Cloning and expression of a type 1 fimbrial subunit of Actinomyces viscosus T14V

Abstract

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...

Immunochemical and functional studies of Actinomyces viscosus T14V type 1 fimbriae with monoclonal and polyclonal antibodies directed against the fimbrial subunit

“…0. Cisar and others cloned A. viscosus T14V gene (Yeung et al, 1987). An antibody raised against the cloned protein also reacted with type 1 fimbriae, and the pattern observed by Western blotting was comparable to the ladder of bands seen with antibody against intact fimbriae.…”

“…All buffers contained 0.1 mM-PMSF (Sigma) and 0.25% CHAPS (Sigma) rather than Tween 20 which had been used previously (Yeung et al, 1987). X-irradiated syngeneic recipients (Fox et al, 1981) and one day later, each recipient was boosted with 50 pg fimbriae administered intravenously.…”

“…The cloned A. viscosus T14V type 1 fimbrial subunit was isolated from a soluble extract of E. coli MY3833 by immunoaffinity chromatography as described above and further purified by Sephacryl S200 (Pharmacia) gel filtration (Yeung et al, 1987). All buffers contained 0.1 mM-PMSF (Sigma) and 0.25% CHAPS (Sigma) rather than Tween 20 which had been used previously (Yeung et al, 1987).…”

Molecular Strategies for Fimbrial Expression and Assembly

“…Analysis of these data demonstrates that the cell-wall sorting signal is important for FapI and fimbriae presentation on the cell surface and for adhesion of S. parasanguis FW2 13 to SHA Moreover, the anchor-domain-deficient mutant secretes increased amounts of Fapl into the growth medium, providing additional evidence that the cell-wall sorting signal of Fapl is important in Fapl anchorage (Wu and Fives-Taylor, 1999 . Almost nothing is known regarding fimbriae biogenesis in Gram-positive bacteria in general, since their fimbriae have been exceedingly difficult to dissociate (Elder and Fives-Taylor, 1986;Yeung et al, 1987). Biogenesis of fimbriae of the Gram-positive S. parasanguis FW213 appears to be unique in several aspects.…”

“…(Donkersloot et al, 1985;Yeung et al, 1987 (Yeung and Ragsdale, 1997;Yeung et al, 1998 (2) Presence of cell-wall sorting signals in fimbrial subunits and a sortase-like protein in a fimbrial locus Both Actinomyces fimbrial subunits display a cell-wall sorting signal at their carboxyl-termini. This LPXTG region is highly conserved in Gram-positive bacterial cell-surface proteins and functions as an anchor to present the proteins on the surface of the cell.…”