformed. The purified plasmid was incubated with HindIII and treated with Klenow (Sambrook et al., 1989
). Following treatment of the plasmid with shrimp alkaline phosphatase (Roche Diagnostics), the DNA was incubated with aad9, transformed into E. coli JM109 and grown on LB agar plates containing 50 µg spectinomycin ml1. The DNA was released by restriction with EcoRI and ligated with the mobilizable plasmid pVT1460 for conjugation (Mintz et al., 2002
). Spectinomycin-resistant colonies were replica-plated on TSBYE plates containing 100 µg kanamycin ml1. Bacteria that were Spcr, Kans were analysed by colony PCR to confirm the presence of mutant emaA in the chromosome. The complete emaA sequence was submitted to GenBank (accession no. AY344064).
RT-PCR.
RNA was isolated (Gentra Systems) following the manufacturer's instructions, and treated with amplification grade DNase I (Invitrogen) to degrade any contaminating DNA. RNA was quantified spectrophotometrically, and equal amounts of RNA from the mutant and the parent strain were used as template for RT-PCR. Sequences within the putative long-chain fatty acid CoA ligase upstream of emaA, (5'-TACGGCATGACGGAAACCAC-3' and 5'-CGCATAGCAAGGCACGATAAG-3', corresponding to the forward and reverse sequences, respectively) and the putative downstream gene, an anaerobic ribonucleoside-triphosphate reductase, (5'-GACTATTCGCCCTTCTTCCC-3' and 5'-TTGTGCCCAAAGGCAAACC-3', corresponding to the forward and reverse sequences, respectively) were used as the primers, and conditions were optimized for use with SuperScript one-step RT-PCR using Platinum Taq (Invitrogen).
Production of recombinant EmaA and generation of polyclonal antibodies.
A 21 kDa expressed fragment of EmaA, corresponding to amino acids 420631, was used to generate polyclonal antibodies in New Zealand white rabbits (Cocalico Biologicals Inc.). The protein was expressed in E. coli BL2(DE3) cells as a GST fusion protein from pGEX-6P-1 (Amersham Biosciences). The 636 bp fragment was amplified using Vent polymerase and chromosomal DNA as the template containing engineered restriction sites for directionally cloning into the EcoRI and BamHI sites of the plasmid. Maximum protein expression was achieved by induction of exponential-phase E. coli cells with 0·1 mM IPTG after 2 h culture at 37 °C. Following induction, the cells were collected by centrifugation, the cell pellet resuspended in PBS, and the cells lysed using a French pressure mini-cell at 18 000 p.s.i. (124 200 kPa) in the presence of a protease inhibitor cocktail for bacterial lysates (Sigma). The GST-hybrid protein was purified by affinity chromatography with Sepharose 4B-glutathione resin, using the column method detailed in the manufacturer's instructions (Amersham Biosciences) at 4 °C. The immobilized GST fusion protein was cleaved with PreScission protease (Amersham Biosciences) overnight, and the GST-free recombinant protein was collected and resolved by SDS-PAGE (Laemmli, 1970). The gel was briefly stained with Coomassie Brilliant Blue, destained, and the band corresponding to the EmaA fragment was excised, soaked in water and used as the immunogen. Amino-terminal sequence analysis was performed, and the sequence was found to be identical to the predicted protein sequence.
The expressed protein immobilized on nitrocellulose was used to affinity-purify antibodies specific for the protein fragment, using a modification of the method of Talian et al. (1983). Briefly, the protein was resolved by SDS-PAGE, transferred to nitrocellulose (Towbin et al., 1979
), the region of the membrane corresponding to the protein excised and treated as described previously, and blocked with 5 % non-fat dried milk dissolved in TBS. The antiserum was applied to the nitrocellulose and incubated for 2 h at room temperature. The antibodies were dissociated from the protein by incubation with 0·2 M glycine, pH 2·7, and neutralized by the addition of 5 M NaOH.
Isolation of outer-membrane proteins.
Cells from a 200 ml overnight culture of A. actinomycetemcomitans were collected by centrifugation (5860 g, 15 min) and washed with PBS (10 mM phosphate, 150 mM NaCl, pH 7·4). The resulting cell pellet was resuspended in 3 ml 10 mM HEPES, pH 7·4, containing 1 mM phenylmethanesulfonyl fluoride. Bacterial membranes were isolated by disruption of the bacteria using a French pressure mini-cell at 18 000 p.s.i. (124 200 kPa), following the protocol of Nikaido et al. (1997). Intact bacteria and debris were collected by centrifugation at 7650 g for 10 min. The supernatant was carefully removed and centrifuged at 100 000 g for 30 min. The pellet was resuspended in 10 mM HEPES, pH 7·4, and recentrifuged. The membranes were resuspended in 10 mM HEPES, pH 7·4, and sodium N-lauroylsarcosine was added to a final concentration of 1 % and incubated at room temperature for 30 min. Based on bacterial membrane protein solubility in sarcosinate (Filip et al., 1973
; Nikaido, 1997
), outer-membrane proteins are insoluble in the detergent. The mixture was centrifuged at 15 600 g for 30 min and the pellet was resuspended in 10 mM HEPES, pH 7·4, and centrifuged as above. The final pellet was resuspended in 10 mM HEPES, pH 7·4. Protein concentration was determined by absorbance at 280 nm. The insoluble proteins were solubilized in 1 % SDS before determination of the protein concentration.
Polyacrylamide gel electrophoresis and immunoblot analysis of proteins.
Equal concentrations of proteins were boiled for 10 min in Laemmli sample buffer, before application to 515 % polyacrylamide-SDS gels. The separated proteins were transferred to nitrocellulose and probed with the affinity-purified antibodies. The immune complexes were detected using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins (Jackson Laboratories) and visualized using the ECL detection system (Amersham Life Sciences) and exposure to photographic film (XAR-5, Eastman Kodak).
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RESULTS |
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Initial conjugation studies with this plasmid and the recipient A. actinomycetemcomitans strain VT1169 resulted in the generation of thousands of transconjugants. However, the frequency of transposition events decreased with repeated studies. The reason for this phenomenon remains elusive. To generate consistent numbers of transconjugants in the A. actinomycetemcomitans strain used in this study, it was necessary to exchange the antibiotic cassette encoding kanamycin resistance for a cassette encoding spectinomycin resistance. The kanamycin gene in the mini-Tn10 was replaced with the spectinomycin gene (aad9) from Enterococcus faecalis, which has been used extensively in this strain of A. actinomycetemcomitans. This modified plasmid, pLOF/Sp (pVT1542), gave consistent high levels of transposon mutants not obtained with pLOF/Km. Characterization of this transposon system in A. actinomycetemcomitans is described below.
Transposon insertion was efficient and appeared random. Of the transconjugants, 98 % were sensitive to ampicillin (plasmid marker), which indicated that the plasmid was not present in the majority of the transconjugants. Southern analysis of 15 randomly selected transconjugants demonstrated that the transposon integrated randomly into the chromosome of these transconjugants (Fig. 1). In most instances, a single copy of the transposon was present in the chromosome. However, infrequent integration of the plasmid was observed (Fig. 1
, lane 12, the faster-migrating hybridizing band corresponded to DNA containing vector sequence following Southern analysis with the vector as the probe). The stability of the transposon within the chromosome of the transconjugants was determined by growing 10 transconjugants in non-selective media for 8 days, followed by replica plating on TSBYE agar plates with or without spectinomycin and Southern blot analysis of the same strains at the start and end of the incubation period. The hybridization pattern of the strains grown in the absence of antibiotic was identical to the same strains grown in the presence of spectinomycin. Collectively, the spc-modified transposon integrates as a stable, single copy genetic element, randomly in the genome. The apparent transposition frequency was calculated to be 2·1x104. This is similar to the frequency of transposition determined for another transposon mutagenesis system developed for A. actinomycetemcomitans (Thomson et al., 1999
).
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ECM-binding mutants reduced for both collagen and fibronectin (col fn) were associated with insertion of the transposon in genes transcribing global regulatory proteins most closely related to P. multocida and Haemophilus somnus cAMP receptor protein (Crp), also known as catabolite activator protein (CAP), and Mlc. These proteins have been thoroughly studied in the regulation of carbohydrate metabolism (Bruckner & Titgemeyer, 2002). However, they may be pleiotropic regulators for other biosynthetic pathways.
Several mutants were identified where insertion resulted in a gain of function: hyper-binding to both substrates (col++ fn++). These proteins include: 1) the heat-modifiable protein (OMP34) of A. actinomycetemcomitans (Wilson, 1991), 2) a protein homologous to the putative membrane protein (YbjE) of Shigella flexneri, 3) a protein homologous to a putative N6-adenine-specific DNA methylase of Salmonella typhimurium, and 4) adenylate cyclase (CyaA) of multiple bacterial taxa, which generates cAMP, a common global regulatory molecule in biological systems (Botsford & Harman, 1992
).
The mutants that displayed a decrease in collagen binding, without an appreciable effect on fibronectin binding (col fn+), were found to have the transposon inserted in the same ORF. Two distinct integration sites were identified, with one 184 bp downstream to the other, within the amino terminus of the predicted protein. The gene associated with the phenotype of these mutants was designated emaA (extracellular matrix protein adhesin A).
The emaA sequence from VT1169 was determined to be 99 % identical to the sequence found in the genomic sequence for the prototypic A. actinomycetemcomitans strain HK1651. The gene encodes a putative 201 kDa protein. The protein sequence (Fig. 3) shares sequence homology with the well-characterized collagen-binding protein, YadA, of Yersinia enterocolitica. The amino terminus of the EmaA protein contains sequences that are identical to those required for the binding of YadA to collagen, or contains conserved amino-acid substitutions within these sequences (Tahir et al., 2000
). The carboxyl terminus of EmaA contains a conserved domain homologous with the cell-membrane anchor domain of YadA (Tamm et al., 1993
). Other conserved features or structural elements, similar to those of the Oca family of adhesins (which includes YadA), are contained in this sequence (Roggenkamp et al., 2003
).
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Characterization of a collagen-binding protein of A. actinomycetemcomitans
The loss-of-function phenotype related to emaA : : Tn10dSpc was confirmed by generating an emaA-directed insertion mutant. An allelic replacement mutant was generated and tested for binding to collagen and fibronectin. The emaA : : spc and emaA : : Tn10dSpc mutants both displayed a similar decrease in binding to collagen, as compared with the parent strain (51±6 and 46±4 %, respectively), while maintaining wild-type levels of binding to fibronectin (91±10 and 105±15 %, respectively). The binding of the emaA : : spc mutant to other fibrillar collagen types (I, II and III) was also reduced when compared with the parent strain (data not shown). These data indicate that inactivation of emaA is not attributable to pleiotropic effects of the integration of the transposon, and that disruption of this gene results in a general defect for collagen binding.
Putative polar effects of the inactivation of emaA on the transcription of upstream and downstream genes were investigated by RT-PCR. Primer sets selected to generate 400 bp internal fragments were used in these reactions with RNA extracted from emaA : : spc and the parent strain. Products were generated for the putative long-chain fatty acid CoA ligase (AAC21681), upstream of the start codon of emaA, and a putative anaerobic ribonucleoside triphosphate reductase, NrdD (AAK03024), downstream of emaA, from RNA extracted from both the emaA mutant and the parent strain (Fig. 4
). In addition, analysis of the emaA sequence indicates the presence of a stemloop structure (
G=13 kcal mol1) immediately following the transcriptional stop codon, consistent with a Rho-independent transcriptional termination signal. These data suggest that the disruption of emaA does not interfere with the gene expression of the surrounding genes, and that the reduction in the binding to collagen is exclusively associated with the inactivation of emaA.
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Membrane fractions of both the wild-type and mutant emaA strain were generated and the outer-membrane proteins recovered by differential solubilization in sodium lauroylsarcosine. Equivalent amounts of outer-membrane proteins were separated by SDS-PAGE and either stained for protein (Fig. 5, panel A) or transferred to nitrocellulose and probed with affinity-purified antibodies (Fig. 5
, panel B). Protein staining (Coomassie Brilliant Blue) revealed very little difference between the profiles of the membrane preparations, which contained few proteins with a molecular mass greater than 90 kDa. However, the immunoblots clearly showed a difference between the two strains. An immunoreactive species greater than 200 kDa was found in the outer-membrane fraction of the parent strain, but was completely absent in the emaA mutant. Some cross-reactivity of the anti-EmaA antibodies with other membrane proteins was present. The band between the 29 kDa and 36 kDa markers was the heat-modifiable protein Omp34, which has been demonstrated to bind the Fc portion of immunoglobulins (Mintz & Fives-Taylor, 1994
). Collectively, these data suggest that emaA encodes a
200 kDa outer-membrane protein, which mediates the adhesion of A. actinomycetemcomitans to collagen.
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DISCUSSION |
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Excluding emaA, the insertion of the transposon in the other mutants can have polar effects on the transcription of surrounding genes. Transcriptional analysis must be performed to define the effects of these insertional mutations. With this caveat stated, the ECM protein binding mutants identified by this genetic screen are indicative of structural proteins and proteins which may act directly or indirectly to regulate adhesin gene expression and/or synthesis. The transposon insertions associated with a defect exclusively in fibronectin adhesion were found in distinct, non-contiguous operons associated with molybdenum cofactor (MoCo) biosynthesis. MoCo is an important cofactor of a number of biosynthetic pathways in which the cofactor itself may be required for adhesin synthesis. Alternatively, the metal moiety of the cofactor (Mo2+) may act as a signalling molecule, similar to other metal ions that modulate virulence-associated factors (Groisman, 2001; Hantke, 2001
; Kehres & Maguire, 2003
; Straley et al., 1993
). In addition, MoeA, which is structurally related to the carboxyl-terminal domain of gephyrin (Sola et al., 2001
), may serve a structural role in fibronectin adhesion. Gephyrin regulates receptor clustering by forming a subsynaptic protein scaffolding, which anchors the receptors to the cytoskeleton (Kneussel & Betz, 2000
). This implies that MoeA may act to stabilize or anchor the fibronectin adhesin or associated molecules.
Loci that reduce or enhance both fibronectin and collagen adhesion (mlc, crp, cyaA) are associated with global regulatory molecules that impact the expression of the actual adhesin. Both Mlc and CRP are usually associated with carbohydrate metabolism, but can activate or repress a large number of promoters outside carbohydrate regulation (Bruckner & Titgemeyer, 2002). The adenylate cyclase and the receptor protein signalling pathway are essential for the global regulation of transcription of the type III secretion systems of both Pseudomonas aeruginosa and Y. enterocolitica (Petersen & Young, 2002
; Wolfgang et al., 2003
). The inactivation of this signalling pathway in Y. enterocolitica results in a reduction in the virulence of this pathogen (Petersen & Young, 2002
).
Several other loci that impacted ECM protein adhesion cannot be readily classified by probable function, but speculation is still warranted. Increased collagen and fibronectin adhesion was associated with loci encoding outer-membrane proteins. It is speculated that absence of the proteins influences the membrane adhesins or allows more adhesins to be integrated into the outer membrane. These mutants may prove to be of value in identifying additional ECM protein mediators if a greater number of adhesins are expressed.
The collagen adhesin identified in this study is likely a member of a novel class of non-fimbrial oligomeric coiled-coil adhesins (Oca) (Roggenkamp et al., 2003). The members of this family, in addition to YadA, include UspA1 and UspA2 of Moraxella catarrhalis, and the autotransporter Hia of H. influenzae (Hoiczyk et al., 2000
). These proteins share conserved features or structural elements. These include: (i) an amino-terminal Sec-dependent secretion signal, (ii) a head domain consisting of degenerate 14-residue repeats, (iii) a highly conserved neck region(s), (iv) a stalk domain of variable length with a high probability of coiled-coil formation, and (v) a carboxyl-terminal membrane-anchor domain with conserved structural features. In addition, members of this protein class do not contain cysteine residues in the mature polypeptide. The known functions of these adhesins are binding to eukaryotic cells and ECM proteins. The binding domains mediating adherence are usually located in non-conserved regions of the amino terminus of the protein (Roggenkamp et al., 2003
).
The EmaA protein consists of 1965 amino acids, and there are no cysteine residues in the mature protein. A single cysteine is present in the putative signal sequence. The amino terminus of this protein contains the approximately 14-residue degenerate repeats (alternating pattern of branched aliphatic and small residues, followed by a position consisting mainly of Ala, Gly, Ser or Thr), as found in the head region of YadA (Hoiczyk et al., 2000). Surrounding and included within this region are multiple sequences identical to the collagen-binding consensus sequence NSVAIG-S, or displaying conserved amino-acid substitutions (Tahir et al., 2000
). Juxtaposed to the putative collagen-binding domain is the sequence TDAVNVAQL, which is identical to the neck sequence of YadA (Hoiczyk et al., 2000
). A putative stalk region is present, which contains one region with a high probability of a coiled-coil domain, as determined by COILS version 2.1 (Window=21 score of 0·62). The carboxyl terminus is separated from the stalk region by another neck sequence followed by a coiled-coil segment (Window=21 score of 0·75) and a membrane-anchor domain formed by four transmembrane
-strands, as suggested by the A. actinomycetemcomitans sequence alignment with the YadA sequence listed in Hoiczyk et al. (2000)
.
The emaA sequence predicts a protein with a molecular mass of 201 kDa, and a protein greater than 200 kDa was observed in immunoblots of membrane proteins. Based on the protein staining, EmaA is not a significant protein component of the bacterial membrane. Although this may be indicative of the relative abundance of the adhesin within the membrane of the bacterium, it does not reflect the protein's biological significance. The position of the protein in the immunoblot makes estimation of the molecular mass unreliable. Therefore, EmaA may exist as heat-stable aggregates in SDS-PAGE, which is a characteristic of Oca proteins (Roggenkamp et al., 2003). Alternatively, posttranslational modification of the protein may alter the protein's electrophorectic mobility, which implies that the ORF encodes a single protein. Further biophysical and structural studies are warranted to resolve these issues.
Surface ultrastructual analysis of the A. actinomycetemcomitans strain used in this study demonstrates the presence of small vesicles and fibrillar membranous extensions with knob-like ends on the surface of the bacterium (Meyer & Fives-Taylor, 1994). The YadA and UspA adhesins form lollipop-shaped structures on the outer membrane of the bacterium, as detected by electron microscopy (Hoiczyk et al., 2000
). Based on the structural homology of EmaA with these proteins, one would predict the presence of similar structures on the surface of A. actinomycetemcomitans. Investigations into the surface structures of the wild-type and emaA mutant strains are being conducted.
Bacterial adhesins that specifically recognize ECM proteins have been characterized for a number of pathogens (Patti et al., 1994). The results of this study are consistent with the existence of distinct mediators for fibronectin and collagen adhesion. However, it is possible that an adhesin that mediates the binding to both ECM proteins is also present. A collagen-specific adhesin, EmaA, has been identified which contains protein sequence information and structural characteristics that are analogous to a family of established ECM-binding proteins. In addition, ubiquitous signalling molecules have also been identified that regulate the adhesion of this pathogen to these substrates. Further studies will be required to determine if these pathways are implicated in the regulation of other virulence determinants expressed by this pathogen.
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ACKNOWLEDGEMENTS |
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Received 20 February 2004;
revised 23 April 2004;
accepted 11 May 2004.
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