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.

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 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.

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.

Aggregatibacter actinomycetemcomitansarcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite / arcB em Aggregatibacter actinomycetmcomitans influencia propriedades hidrofóbicas, formação de biofilme e aderência a hidroxiapatita

The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly. This study investigated in A. actinomycetemcomitans the role of arcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitansarcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis. The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase arcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces.

A regulação da expressão gênica do patógeno oral Aggregatibacter actinomycetemcomitans não está completamente descrita. O sistema de dois componentes ArcAB permite que bactérias anaeróbias facultativas percebam diferenças nas condições respiratórias durante sua multiplicação e adaptem a expressão de genes à estas condições. Este estudo investigou em A. actinomycetemcomitans o papel de arcB na regulação da formação de biofilme, aderência à hidroxiapatita recoberta por saliva (SHA) e nas propriedades hidrofóbicas celulares. Estas características fenotípicas foram determinadas para uma linhagem de A. actinomycetemcomitans deficiente em arcB e para uma linhagem selvagem. Foram observadas diferenças nas propriedades hidrofóbicas entre as linhagens quando estas foram cultivadas em ambiente microaerófilo até início e final de fase exponencial e quando foram cultivadas em ambiente anaeróbio até final de fase exponencial. A linhagem arcB mutante formou menos biofilme do que a linhagem selvagem quando estas foram cultivadas sob incubação anaeróbica, porém, apresentou maior formação de biofilme quando a incubação foi realizada em condições de microaerofilia. A aderência à SHA apresentada pela linhagem mutante foi significantemente menor do que a observada pela linhagem selvagem. Estes estudos sugerem que a quinase sensora arcB, em A. actinomycetemcomitans, percebe as condições redox de multiplicação e regula a expressão de componentes de superfície bacterianos relacionados à formação de biofilme e adesão a superfícies recobertas com saliva.

Aggregatibacter actinomycetemcomitans arcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite.

The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly.This study investigated in A. actinomycetemcomitans the role of ArcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitans arcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis.The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase ArcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces.

impA, a gene coding for an inner membrane protein, influences colonial morphology of Actinobacillus actinomycetemcomitans.

Directed mutagenesis of a gene coding for a membrane protein of the periodontopathogen Actinobacillus actinomycetemcomitans was achieved by conjugation. The gene was disrupted by insertion of an antibiotic cassette into a unique endonuclease restriction sequence engineered by inverse PCR. The disrupted gene was cloned into a conjugative plasmid and transferred from Escherichia coli to A. actinomycetemcomitans. The allelic replacement mutation resulted in the loss of a 22-kDa inner membrane protein. The loss of this protein (ImpA) resulted in changes in the outer membrane protein composition of the bacterium. Concurrent with the mutation in impA was a change in the pattern of growth of the mutant bacteria in broth cultures. The progenitor bacteria grew as a homogeneous suspension of cells compared to a granular, autoaggregating adherent cell population described for the mutant bacteria. These data suggest that ImpA may play a regulatory role or be directly involved in protein(s) that are exported and associated with colony variations in A. actinomycetemcomitans.

Virulence factors of Actinobacillus actinomycetemcomitans.

A. actinomycetemcomitans has clearly adapted well to its environs; its armamentarium of virulence factors (Table 2) ensures its survival in the oral cavity and enables it to promote disease. Factors that promote A. actinomycetemcomitans colonization and persistence in the oral cavity include adhesins, bacteriocins, invasins and antibiotic resistance. It can interact with and adhere to all components of the oral cavity (the tooth surface, other oral bacteria, epithelial cells or the extracellular matrix). The adherence is mediated by a number of distinct adhesins that are elements of the cell surface (outer membrane proteins, vesicles, fimbriae or amorphous material). A. actinomycetemcomitans enhances its chance of colonization by producing actinobacillin, an antibiotic that is active against both streptococci and Actinomyces, primary colonizers of the tooth surface. The fact that A. actinomycetemcomitans resistance to tetracyclines, a drug often used in the treatment of periodontal disease, is on the rise is an added weapon. Periodontal pathogens or their pathogenic products must be able to pass through the epithelial cell barrier in order to reach and cause destruction to underlying tissues (the gingiva, cementum, periodontal ligament and alveolar bone). A. actinomycetemcomitans is able to elicit its own uptake into epithelial cells and its spread to adjacent cells by usurping normal epithelial cell function. A. actinomycetemcomitans may utilize these remarkable mechanisms for host cell infection and migration to deeper tissues. A. actinomycetemcomitans also orchestrates its own survival by elaborating factors that interfere with the host's defense system (such as factors that kill phagocytes and impair lymphocyte activity, inhibit phagocytosis and phagocyte chemotaxis or interfere with antibody production). Once the organisms are firmly established in the gingiva, the host responds to the bacterial onslaught, especially to the bacterial lipopolysaccharide, by a marked and continual inflammatory response, which results in the destruction of the periodontal tissues. A. actinomycetemcomitans has at least three individual factors that cause bone resorption (lipopolysaccharide, proteolysis-sensitive factor and GroEL), as well as a number of activities (collagenase, fibroblast cytotoxin, etc.) that elicit detrimental effects on connective tissue and the extracellular matrix. It is of considerable interest to know that A. actinomycetemcomitans possesses so many virulence factors but unfortunate that only a few have been extensively studied. If we hope to understand and eradicate this pathogen, it is critical that in-depth investigations into the biochemistry, genetic expression, regulation and mechanisms of action of these factors be initiated.

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.

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.

Isolation and characterization of Fap1, a fimbriae-associated adhesin of Streptococcus parasanguis FW213.

An adhesin of Streptococcus parasanguis FW213, a primary colonizer of the tooth surface, has been purified from the culture medium by immunoaffinity chromatography. The purified protein has a molecular mass of 200 kDa and stains positively for carbohydrate. The amino-terminal sequence indicated that this protein represented a unique streptococcal surface protein. Immunogold labelling of the bacterium indicated that this protein was associated with fimbriae and designated Fap1 (fimbriae-associated protein). A polymerase chain reaction (PCR) product based on the amino terminus of Fap1 was used to probe an FW213 genomic library. A 9 kb fragment containing the fap1 gene was isolated and 2.5 kb have been sequenced. Generation of fap1 mutants by a single cross-over (Campbell insertion) or a non-polar allelic exchange abolished the expression of Fap1. The inactivation of fap1 resulted in a dramatic reduction in the expression of the long peritrichous fimbriae and adhesion to saliva-coated hydroxylapatite (SHA). Northern blots probed with an internal gene fragment of fap1 hybridized to a 9 kb transcript, which suggests that fap1 is transcribed as a polycistronic message. These data demonstrate that Fap1 is a unique streptococcal adhesin that is involved in the assembly of S. parasanguis FW213 fimbriae and adhesion to SHA.

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.

Models of invasion of enteric and periodontal pathogens into epithelial cells: a comparative analysis.

Bacterial invasion of epithelial cells is associated with the initiation of infection by many bacteria. To carry out this action, bacteria have developed remarkable processes and mechanisms that co-opt host cell function and stimulate their own uptake and adaptation to the environment of the host cell. Two general types of invasion processes have been observed. In one type, the pathogens (e.g., Salmonella and Yersinia spp.) remain in the vacuole in which they are internalized and replicate within the vacuole. In the other type, the organism (e.g., Actinobacillus actinomycetemcomitans, Shigella flexneri, and Listeria monocytogenes) is able to escape from the vacuole, replicate in the host cell cytoplasm, and spread to adjacent host cells. The much-studied enteropathogenic bacteria usurp primarily host cell microfilaments for entry. Those organisms which can escape from the vacuole do so by means of hemolytic factors and C type phospholipases. The cell-to-cell spread of these organisms is mediated by microfilaments. The investigation of invasion by periodontopathogens is in its infancy in comparison with that of the enteric pathogens. However, studies to date on two invasive periodontopathogens. A actinomycetemcomitans and Porphyromonas (Bacteroides) gingivalis, reveal that these bacteria have developed invasion strategies and mechanisms similar to those of the enteropathogens. Entry of A. actinomycetemcomitans is mediated by microfilaments, whereas entry of P. gingivalis is mediated by both microfilaments and microtubules. A. actinomycetemcomitans, like Shigella and Listeria, can escape from the vacuole and spread to adjacent cells. However, the spread of A. actinomycetemcomitans is linked to host cell microtubules, not microfilaments. The paradigms presented establish that bacteria which cause chronic infections, such as periodontitis, and bacteria which cause acute diseases, such as dysentery, have developed similar invasion strategies.

Virulence Factors of the Periodontopathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is an important periodontopathogen that has been implicated in juvenile and adult periodontitis. This capnophilic coccobacillus elaborates a number of virulence factors such as a bacteriocin, chemotaxis-inhibiting factor, immunosuppressive factors, cytotoxic factors, Fc binding proteins, lipopolysaccharide (LPS), collagenases, antibiotic resistant determinants, adhesins, and invasins. The adhesins are associated with the bacterial surface or released into the surrounding environment in the form of vesicles. Adhesion to epithelial cells involves multiple determinants and is influenced by both bacterial and host environmental conditions. Invasion is a rapid process which occurs within 15 minutes post-infection. Host cell actin is polymerized around the invading bacteria. Bacteria enter through a membraneruffled region of the host cell. This internalization process triggers vesicle formation on the bacteria. A. actinomycetemcomitans is taken up in a vacuole but is seen free in the cytoplasm in a short period of time. The number of internal organisms does not increase with time. A. actinomycetemcomitans can transcytose through the epithelial cells and is released from the host cell in 5 hours. We hypothesize that A. actinomycetemcomitans uses invasion of the epithelial cell as a mechanism to penetrate deeper tissues by cell-to-cell spread. J Periodontol 1996;67:291-297.

Characteristics of Actinobacillus actinomycetemcomitans invasion of and adhesion to cultured epithelial cells.

Actinobacillus actinomycetemcomitans (A.a.) is highly implicated in periodontitis. We have developed several in vitro models using the KB oral cell line to examine A.a.-epithelial cell interactions. In support of the use of KB cell line model systems is our finding that A.a. invaded KB and primary gingival cells to the same extent. Invasion is an active event which requires new protein synthesis by both KB and A.a. Like many other intracellular parasites, A.a. invade by receptor-mediated endocytosis. We observed that internalized A.a. were surrounded by foci of actin which had been transported from the periphery of the KB cell. Adhesion of A.a. to KB cells occurred rapidly and stimulated the formation of microvilli. Adhesion is affected by both host factors (saliva, serum, [NaCl]) and culture conditions. Multiple determinants [fimbriae, outer membrane proteins, vesicles, and/or an extracellular amorphous material (ExAmMat)] which are either associated with the A.a. surface or are released into the milieu are involved. We determined that ExAmMat can convey adhesiveness to weakly adherent A.a. and to at least one other oral species (Streptococcus parasanguis).

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.

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.

Chlorate-induced inhibition of tyrosine sulfation on bone sialoprotein synthesized by a rat osteoblast-like cell line (UMR 106-01 BSP).

Bone sialoprotein (BSP) is a major noncollagenous, RGD-containing glycoprotein found in the extracellular matrix of bone. The RGD sequence is flanked by two tyrosine-rich regions, which fit the established consensus requirements for tyrosine sulfation. Tyrosine sulfation is suggested to be important in the regulation of protein secretion and function. The role of this post-translational modification on the cell attachment activity and secretion of a highly sulfated form of BSP isolated from a rat osteoblast-like cell line (UMR 106-01 BSP) was investigated by inhibiting sulfation with chlorate. [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to monitor biosynthetic products. Chlorate was effective in inhibiting total [35S]sulfate incorporation by 90% without altering overall protein synthesis and secretion in cultures up to 72 h under serum-free conditions. Isolated proteoglycans and purified BSP were analyzed for sulfate incorporation. Proteoglycans isolated from the medium of cells treated with chlorate displayed a difference in the hydrodynamic properties of the molecules as compared with control cultures. An increase in the specific activity of proteoglycans labeled with [3H]glucosamine isolated from chlorate-treated cells was also observed suggesting a change in hexosamine metabolism induced by chlorate. BSP purified from the medium of chlorate-treated cells contained approximately 7% of the 35S incorporation as compared with nontreated control cultures. Quantification of sulfate incorporation into glycoconjugates versus tyrosine sulfate of BSP indicates that the amount of sulfate associated with N- and O-linked oligosaccharides was reduced by approximately 97%, while that on tyrosine residues was reduced by approximately 90%. Using normal human bone cells, the cell attachment activity of the reduced sulfate form of BSP was nearly equivalent to that of the fully sulfated product.

Interactions of matrix proteins from mineralized tissues with octacalcium phosphate.

Acidic matrix macromolecules, present in many mineralized tissues, including those of vertebrates, are thought to be involved in controlling crystal formation. Little, however, is known about their in vivo functions, particularly in relation to calcium-phosphate-containing crystals. The manner in which a variety of synthetic and natural acidic macromolecules interact in vitro with crystals of octacalcium phosphate (OCP) has been studied. Interactions were assessed by examining changes in morphology of the crystals resulting from preferential interaction of the additive with some crystal faces and not others. Macromolecules rich in acidic amino acids, with or without polysaccharides, such as polyaspartate and mollusk shell proteins respectively, were shown to interact preferentially with rows of Ca ions exposed on the hydrated plate surface of OCP crystals. In contrast, the phosphorylated proteins, phosphophoryn and phosvitin, interacted specifically with the apatite-like motifs on the OCP side faces. BSP did not interact specifically with OCP, under the experimental conditions used. The observation that these classes of acidic macromolecules recognize different crystal faces should be taken into account when evaluating functions of acidic matrix macromolecules in mineralized tissues.