Bacterial strains and culturing conditions
The A. naeslundii genospecies 1 strains Pn-22-E (CCUG 34193), Pn-6-N (CCUG 33519), P-5-N (CCUG 33914), ATCC 12104 (CCUG 32832), and A. naeslundii genospecies 2 strains P-1-N (CCUG 33910), P-1-K (CCUG 32838), LY7 (CCUG 33934) and T14V used were generally from the Culture Collection, University of Göteborg (CCUG), Sweden. A.odontolyticus PK984 was provided by Dr. Kolenbrander, National institute of health/NIDCR, Bethesda, USA. The strains were cultured on Columbia-II-agar base plates (Becton Dickinson and Co., Cockeysville, MD), supplemented with 3 % human red blood cells, at 37°C in candle jar.
Cloning and sequencing of fimA genes
Chromosomal DNA was isolated from bacteria and purified as described [14]. Gene segments were amplified by PCR, using standard protocols, by use of primers both inside and outside the fimA open reading frame (primer sequences are available upon request). All fimA PCR fragments were cloned into pGEM-T vectors using T4 DNA ligase (Promega Corp., Madison, WI), except for fimA from A. odontolyticus for which fragments were cloned into pCR 2.1-TOPO vectors using Invitrogen TOPO TA Cloning kit (Invitrogen, Carlsbad, CA).
Plasmid DNA was isolated using the QIAprep® Spin Miniprep Kit (Qiagen GmbH, Hilden, Germany) and the size of DNA inserts were subsequently confirmed by SalI I and Nco I or only EcoR1 (Invitrogen) cleavage. Sequencing was done using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Life Science, Cleveland, OH), and were performed according to the manufacturer's instruction. The DNA fragments were sequenced in both directions.
Sequence and motif analysis
Complete open reading frames of fim A nucleotide sequences were analyzed using the Wisconsin Package (version 9.0) from the Genetics Computer Group (GCG, University of Wisconsin, Madison, WI), except for fimA from A. odontolyticus which was analysed with The Molecular Toolkit [34].
Sequence alignments and dendrogram was made using Clustal W (1.83) multiple sequence alignment [35].
Signal peptide, LPXTG motif and proline-rich region were analyzed using bioinformatics tools [36–38].
Nucleotide sequence accession numbers
The GenBank accession numbers for genes presented in this paper are: for fim A: strain Pn-22-E [GenBank: DQ425098], strain Pn-6-N [GenBank: DQ425100], strain P-5-N [GenBank: DQ425097], strain P-1-N [GenBank: DQ425102], strain P-1-K [GenBank: DQ425099], strain LY7 [GenBank: DQ425101], strain ATCC 12104 [GenBank: M 21976], strain T14V [GenBank: AF019629], and strain PK984 [GenBank: DQ425103]; for fimP; strain P-1-K [GenBank: AF107019], strain LY7 [GenBank: AF10720], A. viscosus 19246 [GenBank: M 21976]; for SpaH: C. diphteriae NCTC 13129 [GenBank: NP_940533].
Agglutination of Actinomyces by antisera
Agglutination of Actinomyces cells by rabbit antiserum R70-3 specific for type-2 fimbriae of A. naeslundii strain ATCC 12104 and pre-immunization serum R70-0 was a gift from Dr. Cisar, National institute of health/NIDCR, Bethesda, USA. Antisera (1 μml) was added to Actinomyces cells (1 ml, OD550 = 0.7) suspended in coaggregation buffer (150 mM NaCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 1.0 mM Tris pH 8.0 and 0.02 % sodium azide). After incubation for one hour at room temperature on slow rotation, agglutination was measured by recording the optical density at 550 nm for 90 min, giving a total agglutination time of 150 min.
Cloning, expression and purification of recombinant FimA
The fimA gene was PCR-amplified from genomic DNA from strain 12104 and cloned into the expression vector pETM11 (EMBL, Hamburg, Germany). The resulting construct encodes a protein (recombinant FimA or rFimA) with an N-terminal hexa-histidine tag, an 18 residue long linker and the FimA protein excluding the N-terminal signal sequence and the C-terminal transmembrane helix. E. coli BL21 (DE3) (Novagen, Madison, WI) was transformed with the pETM11-rFimA construct and grown at 37°C to optical density (OD600) of 0,5. Protein expression was induced with 0.5 mM isopropyl β-D-thiogalactoside for four hours at 30°C. Cultures were harvested and the cells lysed by sonication. rFimA was purified by Ni-agarose chromatography (Quiagen) and elution with imidazole. The protein was further purified by a size exclusion on a Superdex 26/60 column (Amersham, Uppsala, Sweden). The rFimA fractions were analyzed by SDS-PAGE and by Western blot.
Western blot of whole cell extracts and recombinant FimA
A suspension (300 μml) of bacterial cells (3 × 109 cells) was sonicated 4 × 15 seconds using a Branson sonicator (Branson ultrasonics corporation, Danbury, CT). Proteins were precipitated with acetone for 1 hour at -20°C, centrifuged and dissolved in 50 μml sample buffer (62.5 mM Tris, 10.1% glycerol, 2.0 % SDS, 5 mM dithiothreitol and 0.01% pyronin) by boiling for 10 min. After centrifugation, the supernatant was electrophoretically separated on a 4 to 20 % or 4 to 15 % (whole cell extracts and recombinant FimA, respectively) polyacrylamid gel using Tris-glycine buffer (25 mM Tris, 192 mM glycine, and 0.1 % SDS, pH 8.3) at 15 mA. The separated proteins were blotted onto membranes (Immobilon PVDF membrane, Millipore Corp., Bedford, MA) using a transblotter. The membranes were blocked with 5 % non-fat dried milk overnight and incubated with antisera R70-3 (diluted 1:20,000) overnight followed by five washes in 10 mM phosphate buffered saline (PBS), pH 6.8, 0.5 % Tween. Peroxidase conjugated goat anti-rabbit IgG was used as secondary antibody (Dakopatts a/s, Glostrup, Denmark) and a chemiluminescent substrate (Supersignal Substrate; Pierce, Rockford, Il) was used to visualize immobilized antibodies.
Hemagglutination
Equal volumes (10 μml of each) of suspensions of bacterial cells (3 × 109 cells/ml PBS or reciprocal dilutions) and human, goat or chicken erythrocytes (4 % erythrocyte suspension in PBS) were mixed and agitated gently for 1 min on a glas slide. Erythrocytes were depleted of sialic acid by incubation with 1 unit/ml Clostridium perfringens neuraminidase (Sigma Chemical Co, St Louis, MO) for 30 min. at 37°C. Agglutination was scored visually; 0 = no agglutination, 1 = weak agglutination, 2 = moderate agglutination, 3 = strong agglutination, and 4 = very strong agglutination.