The extended signal peptide of the trimeric autotransporter EmaA of Aggregatibacter actinomycetemcomitans modulates secretion.

The extracellular matrix protein adhesin A (EmaA) of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a fibrillar collagen adhesin belonging to the family of trimeric autotransporters. The protein forms antenna-like structures on the bacterial surface required for collagen adhesion. The 202-kDa protein monomers are proposed to be targeted and translocated across the inner membrane by a long signal peptide composed of 56 amino acids. The predicted signal peptide was functionally active in Escherichia coli and A. actinomycetemcomitans using truncated PhoA and Aae chimeric proteins, respectively. Mutations in the signal peptide were generated and characterized for PhoA activity in E. coli. A. actinomycetemcomitans strains expressing EmaA with the identical mutant signal peptides were assessed for cellular localization, surface expression, and collagen binding activity. All of the mutants impaired some aspect of EmaA structure or function. A signal peptide mutant that promoted alkaline phosphatase secretion did not allow any cell surface presentation of EmaA. A second mutant allowed for cell surface exposure but abolished protein function. A third mutant allowed for the normal localization and function of EmaA at 37°C but impaired localization at elevated temperatures. Likewise, replacement of the long EmaA signal peptide with a typical signal peptide also impaired localization above 37°C. The data suggest that the residues of the EmaA signal peptide are required for protein folding or assembly of this collagen adhesin.

The conserved carboxyl domain of MorC, an inner membrane protein of Aggregatibacter actinomycetemcomitans, is essential for membrane function.

Morphogenesis protein C (MorC) of Aggregatibacter actinomycetemcomitans is important for maintaining the membrane morphology and integrity of the cell envelope of this oral pathogen. The MorC sequence and operon organization were found to be conserved in Gammaproteobacteria, based on a bioinformatic analysis of 435 sequences from representative organisms. Functional conservation of MorC was investigated using an A. actinomycetemcomitans morC mutant as a model system to express MorC homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. The A. actinomycetemcomitans strains expressing the homologous proteins were assessed for sensitivity to bile salts, leukotoxin secretion, autoaggregation and membrane morphology. MorC from the most closely related organism (H. influenzae) was functionally identical to MorC from A. actinomycetemcomitans. However, the genes from more distantly related organisms restored some but not all A. actinomycetemcomitans mutant phenotypes. In addition, deletion mutagenesis indicated that the most conserved portion of the protein, the C-terminus DUF490 domain, was necessary to maintain the integrity of the membrane. Deletion of the last 10 amino acids of this domain of the A. actinomycetemcomitans MorC protein was sufficient to disrupt membrane stability and leukotoxin secretion. The data suggest that the MorC sequence is functionally conserved across Gammaproteobacteria and the C-terminus of the protein is essential for maintaining membrane physiology.

Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies.

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.

The cell envelope proteome of Aggregatibacter actinomycetemcomitans.

The cell envelope of gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 27% of the predicted open reading frames in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. A total of 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, whereas others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity.

Characterization of native and recombinant bone sialoprotein: delineation of the mineral-binding and cell adhesion domains and structural analysis of the RGD domain.

Bone sialoprotein is a small, sulfated, and phosphorylated integrin-binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106-01 BSP) culture media is shown to have a hydroxyapatite Kd approximately 2.6 x 10(-9) M, perhaps the strongest affinity for this mineral of any of the matrix proteins. Both native BSP and a 47 kD fragment of UMR-BSP (Fragment 1 approximately 133A- approximately 265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post-translational modifications. The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral-binding properties are encoded directly within the protein sequence itself. BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence. Nuclear magnetic resonance (NMR) analysis of a 15N-enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium. Previous work has also shown that an endogenous fragment of UMR-BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence. In this report, non-RGD cell adhesion sites are localized within conserved amino- and carboxy-terminal tyrosine-rich domains of recombinant human BSP. Given the proximity of the latter non-RGD cell adhesion site to the RGD tripeptide, a model of BSP-receptor interactions is presented.

Purification and fragmentation of nondenatured bone sialoprotein: evidence for a cryptic, RGD-resistant cell attachment domain.

Bone sialoprotein (BSP), a small (approximately 80,000 M(r)) integrin binding, RGD-containing bone matrix glycoprotein, has been purified in milligram quantities from the serum-free medium of the rat osteosarcoma cell line UMR-106-BSP using nondenaturing conditions. Routine protein purification without serine protease inhibitors or reducing agents consistently resulted in three major fragments. The largest fragment (E1) started at amino acid 117 and did not bind to antibodies made to the RGD region of the protein. Furthermore, the smallest fragment (E3), was shown by sequencing to contain the RGD region of the protein. Digestion of intact BSP with highly purified chymotrypsin also resulted in a large fragment (C1) with properties nearly identical to those of E1. The large, non-RGD-containing fragments, E1 and C1, as well as the intact BSP, supported attachment by normal human bone cells and human skin fibroblasts in vitro. Attachment to the intact BSP was totally blocked by 0.4 mM GRGDS peptide. Both preparations of skin fibroblasts and approximately half of the preparations of normal human bone cells, however, also would not attach to the E1 and C1 fragments in the presence of 0.4 mM GRGDS peptide. In contrast, half of the bone cell preparations had significant attachment activity to E1 (> 50%) and C1 (> 25%) in the presence of 0.4 mM GRGDS peptide. These data suggest that cleavage of the BSP results in either (1) the exposure of a previously unavailable or cryptic cell attachment site or (2) a conformational change that increases the affinity of the complex between a non-RGD-encoded binding region of the E1 and C1 fragments and at least one receptor. The possible homology of the second, non-RGD-suppressible site of BSP with the second cell attachment site on the gamma chain of fibrinogen is discussed.

Osteogenesis imperfecta: changes in noncollagenous proteins in bone.

The noncollagenous proteins osteonectin, bone sialoprotein, osteocalcin, the small proteoglycan decorin (PG II), and alpha 2-HS glycoprotein (which is synthesized in the liver but highly concentrated in bone) were measured in extracts of cortical bone from 3 type I, 2 type II, 8 type III and 13 type IV patients with osteogenesis imperfecta (OI) and from 7 control subjects. Osteonectin was found to be reduced in the bone of all OI patients. The bone from severely affected type III OI patients contained the lowest levels of osteonectin. In contrast, bone sialoprotein was found to be elevated in the bones of OI patients. The highest levels were found in individuals classified as type IV patients. Osteocalcin and alpha 2-HS glycoprotein concentrations were increased in all OI patients. Decorin levels were not significantly altered in OI bones compared to controls. These changes in the concentrations of the noncollagenous proteins may contribute to the fragility of the OI bone by interfering with complete mineralization and/or normal tissue architecture.

Structure and molecular regulation of bone matrix proteins.

The organic matrix of bone contains several protein families, including collagens, proteoglycans, and glycoproteins, all of which may be extensively modified by posttranslational events, such as phosphorylation and sulfation. Many of the glycoproteins contain Arg-Gly-Asp (RGD), the integrin-binding sequence, within their structure, whereas other constituent proteins contain gamma-carboxyglutamic acid. The deposition of bone matrix by cells in the osteoblastic lineage is regulated by extrinsic factors, such as systemic and local growth factors and physical forces, and factors that are intrinsic to the cell, such as position in the cell cycle, maturational stage, and developmental age of the donor. Recent studies of several bone matrix gene promoters have identified cis- and trans-acting elements that are responsible for gene activity, although the precise sequence of regulatory events is not known. Development of in vitro assays, coupled with studies of the appearance of these proteins during development in vivo, provides insight into the functions of these proteins during the various stages of bone metabolism. Potential roles for these proteins include proliferation and maturation of stem cells, formation of matrix scaffolding elaborated by bone-forming cells, modeling, and remodeling. Changes in the functional properties of the extracellular matrix may be involved in a variety of disease processes, including osteoporosis and oral bone loss.

Dansylarginine N-(3-ethyl-1.5-pentanediyl)amide. A potent and selective fluorescent inhibitor of butyrylcholinesterase.

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase (IC50 = 2 x 10(-7) M) but not of acetylcholinesterase (IC50 = 4 x 10(-4) M). For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate Ki unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a Kd of 4.5 x 10(-7) M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.

Characterization of the secretion pathway of the collagen adhesin EmaA of Aggregatibacter actinomycetemcomitans.

The extracellular matrix protein adhesin A (EmaA) surface antennae-like structures of the periodontal pathogen Aggregatibacter actinomycetemcomitans are composed of three identical protein monomers. Recently, we have demonstrated that the protein is synthesized with an extended signal peptide of 56 amino acids necessary for membrane targeting and protein translocation. In this study, EmaA secretion was demonstrated to be reliant on a chaperone-dependent secretion pathway. Deletion of secB partially reduced but did not abolish the amount of EmaA in the membrane. This observation was attributed to an increase in the synthesis of DnaK in the ΔsecB strain. Overexpression of a DnaK substitution mutant (A174T), with diminished activity, in the ΔsecB strain further reduced the amount of EmaA in the membrane. Expression of dnaK A174T in the wild-type strain did not affect the amount of EmaA in the membrane when grown under optimal growth conditions at 37°C. However, EmaA was found to be reduced when this strain was grown at heat-shock temperature. A chromosomal deletion of amino acids 16-39 of the EmaA extended signal peptide, transformed with either the wild-type or dnaK A174T-expressing plasmid, did not affect the amount of EmaA in the membrane. In addition, the level of EmaA in a ΔsecB/emaA(-) double mutant strain expressing EmaAΔ16-39 was unchanged when grown at both temperatures. The data suggest that chaperones are required for the targeting of EmaA to the membrane and a specific region of the signal peptide is necessary for secretion under stress conditions.

Lipopolysaccharides mediate leukotoxin secretion in Aggregatibacter actinomycetemcomitans.

We previously reported that lipopolysaccharide (LPS) -related sugars are associated with the glycosylation of the collagen adhesin EmaA, a virulence determinant of Aggregatibacter actinomycetemcomitans. In this study, the role of LPS in the secretion of other virulence factors was investigated. The secretion of the epithelial adhesin Aae, the immunoglobulin Fc receptor Omp34 and leukotoxin were examined in a mutant strain with inactivated TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase (rmlC), which resulted in altered O-antigen polysaccharides (O-PS) of LPS. The secretion of Aae and Omp34 was not affected. However, the leukotoxin secretion, which is mediated by the TolC-dependent type I secretion system, was altered in the rmlC mutant. The amount of secreted leukotoxin in the bacterial growth medium was reduced nine-fold, with a concurrent four-fold increase of the membrane-bound toxin in the mutant compared with the wild-type strain. The altered leukotoxin secretion pattern was restored to the wild-type by complementation of the rmlC gene in trans. Examination of the ltxA mRNA levels indicated that the leukotoxin secretion was post-transcriptionally regulated in the modified O-PS containing strain. The mutant strain also showed increased resistance to vancomycin, an antibiotic dependent on TolC for internalization, indicating that TolC was affected. Overexpression of TolC in the rmlC mutant resulted in an increased TolC level in the outer membrane but did not restore the leukotoxin secretion profile to the wild-type phenotype. The data suggest that O-PS mediate leukotoxin secretion in A. actinomycetemcomitans.

Unimpaired energy metabolism in experimental neuropathy induced by p-bromophenylacetylurea.

The contribution of defective energy metabolism to the induction of neuronal pathology by p-bromophenylacetylurea (BPAU) was examined in several ways. It was found that a saturated aqueous solution of BPAU had no effect on the activity of crystalline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or phosphofructokinase (PFK). In rats with total hindlimb paralysis from treatment with BPAU (400 mg/kg), the endogenous GAPDH and PFK of sciatic nerve showed normal activity. Endogenous enolase and nerve-specific enolase activities were likewise unaffected. Consequently, it appeared improbable that BPAU neuropathy involves impaired glycolysis. This conclusion was supported by the failure to prevent hindlimb weakness by feeding pyruvate, a substrate for the Krebs cycle. To test for interference with glycolysis at other steps, or for an impairment in oxidative phosphorylation, adenosine triphosphate (ATP) and creatine phosphate were measured. The amounts of high energy phosphates in nerves of paralyzed animals were found to be the same as in nerves of untreated and vehicle-treated controls. A similar observation was made in nerves regenerating from a crush injury. To test turnover, ATP and creatine phosphate were measured in nerves exposed to an N2 atmosphere in vitro. Since the high energy phosphates disappeared at the same rates in all groups, it was concluded that BPAU neuropathy does not alter energy utilization. In our view, BPAU neuropathy arises by a mechanism that does not depend on altered energy metabolism.

Detection of procollagen biosynthesis using peptide-specific antibodies.

Peptides corresponding to selected sequences of the alpha 1 chain of the COOH propeptide of type I and type III human procollagen were synthesized and used as antigens to develop polyclonal and monoclonal antibodies. The antibodies were shown to be epitope specific using a peptide-based solid phase enzyme-linked immunoadsorbent assay. The antibodies were specific for the appropriate procollagens and the COOH propeptides isolated from serum-free culture supernatants of human skin fibroblasts. The rabbit antisera directed to the type I synthetic peptide bound the intact procollagen molecule and both the procollagen alpha 1(I) and alpha 2(I) chains after the reduction of the disulfide bonds. In addition, the antisera bound intact type I COOH propeptide, generated by bacterial collagenase treatment of procollagen, and the individual chains of the propeptide after reduction. In contrast, a monoclonal antibody to the type I peptide was able to bind only to the reduced form of the COOH propeptide. Both rabbit polyclonal and murine monoclonal antibodies directed to the type III synthetic peptide bound the intact and the individual chains of type III procollagen as well as the intact and reduced forms of the type III COOH propeptide. The antibodies have been used to detect procollagen synthesis in two human osteosarcoma cell lines and the differential expression of procollagen in the culture medium of rat lung fibroblasts grown in the presence or absence of glucocorticoids.

Two-step immunoaffinity purification of acetylcholinesterase from rabbit brain.

Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X-100 from rabbit brain was purified 2,000-fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium-Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver-staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogeneous AChE to be completed in fewer than 7 days, at yields exceeding 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 showed little difference between the properties of the native and the purified enzyme. The molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (Km = 0.16 +/- 0.01 mM) were close to those of the native enzyme (Km = 0.12 +/- 0.01 mM) when examined with acetylthiocholine iodide as substrate. The two-step immunopurification procedure presented in this communication offers a convenient route to homogeneous neural AChE in quantities useful for detailed biochemical and immunochemical study.

Monoclonal antibodies to rabbit brain acetylcholinesterase: selective enzyme inhibition, differential affinity for enzyme forms, and cross-reactivity with other mammalian cholinesterases.

Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.

Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line.

Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells. The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A. actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or [3H]thymidine-labeled bacteria. Adhesion was found to be time dependent and increased linearly with increasing numbers of bacteria added. Variation in the level of adhesion was noted among strains of A. actinomycetemcomitans. Adhesion was not significantly altered by changes in pH (from pH 5 to 9) but was sensitive to sodium chloride concentrations greater than 0.15 M. Pooled human saliva was inhibitory for adhesion when bacteria were pretreated with saliva before being added to the cells. Pretreatment of the KB cells with saliva did not inhibit adhesion. Protease treatment of A. actinomycetemcomitans reduced adhesion of the bacteria to KB cells. The data are consistent with the hypothesis that a protein(s) is required for bacterial adhesion and that host components may play a role in modulating adhesion to epithelial cells.

Evolution of butyrylcholinesterase in higher primates: an immunochemical study.

Serum butyrylcholinesterase (BuChE; EC 3.1.1.8) of man and the higher primates was tested enzymatically and immunochemically, with the aid of monoclonal antibodies (McAb) developed against the enzyme isolated from human blood. Enzyme activities showed great differences across species and among individuals, but all samples tested were dibucaine-sensitive. One McAb showed similar affinities for BuChE of each species, but another showed marked differences in affinity, preferring species in the order: man greater than chimpanzee = pygmy chimpanzee greater than gorilla much greater than orangutan greater than gibbon. We conclude that at least one epitope of BuChE underwent progressive modification during the later stages of primate evolution.

Identification of an immunoglobulin Fc receptor of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans expresses proteins that bind to the Fc portion of immunoglobulins. The immunoglobulin Fc receptors on the surface of A. actinomycetemcomitans were detected by the binding of biotinylated human or murine Fc molecules to strain SUNY 465 adsorbed to the bottom of microtiter wells. Biotinylated Fc binding was inhibited by unlabeled Fc molecules and human plasma. Fc receptors were identified by the binding of biotinylated Fc molecules to bacterial membrane proteins separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose. Multiple bands were identified, and the major Fc-binding protein was determined to be a heat-modifiable protein. This protein migrated with approximate molecular weights of 25,000 and 32,000 (unheated and heated, respectively). Amino-terminal sequence analysis of this protein revealed a sequence identical to the heat-modifiable protein described for A. actinomycetemcomitans ATCC 43718. This protein sequence exhibits significant homology with the N termini of outer membrane protein A (OmpA) of Escherichia coli and related OmpA-like proteins from other gram-negative bacteria.

Binding of the periodontal pathogen Actinobacillus actinomycetemcomitans to extracellular matrix proteins.

The interaction of Actinobacillus actinomycetemcomitans, an important pathogen implicated in juvenile and adult periodontitis, with collagenous and noncollagenous proteins of the extracellular matrix was investigated. A. actinomycetemcomitans SUNY 465 bound to immobilized type I, II, III and V but not type IV collagen. Binding to immobilized collagen was saturable and concentration dependent. This interaction could not be inhibited by soluble collagen, suggesting that binding was dependent on a specific collagen conformation. Bacteria grown anaerobically exhibited decreased collagen-binding activity as compared with organisms grown acrobically. Bacterial outer membrane proteins were essential for binding to collagen. A actinomycetemcomitans SUNY 465 also bound to immobilized fibronectin. In contrast, bacteria did not bind to fibrinogen, bone sialoprotein, alpha 2-HS glycoprotein or albumin. The mechanism of the interaction with fibronectin was more complex, possibly involving both protein and nonproteinaceous components. The majority of other A. actinomycetemcomitans strains tested bound to extracellular matrix proteins in a manner similar to SUNY 465 but with minor variation. These results demonstrate that A. actinomycetemcomitans binds to proteins found in connective tissue. The interaction with extracellular matrix proteins may contribute to the virulence of this pathogen at oral and extraoral sites of infection.

Identification of genes coding for exported proteins of Actinobacillus actinomycetemcomitans.

Random fusions of genomic DNA fragments to a partial gene encoding a signal sequence-deficient bacterial alkaline phosphatase were utilized to screen for exported proteins of Actinobacillus actinomycetemcomitans in Escherichia coli. Twenty-four PhoA(+) clones were isolated and sequenced. Membrane localization signals in the form of signal sequences were deduced from most of these sequences. Several of the deduced amino acid sequences were found to be homologous to known exported or membrane-associated proteins. The complete genes corresponding to two of these sequences were isolated from an A. actinomycetemcomitans lambda phage library. One gene was found to be homologous to the outer membrane lipoprotein LolB. The second gene product had homology with a Haemophilus influenzae protein and was localized to the inner membrane of A. actinomycetemcomitans.