Expression of Actinomyces viscosus antigens in Escherichia coli: cloning of a structural gene (fimA) for type 2 fimbriae

Abstract

A cosmid gene library of Actinomyces viscosus T14V was prepared in Escherichia coli to examine the expression ofA. viscosus antigens and to gain insight idto the structure ofA. viscosus type 1 and type 2 fimbriae. Out of this library of 550 clones, 28 reacted in a colony immunoassay with antibodies against A. viscosus cells. The proteins responsible for these reactions were identified in three clones. Clones AV1209 and AV2009 displayed nonfimbrial antigens with subunits of 40 and 58 kilodaltons, respectively. Clone AV1402 showed a 59-kilodalton protein that reacted with monospecific antibody against type 2 fimbriae and that comigrated with a subunit of type 2 fimbriae during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that AV1402 expresses a gene (fimA) for a subunit of A. viscosus type 2 fimbriae.Actinomyces viscosus is a gram-positive bacterium that is often found in high numbers in human dental plaque, where it may play a role in periodontal infections (reviewed in reference 14). The colonization of teeth by this microbe involves two antigenically distinct types of fimbriae (type 1 and type 2). Type 1 fimbriae selectively promote adherence to saliva-coated hydroxyapatite (9, 28). In contrast, type 2 fimbriae mediate attachment of A. -viscosus to certain streptococci that predominate in developing dental plaque (7). This latter property depends on a lactose-sensitive lectin activity associated with type 2 fimbriae (6, 8, 25) and complementary carbohydrate-containing receptors on the streptococcal surface. Whereas the existence and function of each type of fimbriae has been clearly demonstrated, little is known about their subunits. This is largely due to their resistance to dissociation by methods that will extensively depolymerize fimbriae from gram-negative organisms (23,28). To identify and characterize putative subunits and precursors of A. viscosus fimbriae, a study to clone the respective genes was undertaken.MATERIALS AND METHODS Strains. Escherichia coli HB101 (3) and A. viscosus T14V (6, 8) have been described previously.Preparation of A. viscosus T14V chromosomal DNA. Cells were grown in 400 ml of medium and treated with lysozyme as described (5). They were suspended in 40 ml of 10 mM Tris-1 mM EDTA (pH 8.0), and lysed by adding 4.4 ml of 10% sodium dodecyl sulfate (SDS) with a 15-min incubation at 65°C. In this and subsequent steps care was taken not to shear the DNA. The viscous lysate was extracted with an equal volume of phenol saturated with 3% NaCl and then with chloroforn-isoamyl alcohol (24:1 [vol/vol]). After addition of 2 volumes of ethanol, the precipitate was collected by centrifugation and dissolved in 25 ml of 10 mM Tris-1 mM EDTA-10 mM NaCl (pH 8.0). After treatment with RNase A (100 p.g/ml) and RNase Ti (100 U/ml) for 1 h at 37°C, proteinase K was added (100 ,ug/ml), and the incubation was continued at 55°C for another hour. The solution was extracted with phenol and chloroform as described previously, adjusted to 3 M ammonium acetate, and left at 4°C for ...

Expression of Streptococcus sanguis antigens in Escherichia coli: cloning of a structural gene for adhesion fimbriae

“…Many higher-molecular-mass polypeptides were noted in the Western immunoblot of type 1 fimbriae. Type 2 fimbriae of A. viscosus have been similarly purified and cloned, and a similar pattern of multiple bands also has been noted (6). These are the only two reports on the biochemistry of gram-positive fimbriae, but the data do suggest that grampositive fimbriae are very distinct from gram-negative fimbriae.…”

Metal-dependent repression of siderophore and biofilm formation inActinomyces naeslundii

“…This ORF was designated AmdR gene or amdR. Chromosomal DNA was isolated from A. naeslundii as described by Donkersloot et al (1985). Primers based on the sequence of the amdR gene were designed to incorporate a 5 0 BamHI site and a 3 0 KpnI site flanking the amdR orf 5 0 -CACCTCAAGCAGGATCCCACCCCATGACC-3 0 and 5 0 -GCCGTCAGCGGTACCGCGAGACAG-3 0 .…”

Mutants of Actinomyces viscosus T14V lacking type 1, type 2, or both types of fimbriae

“…Each fimbria-specific monoclonal antibody used in these determinations reacts with the corresponding fimbrial subunit. These proteins have molecular weights near 60,000 (11,29), and each has been detected by Western blotting (immunoblotting) of crude soluble extracts prepared from cells that express specific fimbriae but not from those that are fimbria deficient (results not presented). Thus, the genetic defects in the various mutant strains seem to affect the biosynthesis of either the type 1 or type 2 fimbrial subunit.…”