Members of the genus Actinomyces are predominant primary colonizers of the oral cavity and play an important role in initiating plaque development. These bacteria have evolved unique mechanisms that favor colonization and persistence in this micro-environment. The expression of cell-surface fimbriae is correlated with the ability of these bacteria to adhere to specific receptors on the tooth and mucosal surfaces, and to interact with other plaque bacteria. The elaboration of sialidase is thought to enhance fimbriae-mediated adherence by unmasking the fimbrial receptors on mammalian cells. The presence of certain cell-associated or extracellular enzymes, including those involved in sucrose or urea metabolism, may provide the means for these bacteria to thrive under conditions when other growth nutrients are not available. Moreover, these enzyme activities may influence the distribution of other plaque bacteria and promote selection for Actinomyces spp. in certain ecological niches. The recent development of a genetic transfer system for Actinomyces spp. has allowed for studies the results of which demonstrate the existence of multiple genes involved in fimbriae synthesis and function, and facilitated the construction of allelic replacement mutants at each gene locus. Analyses of these mutants have revealed a direct correlation between the synthesis of assembled fimbriae and the observed adherence properties. Further genetic analysis of the various enzyme activities detected from strains of Actinomyces should allow for an assessment of the role of these components in microbial ecology, and their contribution to the overall success of Actinomyces spp. as a primary colonizer and a key player in oral health and disease.
Bacteriol. 170:3803-3809, 1988). This homology included several different conserved sequences of up to eight identical amino acids that were distributed in both the amino-and carboxy-terminal thirds of each Actinomyces fimbrial subunit. These findings indicate that the different types of fimbriae on these gram-positive bacteria share a common ancestry.
The type 1 fimbriae of Actinomyces viscosus mediate the adherence of this organism to saliva-treated hydroxyapatite. The gene encoding a putative subunit of this fimbrial adhesin was cloned in Escherichia coli, and its product was examined. A. viscosus T14V chromosomal DNA was partially restricted with Sau3AI and cloned into E. coli JM109 by using the plasmid vector pUC13. Two Two immunologically distinct types of fimbriae on the gram-positive oral bacterium Actinomyces viscosus T14V have been identified (5). Those designated as type 1 are thought to be the principal adhesin for bacterial attachment to the tooth surface. This has been inferred from the ability of specific type 1 fimbria antibody to inhibit the attachment of A. viscosus to saliva-treated hydroxyapatite (8) and by the demonstration that mutants lacking type 1 fimbriae fail to attach (J. 0. Cisar, A. E. Vatter, W. B. Clark, S. H. Curl, S. Hurst-Calderone, and A. L. Sandberg, manuscript in preparation). In contrast to type 1, the type 2 fimbriae are the sites of a lactose-sensitive lectin activity involved in the interactions of Actinomyces spp. with certain streptococci (5) as well as with sialidase-treated mammalian cells (4,24).While functional activities have been associated with each type of A. viscosus fimbria, these adhesins have not been structurally characterized because isolated fimbriae are resistant to complete dissociation by various means (5). Therefore, cloning of A. viscosus genes in Escherichia coli has been initiated as an approach to the identification of the fimbrial subunits. In this respect, a type 2 fimbrial gene has recently been cloned into a cosmid vector by Donkersloot et al. (10) and the encoded protein (molecular weight, 59,000) has been identified by its reaction with anti-type 2 fimbria antibody. The cosmid library was also screened for recombinant clones that expressed the type 1 fimbrial antigen, but none were detected. The present study describes the cloning of the type 1 fimbrial subunit gene of A. viscosus T14V by using pUC as the expression vector. rotor. Fractions of 200 RI were collected, and 25 pI of each fraction was analyzed by agarose gel electrophoresis. Fractions containing DNA fragments of less than 10 kb were pooled, concentrated, and dissolved in 10 mM Tris-1 mM EDTA (pH 8.0) (TE) for ligation. Plasmid pUC13 (restricted with BamHI and dephosphorylated with bacterial alkaline phosphatase) was purchased from Boehringer Mannheim Biochemicals, Indianapolis, Ind. Sau3AI-restricted A. viscosus DNA and BamHIrestricted pUC13 were combined (3:1 molar ratio) and incubated at 14°C with 2 U of T4 DNA ligase (Bethesda Research Laboratories) in 20 pul of ligation buffer (18). The ligation mixture was used to transform E. coli JM109 competent cells prepared by the method of Hanahan (12). Portions of transformed cells were plated onto LB (18) agar plates containing ampicillin (50 pug/ml), 5-bromo-4-chloro-3-indolyl-p-Dgalactopyranoside (X-Gal; 0.005%), and isopropyl-p-Dthiogalactopyranoside (IPTG; 0.25 mM).Plasm...
A genomic library of Actinomyces naeslundii WVU45 DNA in Escherichia coli was screened for antigen expression with rabbit antibody against A. naeslundii fimbriae. Western blotting (inmunoblotting) of one recombinant clone carrying a 13.8-kilobase-pair insert revealed a 59-kilodalton (kDa) immunoreactive protein.A protein of similar electrophoretic mobility was detected from the isolated fimbrial antigen. Expression of the 59-kDa cloned protein in E. coli was directed by a promoter from the insert. The DNA sequence of the subunit gene was determined, and an open reading frame of 1,605 nucleotides was identified which was preceded by a putative ribosome-binding site and followed by two inverted repeats of 14 and 17 nucleotides, respectively. The reading frame encoded a protein of 534 amino acids (calculated molecular weight, 57,074), and the N-terminal sequence resembled that of a signal peptide. The presence of a 32-amino-acid signal peptide was indicated by amino-terminal sequencing of the fimbriae from A. naeslundii. The sequence, as determined by Edman degradation, was identical to that deduced from the DNA sequence beginning at predicted residue 33 of the latter sequence. Moreover, the amino acid composition of the predicted mature protein was similar to that of the isolated fimbriae from A. naeslundii. Thus, the cloned gene encodes a subunit of A. naeslundii fimbriae.Typical human strains of the gram-positive oral bacterium Actinomyces naeslundii elaborate a single type of fimbriae which is antigenically and functionally related to the type 2 but not type 1 fimbriae of Actinomyces viscosus T14V (9). Whereas the type 1 fimbriae of A. viscosus mediate bacterial adsorption to saliva-treated hydroxyapatite (10,11), the type 2 fimbriae of A. viscosus and A. naeslundii are associated with a lectin activity that mediates coaggregation of these organisms with certain oral streptococci (7). Further studies have shown that the fimbrial lectin is also involved in bacterial interactions with sialidase-treated erythrocytes (8), buccal epithelial cells (3, 4), and polymorphonuclear leukocytes (31,32 The present study was initiated to gain insight into the structure of the fimbriae from A. naeslundii WVU45 (ATCC 12104), a strain that is typical of those found on oral mucosal surfaces (3,14). The gene cloning and sequencing experi-* Corresponding author. ments described in this paper identified a 54-kDa protein as the major fimbrial subunit.MATERIALS AND METHODS Bacterial strains and plasmids. A. naeslundii WVU45 (ATCC 12104) (9) was the source of target DNA, and E. coli JM109 (37) was the host strain in all transformation and transfection procedures. Plasmid pUC13 was used as the vector to prepare the A. naeslundii clone bank in E. coli, and pUC18 and pUC19 were used in subsequent subcloning experiments. Bacteriophage vectors M13mpl8 and M13 mpl9 (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) were used in DNA sequencing studies.Antigens and antibodies. Type 2 fimbriae of A. naeslundii WVU45 were prepared as desc...
The nucleotide sequence of the Actinomyces naeslundiiT14V type 2 fimbrial structural subunit gene, fimA, and the 3′ flanking DNA region was determined. The fimA gene encoded a 535-amino-acid precursor subunit protein (FimA) which included both N-terminal leader and C-terminal cell wall sorting sequences. A second gene, designated orf365, that encoded a 365-amino-acid protein which contained a putative transmembrane segment was identified immediately 3′ to fimA. Mutants in which either fimA or orf365 was replaced with a kanamycin resistance gene did not participate in type 2 fimbriae-mediated coaggregation with Streptococcus oralis34. Type 2 fimbrial antigen was not detected in cell extracts of thefimA mutant by Western blotting with anti-A. naeslundii type 2 fimbrial antibody, but the subunit protein was detected in extracts of the orf365 mutant. The subunit protein detected in this mutant also was immunostained by an antibody raised against a synthetic peptide representing the C-terminal 20 amino acid residues of the predicted FimA. The antipeptide antibody reacted with FimA isolated from the recombinant Escherichia coliclone containing fimA but did not react with purified type 2 fimbriae in extracts of the wild-type strain. These results indicate that synthesis of type 2 fimbriae in A. naeslundii T14V may involve posttranslational cleavage of both the N-terminal and C-terminal peptides of the precursor subunit and also the expression oforf365.
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