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.

Role of gap3 in Fap1 glycosylation, stability, in vitro adhesion, and fimbrial and biofilm formation of Streptococcus parasanguinis.

BACKGROUND/AIMS: Streptococcus parasanguinis is a primary colonizer of the tooth surface. Its adhesion is mediated by the long fimbriae, which are composed of multiple subunits of a serine-rich glycoprotein, Fap1. Previous studies revealed that a chromosomal region located downstream of fap1 is involved in the secretion and glycosylation of Fap1. In this study, we investigated the role of a glycosylation-associated gene, gap3, in Fap1 biogenesis. METHODS: A gap3 non-polar mutant was constructed by insertional inactivation. The phenotype of the mutant and the subcellular distribution of its products were investigated. The binding ability of the mutant was tested with saliva-coated hydroxyapatite (SHA). Electron microscopy was used to observe the morphological changes on the mutant cell surface. Confocal microscopy was utilized to determine biofilm formation ability. RESULTS: The gap3 mutant produced a partially glycosylated Fap1 precursor, that was less stable than mature Fap1. The Fap1 precursor was distributed in all subcellular fractions including the cell surface and culture medium although in decreased amounts. These data suggest a role for Gap3 in Fap1 glycosylation as well as a link between glycosylation and secretion of Fap1. The gap3 mutant had reduced binding to saliva-coated hydroxyapatite. Electron microscopy revealed that the gap3 mutant had lost its long fimbriae. Biofilm formation was also inhibited by the gap3 mutation. Fewer gap3 mutant cells adhered to the biofilm surface and microcolony formation was decreased. CONCLUSION: Gap3 is required for the complete glycosylation and secretion of Fap1, which is important for fimbrial assembly, bacterial adhesion, and in vitro biofilm formation.

Human cytomegalovirus enhances A. actinomycetemcomitans adherence to cells.

Adherence of Actinobacillus actinomycetemcomitans to epithelial cells is an important step in periodontal disease pathogenesis. Recent publications describe the subgingival presence of a wide array of viruses [e.g., human cytomegalo-virus (hCMV)]. Since viruses can increase cellular susceptibility for bacterial adherence, we investigated whether hCMV renders epithelial cells more prone to adherence by Actinobacillus actinomycetemcomitans. Cultivated HeLa and primary epithelial cells were shown to be semi-permissive for hCMV infection, which resulted in increased bacterial adherence. This increase correlated with viral concentrations, was evident in all Actinobacillus actinomycetemcomitans strains examined, and increased during the first 24 hrs, followed by a slight decrease. Immediate early antigen expression was not correlated with the increased adherence of Actinobacillus actinomycetemcomitans. The results confirmed our hypothesis that the adherence of Actinobacillus actinomycetemcomitans is influenced by hCMV in vitro.

Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans.

DNA adenine methyltransferase (DAM) plays critical roles in diverse biological pathways in gram-negative bacteria, and specifically in regulating the expression of virulence genes in several organisms. Actinobacillus actinomycetemcomitans plays an important role in the pathogenesis of juvenile and adult periodontal disease, yet little is known about its mechanisms of gene regulation. DAM is shown here to directly or indirectly affect well-known A. actinomycetemcomitans virulence factors. A mutant A. actinomycetemcomitans strain lacking the dam gene was created by homologous recombination and shows normal growth phenotypes when grown exponentially. This mutant strain has four sixfold increased levels of extracellular leukotoxin, altered cellular levels of leukotoxin, and significant changes in bacterial invasion of KB oral epithelial cells. These results provide a basis for further characterization of regulatory mechanisms that control A. actinomycetemcomitans virulence.

Influence of genetic background on transformation and expression of Green Fluorescent Protein in Actinobacillus actinomycetemcomitans.

BACKGROUND/AIMS: The development of an electro-transformation system and the construction of shuttle plasmids for Actinobacillus actinomycetemcomitans have enhanced the molecular analysis of virulence factors. However, inefficient transformation is frequently encountered. This study investigated the efficiency of electro-transformation and expression of Green Fluorescent Protein (GFP) in 12 different A. actinomycetemcomitans strains. The influence of the plasmid vector, serotype, and phenotype were the major factors taken into consideration. MATERIAL AND METHODS: Twelve serotyped A. actinomycetemcomitans strains were independently electro-transformed with two different Escherichia coli-A. actinomycetemcomitans shuttle plasmids (pVT1303 and pVT1304), both containing an identical ltx-GFPmut2 gene construct but a different backbone (pDMG4 and pPK1, respectively). The transformation efficiency, transformation frequency, and electro-transformation survival rate were determined by culture techniques. GFP expression was observed at the colony level by fluorescence microscopy. RESULTS: All strains could be transformed with both plasmids. However, major differences were observed for the transformation efficiency, transformation frequency, and electro-transformation survival rate between strains. The data demonstrated that plasmid vector, serotype, and phenotype are key players for obtaining a successful transformation. An inverted relationship between the electro-transformation survival rate and tranformation frequency was also observed. GFP expression was also influenced by phenotype, serotype and plasmid vector. CONCLUSIONS: The serotype of A. actinomycetemcomitans has an important influence on its survival after electro-transformation and on transformation frequency. The expression of GFP is strain and plasmid vector dependent.

Molecular strategies for fimbrial expression and assembly.

Fimbriae or pili are long, filamentous, multimeric macromolecules found on the bacterial cell surface. Bacteria express a diverse array of fimbriae or pili that are involved in bacterial adherence and invasion. Fimbriae can be categorized based on their modes of expression and assembly. Type I fimbriae and P pili are distributed peritrichously and translocated to the cell surface by a chaperone/usher pathway. Type 4 pili are located at the pole of the cell and assembled via the type II secretion system. Curli fimbriae are coiled surface structures assembled by an extracellular nucleation/precipitation pathway. Fimbriae of oral gram-negative and gram-positive bacteria have not been well-studied as compared with the fimbriae of enteric pathogens. Oral pathogens, such as Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Porphyromonas gingivalis, possess fimbriae that have been implicated in bacterial adhesion and invasion. These fimbriae are potential virulence factors in oral infectious processes. A. actinomycetemcomitans and E. corrodens have Type 4-like fimbriae, whereas P. gingivalis displays a unique type of fimbriae. To date, fimbriae of the oral primary colonizers, Actinomyces naeslundii and Streptococcus parasanguis, represent the only fimbriae characterized for any gram-positive bacteria. The putative major fimbrial subunits, FimA and FimP of A. naeslundii and Fap1 of S. parasanguis, contain a signal sequence and cell-wall-sorting signal. The presence of extensive dipeptide repeats in Fap1 makes it unique among fimbrial molecules. Based on experimental data, a nucleation/precipitation pathway is proposed for fimbrial biogenesis of both S. parasanguis and A. naeslundii, although we cannot rule out an alternative covalent linkage model. The model systems described in this review served as a framework for hypotheses for how the known molecular factors of fimbriae on oral bacteria may be expressed and assembled.

Streptococcus parasanguis fimbria-associated adhesin fap1 is required for biofilm formation.

The sanguis streptococci are primary colonizers of the tooth surface and thus form the foundation for the complex multiple species biofilm known as dental plaque. In addition, these bacteria can colonize native and prosthetic heart valves and are a common cause of endocarditis. Little is known about the molecular mechanisms governing multiple or single species biofilm development within this group of organisms. Using an in vitro assay for biofilm formation, we determined that (i) Streptococcus parasanguis FW213 can form biofilms on inert surfaces such as polystyrene and (ii) environmental and nutritional factors, such as glucose, affect S. parasanguis biofilm formation. Several isogenic mutants of FW213 were tested in the biofilm assay. Strains containing mutations in fap1, a gene encoding a protein required for assembly of fimbriae, were deficient in biofilm formation. Mutants defective in recA, PepO endopeptidase activity, or the production of a fimbriae-associated protein, FimA, were still capable of biofilm formation. Phase-contrast microscopy was used to follow biofilm development by wild-type and fap1 mutant strains on plastic coverslips over time. Wild-type FW213 attached to the surface, formed aggregates of cells, and eventually formed a dense layer of cells that included microcolonies. In contrast, few fap1 mutant cells were observed attached to the surface, and no cell aggregates or microcolonies were formed. These results suggest that the long peritrichous fimbriae of FW213 are critical for the formation of biofilms on solid surfaces.

Characterization of a streptococcal endopeptidase with homology to human endothelin-converting enzyme.

A gene encoding an endopeptidase from Streptococcus parasanguis FW213 has been cloned and shown to have high sequence homology to genes encoding mammalian metalloendopeptidases. The gene, designated S. parasanguis pepO, was cloned into the pET28a expression vector, resulting in a fusion of vector sequences encoding a hexahistidine tag at the carboxyl terminus. The recombinant PepO (rPepO) was expressed in Escherichia coli and purified using an Ni(2+) affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and immunoblot analysis revealed that other oral streptococci contain regions of DNA with homology to pepO and produce a protein with antigenic properties similar to that of FW213 PepO. Enzymatic activity assays indicated that only S. parasanguis species possess the ability to cleave metenkephalin, the natural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that S. parasanguis PepO is a member of the M13 category of metalloendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE-1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that S. parasanguis PepO is more similar to ECE-1 than to NEP.

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.

Identification of dipeptide repeats and a cell wall sorting signal in the fimbriae-associated adhesin, Fap1, of Streptococcus parasanguis.

Fap1, a fimbriae-associated protein, is involved in fimbriae assembly and adhesion of Streptococcus parasanguis FW213 (Wu et al., 1998). In this study, the sequence of the fap1 gene was resolved using a primer island transposition system. Sequence analysis indicated that fap1 was composed of 7659 nucleotides. The predicted Fap1 protein contains an unusually long signal sequence (50 amino acid residues), a cell wall sorting signal and two repeat regions. Repeat regions I and II have a similar dipeptide composition (E/V/I)S, composed of 28 and 1000 repeats respectively. The two regions combined accounted for 80% of the Fap1 coding region. The experimental amino acid composition and isoelectric point (pI) of Fap1 were similar to that predicted from the deduced Fap1 protein. Results of Northern analyses revealed that the fap1 open reading frame (ORF) was transcribed as a 7.8 kb monocistronic message. Insertional inactivation at the 3' end, downstream of the fap1 ORF, did not affect Fap1, fimbrial expression or bacterial adhesion. Insertional inactivation of fap1 immediately upstream of the repeat region II abolished expression of Fap1 and fimbriae, and was concurrent with a diminution in adhesion of FW213. Inactivation of the cell wall sorting signal of fap1 also eliminated long fimbrial formation and reduced the ability of FW213 to bind to SHA. Fap1 was no longer anchored on the cell surface. Large quantities of truncated Fap1 were found in the growth medium instead. These results suggest that the fap1 ORF alone is sufficient to support Fap1 expression and adhesion, and demonstrate that anchorage of Fap1 on the cell surface is required for long fimbriae formation. These data further document the role of long fimbriae in adhesion of S. parasanguis FW213 to SHA.

Use of the Actinobacillus actinomycetemcomitans leukotoxin promoter to drive expression of the green fluorescent protein in an oral pathogen.

The gene for the green fluorescent protein, gfp, was cloned, under the control of the Actinobacillus actinomycetemcomitans leukotoxin (ltx) promoter, in the A. actinomycetemcomitans shuttle vector, pSU20. A actinomycetemcomitans containing the ltx-gfp construct emitted bright green fluorescence in the standard invasion assay using epifluorescence microscopy. These data demonstrate that the green fluorescent protein will be a useful tool for the live analysis of A. actinomycetemcomitans interactions with host cells, and that the ltx promoter can be used to drive the expression of non-A. actinomycetemcomitans genes.

Microtubules are associated with intracellular movement and spread of the periodontopathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans SUNY 465, the invasion prototype strain, enters epithelial cells by an actin-dependent mechanism, escapes from the host cell vacuole, and spreads intracellularly and to adjacent epithelial cells via intercellular protrusions. Internalized organisms also egress from host cells into the assay medium via protrusions that are associated with just a single epithelial cell. Here we demonstrate that agents which inhibit microtubule polymerization (e.g., colchicine) and those which stabilize polymerized microtubules (e.g., taxol) both increase markedly the number of intracellular A. actinomycetemcomitans organisms. Furthermore, both colchicine and taxol prevented the egression of A. actinomycetemcomitans from host cells into the assay medium. Immunofluorescence microscopy revealed that protrusions that mediate the bacterial spread contain microtubules. A. actinomycetemcomitans SUNY 465 and 652, strains that are both invasive and egressive, interacted specifically with the plus ends (growing ends) of the filaments of microtubule asters in a KB cell extract. By contrast, neither A. actinomycetemcomitans 523, a strain that is invasive but not egressive, nor Haemophilus aphrophilus, a noninvasive oral bacterium with characteristics similar to those of A. actinomycetemcomitans, bound to microtubules. Together these data suggest that microtubules function in the spread and movement of A. actinomycetemcomitans and provide the first evidence that host cell dispersion of an invasive bacterium may involve the usurption of host cell microtubules.

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.

Actinobacillus actinomycetemcomitans may utilize either actin-dependent or actin-independent mechanisms of invasion.

Actinobacillus actinomycetemcomitans is an important pathogen implicated in juvenile and adult periodontal diseases. An important virulence factor of A. actinomycetemcomitans is the ability to invade human oral epithelial cells. A clinical isolate, A. actinomycetemcomitans SUNY 465, has previously been shown to enter epithelial cells by an actin-dependent mechanism. The internalized bacteria are surrounded by an actin halo upon entry. These data are consistent with the mode of entry associated with many enteric pathogens. We tested the effects of cytochalasin D, an inhibitor of the actin microfilament network, on bacterial entry to determine whether this mode of entry was common to other A. actinomycetemcomitans clinical isolates. Cytochalasin D was added prior to infection. A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065 exhibited enhanced ability to enter epithelial cells in the presence of cytochalasin D. Immunofluorescent labeling of bacteria and host cell actin confirmed that actin was not being mobilized by the entry of A. actinomycetemcomitans SUNY 523. Inhibitors of receptor-mediated endocytosis inhibited invasion of A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065. Microtubule effectors did not inhibit invasion of A. actinomycetemcomitans. A. actinomycetemcomitans SUNY 523, but not A. actinomycetemcomitans 4065, was deficient in exit from epithelial cells as determined by the absence of organisms in the assay medium. These data suggest that A. actinomycetemcomitans strains utilize at least two distinct mechanisms for entry into epithelial cells, and that A. actinomycetemcomitans SUNY 523 may be defective in exit and cell-to-cell spread.

Streptococcus parasanguis pepO encodes an endopeptidase with structure and activity similar to those of enzymes that modulate peptide receptor signaling in eukaryotic cells.

Studies in our laboratory have identified two fimbria-associated adhesins, FimA and Fap1, of Streptococcus parasanguis FW213. In this study, we isolated and sequenced DNA fragments linked to fimA to determine if they contained additional factors associated with adherence, virulence, or survival in the host. An open reading frame just upstream and divergently transcribed from the fimA operon was identified and named pepO. Northern hybridization indicated that pepO is transcribed as a monocistronic message. pepO encodes a predicted 631-amino-acid protein with a molecular mass of approximately 70.6 kDa. PepO contains the essential motif HEXXH, typical of many zinc-dependent metalloproteases and metallopeptidases. PepO has significant sequence identity to mammalian metallopeptidases, including endothelin-converting enzyme, which converts a potent vasoconstrictor into its active form, and neutral endopeptidase (NEP), which is involved in terminating the activity of opioid peptides. The opioid peptide metenkephalin is a natural substrate of NEP. Cell extracts of FW213 cleaved metenkephalin at the same site as does NEP, while an extract from an insertionally inactivated pepO mutant did not. These results indicate that FW213 pepO encodes an enzyme with activity similar to that of known mammalian endopeptidases. Phylogenetic analysis of PepO and its homologues suggests lateral genetic exchange between bacteria and eukaryotes.

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.

Construction and analysis of a Streptococcus parasanguis recA mutant: homologous recombination is not required for adhesion in an in vitro tooth surface model.

PCR was used to amplify an internal region of the recA gene from Streptococcus parasanguis FW213. The PCR fragment was used as a probe to recover the entire streptococcal recA gene from an S. parasanguis genomic library, and the sequence of the gene was determined. The deduced product of the S. parasanguis recA gene showed a high degree of amino acid identity with other prokaryotic RecA proteins. The cloned recA sequence was disrupted in vitro by insertional mutagenesis, and the mutated allele was then introduced into the S. parasanguis chromosome by homologous recombination. Results of Southern hybridizations confirmed the replacement of the wild-type recA gene with the mutated allele. The recA mutant strain was considerably more sensitive to UV light than the parental strain, and this phenotype was consistent with a mutation in recA. The S. parasanguis recA mutant showed no reduction in its ability to adhere in the in vitro tooth surface model, saliva-coated hydroxylapatite (SHA), or in its ability to express the fimbria-associated adhesin Fap1. These results demonstrate that in vitro attachment of S. parasanguis FW213 to SHA and expression of Fap1 are recA independent.

Characterization of serologically nontypeable Actinobacillus actinomycetemcomitans isolates.

Our previous studies have shown that Actinobacillus actinomycetemcomitans isolates of a given arbitrarily primed PCR (AP-PCR) genotype belong to the same serotype (of serotypes a through e). In the present study we investigated whether the AP-PCR genotypes of nonserotypeable A. actinomycetemcomitans isolates match those of the serotypeable isolates. The isolates were additionally characterized by restriction analysis of the apaH PCR amplification products. The material included 75 nonserotypeable and 18 serotypeable A. actinomycetemcomitans isolates from 34 epidemiologically unrelated subjects. The serotypeable isolates were obtained from subjects who also harbored nonserotypeable isolates. Eight AP-PCR genotypes were distinguished among the isolates; six genotypes matched those detected in our previous studies, whereas two genotypes were new. Intraindividually, the A. actinomycetemcomitans isolates produced identical AP-PCR banding patterns, regardless of whether they were serotypeable or nonserotypeable, in 22 of 23 subjects participating with multiple isolates. AP-PCR genotype 3, corresponding to serotype c, was by far the most common among the nonserotypeable isolates (62% of subjects). Results obtained with the apaH restriction analysis confirmed the results obtained with AP-PCR for 31 of the 34 subjects. The results suggest that nonserotypeable A. actinomycetemcomitans isolates originate from serotypeable isolates, especially from serotype c isolates, and the likelihood of the existence of additional serotypes is small.