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Actinobacillus actinomycetemcomitans is implicated as a pathogen in one of the most prevalent diseases of humansperiodontitis (9). However, surprisingly little is known about the virulence factors of this organism (16). It is well recognized that bacterial exported proteins play key roles in many bacterial functions and are particularly important in the processes of infection (3). In order to gain some idea of the nature of the genes encoding exported proteins in A. actinomycetemcomitans use has been made of a plasmid-based phoA gene fusion system initially developed for studying protein secretion in Escherichia coli (5). This methodology has been used to identify and characterize exported proteins in a number of gram-negative and gram-positive bacteria (1, 4, 7, 10, 12), including a recent report of exported proteins of A. actinomycetemcomitans (8).

Bacterial strains and media. A. actinomycetemcomitans NCTC 9710 was grown on brain heart infusion agar (Oxoid) supplemented with 5% (vol/vol) horse blood in a carbon dioxide-rich atmosphere for 48 h. E. coli JM107 was grown on nutrient agar (Oxoid). E. coli CC118 was grown on nutrient agar containing erythromycin (ERY) (150 µg/ml).

Isolation of cell-associated proteins. A fraction containing the cell surface-associated proteins of A. actinomycetemcomitans was prepared by gentle saline extraction as described in reference (6).

Production of rabbit antisera to A. actinomycetemcomitans cell surface-associated proteins. Three rabbits were immunized with this saline wash of A. actinomycetemcomitans in a nonulcerogenic adjuvant and boosted with material in Freund's incomplete adjuvant. Animals were bled, and titers of antisera were assessed by enzyme-linked immunosorbent assay at various intervals, until titers peaked. Rabbits were then exsanguinated and sera were prepared using conventional means. The rabbit antisera were pooled and extensively immunoadsorbed with E. coli until binding to this bacterium was extinguished. Animal experimentation was done under United Kingdom Home Office regulations.

Construction of phoA fusion library. Chromosomal DNA was prepared from A. actinomycetemcomitans using standard methods (described in reference 13). Plasmid libraries containing DNA from A. actinomycetemcomitans were prepared in the plasmid vector pHRM104 (23). Chromosomal DNA was partially digested with Sau3AI for 3 h at 37°C. The vector DNA was extracted from 200 ml of overnight cultures of E. coli JM107 using a Midi plasmid preparation kit (Qiagen Ltd, Crawley, United Kingdom). The plasmid was linearized by digestion with BamH1 for 2 h at 37°C. The partially digested chromosomal DNA was ligated with the linearized pHRM104 overnight in a ligation mixture consisting of 40 µl (5 µg) of Sau3AI fragments, 20 µl (1 µg) of linearized plasmid, 6 µl of ligase buffer, and 2 µl of T4 DNA ligase. To confirm that digestion and ligation had taken place, aliquots were removed from the various reactions and analyzed by agarose gel electrophoresis.

Transformation of E. coli CC118. Competent E. coli CC118 cells were prepared and transformed with the ligated DNA. The transformed cells were transferred into 5 ml of nutrient broth and incubated at 37°C with shaking for only 1.5 h. Cells were plated onto nutrient agar containing ERY (150 µg/ml) and 5-bromo-chloro-3-indoyl phosphate (XP) (40 µg/ml) a substrate for alkaline phosphatase, and incubated overnight at 37°C. Alkaline phosphatase-positive colonies were picked and subcultured onto fresh ERY-XP plates, and the presence of alkaline phosphatase activity confirmed. Plasmid DNA was extracted from individual alkaline phosphatase positive colonies and prepared using the Qiaprep spin miniprep kit. The sizes of the inserts in the recombinant plasmids were determined by digesting the DNA with KpnI and running the digests on agarose gels.

Sequencing of alkaline phosphatase-positive clones. An oligonucleotide (5'-CGGTTTTCCAGAACAGG-3') specific to the 5' end of the truncated phoA gene in pHRM104 was used to sequence over the fusion junction and into the 3' end of the A. actinomycetemcomitans insert DNA. Double-stranded plasmid DNA was sequenced using dye terminator chemistry and cycle sequencing using the BigDye terminator kit according to the manufacturer's instructions (ABI Perkin Elmer). The reactions were run on an ABI 377 sequencer.

Bioinformatics. The DNA sequences were analyzed using BLAST searches of the A. actinomycetemcomitans database at the University of Oklahoma (http://www.genome.ou.edu/act.html) and also using the PEDANT database, which contains complete and partial genome sequences of bacteria, including A. actinomycetemcomitans and Haemophilus influenzae (http://pedant.mips.biochem.mpg.de/). PEDANT was also the source of the numbering for the A. actinomycetemcomitans open reading frames (ORFs). The database of all derived protein sequences was also searched at the NCBI database. Segments containing the first 70 amino acids were searched for signal sequences using the SignalP program (http://www.expasy.ch/tools/). For those proteins that were negative on the SignalP programe, the transmembrane protein sequence analysis program DAS, TMpred, TMHMM, and HMMTOP on the Expasy Tools site (http://www.expasy.ch/tools/) were used.

Immunoscreening of phoA clones. A volume of 5 µl of each phoA clone was spotted onto nutrient agar containing ERY (150 µg/ml) and grown for 18 h at 37°C. Colony lifts were made onto 0.45-µm-pore-size nitrocellulose membranes (Nitrocellulose Extra; Sartorius), and these were blocked by immersion for 30 min in phosphate-buffered saline (PBS) containing 0.5% Tween 20 (PBS-T) and 5% skim milk powder. Membranes were washed three times in PBS-T for 10 min per wash. They were then incubated for 1 h at room temperature with immunoadsorbed rabbit antiserum (1:200 dilution) to the saline wash of A. actinomycetemcomitans. Membranes were then washed three times for 10 min each in PBS-T before being incubated for 1 h at room temperature in a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Sigma A2074). Following a further three washes in PBS-T, membranes were incubated with a commercial ECL Western blotting detection reagent (Amersham Pharmacia Biotech), and the enzymic reaction was determined by exposing the treated membranes to X-ray film.

Positive clones and analysis of insert sizes. A total of 80 clones, out of 8,000 screened, producing alkaline phosphatase fusion proteins were identified, with insert sizes ranging from 300 bp to 6 kb. Of these, 77 positive clones were sequenced.

Sequencing of clones. The protein sequences derived from the DNA sequences across the fusion junction were used in BLAST searches of the A. actinomycetemcomitans database and the full-length ORFs were obtained. The derived full-length protein sequence was then used in a BLAST search of all protein sequences at the NCBI database. Data analysis was confirmed by using the PEDANT database, which in most cases provided an annotation of the primary data from the University of Oklahoma A. actinomycetemcomitans genome database. The amount of protein fused to PhoA in each of the recombinants and the size of the full-length ORF, provided from genome databases, is shown in Tables 1 to 3. A number of the clones could not be sequenced, or the sequences showed that two different pieces of A. actinomycetemcomitans genomic DNA had become fused to phoA, and were not interpretable. A number of clones revealed the presence of identical DNA sequences. For example four clones contained the signal sequence of peptide methionine sulphoxide reductase. Tables 1 to 3 and Fig. 1 show the 48 nonredundant clones identified in this study.

View larger version (67K): [in this window] [in a new window]   FIG. 1.   Protein sequences of the four proteins with no homologies to other proteins in current sequence databases. A vertical arrow indicates the site of signal sequence cleavage using the SignalP V1.1 program. A horizontal arrow indicates that the C terminus of the protein is not known and the amino acid sequence shown is fused to the truncated PhoA at this point. Clone 26 ORF 1440 has no signal sequence cleavage site but has a weak membrane-spanning segment just prior to the point of fusion to PhoA. The clones which bound to the rabbit antisera raised to saline washes of A. actinomycetemcomitans have been identified.

Identification of proteins fused to phoA. Proteins fused to phoA could be divided into four groups. The first were those proteins that had homology to known H. influenzae proteins (Table 1). The second were those proteins that had homology to hypothetical proteins in the H. influenzae database (Table 2). The third were those proteins which had no homology to H. influenzae proteins but could be recognized by homology to other bacterial proteins (Table 3). The final group consisted of those proteins that had no homology to any proteins in the current sequence databases (Fig 1).

Immunoscreening of phoA clones. A high-titer polyclonal antiserum to a saline wash of A. actinomycetemcomitans was used to immunoscreen the phoA clones. Negative controls of E. coli CC118 containing unligated vector, as expected, failed to bind the antiserum. This antiserum bound to 22 out of the 48 clones identified in this study (Fig 2). These antibody-binding clones are identified in the tables by the emboldened clone numbers. All four of the clones showing no homology to proteins in sequence databases were recognised by this antiserum (Fig 1).

View larger version (163K): [in this window] [in a new window]   FIG. 2.   Typical results of screening phoA clones with a polyclonal antiserum to a saline wash of A. actinomycetemcomitans. Some clones bind to the antibody (+) while others show no reaction (). Clones which react with this antiserum have been denoted in the tables by emboldened clone numbers.