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Department of Periodontics, Endodontics and Dental Hygiene, University of Louisville School of Dentistry, Louisville, KY 40292, USA
Correspondence
Donald R. Demuth
drdemu01{at}louisville.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Slots et al., 1980; Zambon, 1985; Zambon et al., 1983, 1996), endocarditis (Block et al., 1973; Page & King, 1966) and subcutaneous abscesses (Page & King, 1966). A. actinomycetemcomitans expresses several virulence factors that may contribute to pathogenesis, including two toxins that may play a role in suppressing the host response to infection by targeting various components of the immune system. One of these, a cytolethal distending toxin (Cdt) similar to the Cdt of Haemophilus ducreyi, induces G2 arrest of proliferating human lymphocytes (Shenker et al., 1999, 2000). A. actinomycetemcomitans also expresses a leukotoxin that is a member of the repeats in toxin (RTX) family of host-specific cytolysins (Welch, 1991). The leukotoxin targets cells of the lymphocytic and monomyelocytic lineages expressing the β2-integrin LFA-1 (CD11a/CD18) (Karakelian et al., 1998; Lally et al., 1997, 1999) and a recent report suggests that it may also have haemolytic activity (Balashova et al., 2006). At high doses in vitro, intoxication of target cells by the leukotoxin leads to cell lysis via the development of membrane pores (Iwase et al., 1990; Korostoff et al., 1998, 2000), whereas lower doses of toxin perturb mitochondrial functions, leading to apoptosis of host cells (Korostoff et al., 1998, 2000).
Most A. actinomycetemcomitans strains express relatively low levels of leukotoxin and have been designated minimally leukotoxic (Brogan et al., 1994; Hritz et al., 1996). However, several A. actinomycetemcomitans strains have been isolated that exhibit a hyperleukotoxic phenotype. These strains exhibit significantly increased transcription of the ltx operon and express elevated levels of the leukotoxin (Brogan et al., 1994; He et al., 1999; Kolodrubetz et al., 1996). Hyperleukotoxic strains are also isolated at higher frequency from patients with localized aggressive periodontitis (Bueno et al., 1998; Haraszthy et al., 2000; Haubek et al., 1997, 2001), suggesting that the level of leukotoxin production may in part determine whether A. actinomycetemcomitans contributes to the development of periodontitis or is tolerated as a commensal in the oral flora.
The leukotoxin operon consists of four genes, ltxCABD, and has an arrangement typical of other RTX family toxins (Kraig et al., 1990; Lally et al., 1989). However, immediately upstream of ltxCABD is a co-transcribed ORF of 
450 bp designated orfA (or orfX) (Brogan et al., 1994; Kolodrubetz et al., 1996; Mitchell et al., 2003) that is capable of encoding a polypeptide of approximately 15 kDa of unknown function. Interestingly, the hyperleukotoxic phenotype of A. actinomycetemcomitans is correlated with insertions or deletions that do not affect the major structural genes of the ltx operon (ltxCABD) but occur within or upstream of orfA (Brogan et al. 1994; He et al., 1999). Other than the inserted or deleted segments, all of the remaining sequences in the ltx promoter and in orfA are virtually identical in minimally leukotoxic and hyperleukotoxic strains (Brogan et al., 1994; He et al., 1999). For example, the hyperleukotoxic JP2 strain expresses approximately 10-fold higher levels of cytotoxic activity than minimally leukotoxic strains and has been found extensively in localized aggressive periodontitis patients in United States, Northern African and European populations (Cortelli et al., 2005; Haraszthy et al., 2000; Haubek et al., 1997, 2001, 2004, 2007). This strain possesses a 530 bp deletion that truncates orfA (Brogan et al., 1994), suggesting either that orfA plays a role in regulating ltx expression or that the truncation of orfA in A. actinomycetemcomitans JP2 increases transcription simply by bringing the ltx promoter into closer proximity to ltxCABD (Kolodrubetz et al., 1996).
In contrast, analysis of clinical isolates from periodontitis patients in a Japanese population failed to detect the JP2 clone. Instead, hyperleukotoxic A. actinomycetemcomitans strains were identified that exhibited increased transcription of the ltx operon and possessed an intact copy of orfA. He et al. (1999) showed that these strains contained an insertion sequence related to IS1301 of Neisseria meningitidis in the ltx promoter upstream of orfA. Spontaneous loss of the IS element resulted in reduced expression of leukotoxin, similar to that of minimally leukotoxic strains (He et al., 1999), indicating that the hyperleukotoxic phenotype was dependent on the presence of IS1301. Integration of IS1301 occurred at the 5'-end of an AT-rich positive regulatory sequence and downstream from a cis-acting negative regulatory sequence in the ltx promoter (Mitchell et al., 2003). In addition, the IS element itself possesses an outwardly directed –35 sequence. However, Mitchell et al. (2003) showed that IS1301 does not functionally disrupt the AT-rich regulatory sequence and furthermore, that the outwardly directed –35 element does not influence transcription of ltx genes. These results led the authors to suggest that the hyperleukotoxic phenotype may arise from the uncoupling of the cis-acting negative regulator from the basal elements of the ltx promoter upon the acquisition of IS1301.
In this report, we show that replacing IS1301 with an equal length of random sequence does not affect the transcriptional activity of the ltx promoter, suggesting that physical displacement of the cis-acting negative regulator does not contribute to the hyperleukotoxic phenotype of IS1301 containing strains. Instead, we show that a –10-like sequence upstream of the transposase gene in IS1301 is necessary and sufficient for increased transcription of the ltx operon. In addition, reporter constructs containing frameshift mutations in orfA exhibited significantly reduced ltx promoter activity, suggesting that the OrfA polypeptide itself modulates expression of ltxCABD. Consistent with this, purified OrfA was shown to interact with the ltx promoter. Together, these results suggest that both IS1301 and orfA influence leukotoxin expression and may contribute to the hyperleukotoxic phenotype exhibited by some A. actinomycetemcomitans strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli cultures were grown in Luria–Bertani (LB) broth (Becton Dickinson) at 37 °C with aeration. A. actinomycetemcomitans strains were grown in brain-heart infusion (BHI) broth (Becton Dickinson) that was supplemented with 40 µg NaHCO3 ml–1 at 37 °C in a standing culture. Where indicated, media were supplemented with the appropriate antibiotics at the following concentrations: ampicillin, 100 µg ml–1, kanamycin, 25 µg ml–1, spectinomycin 80 µg ml–1. Plates containing 15 % (w/v) granulated agar (Becton Dickinson) were used where indicated. Plasmids were isolated from A. actinomycetemcomitans strains using the Wizard Plus Mini-Prep DNA purification system (Promega), following the protocol supplied by the manufacturer.
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. To facilitate directional cloning of the PCR amplicons, restriction sites (underlined in Table 2) were incorporated into all primers. The PCR products were initially cloned into pGEM-T Easy vector (Promega) using the TA sites provided. The resulting plasmids were transformed into E. coli DH5
and confirmed both by blue/white colour selection on LB agar containing IPTG and X-Gal, and by restriction digestion with EcoRI to release the insert fragment. Plasmid purifications were carried out using the Wizard Plus Miniprep DNA purification system (Promega). PCR products were subsequently excised using ClaI and XmaI sites and ligated into the acceptor plasmid pBluescript-orfA. pBluescript-orfA contains the 1000 bp ltx promoter of A. actinomycetemcomitans 652 (Brogan et al., 1994) with ClaI and XmaI restriction sites engineered in at the point of IS1301 insertion (Mitchell et al., 2003). Since the ltx promoter of strain JP2 is identical in sequence to that of strain 652, with the exception of the 530 bp deletion, a similar approach was used to produce the truncated ltx promoter of strain JP2 containing the ClaI and XmaI restriction sites. Thus, each of the PCR fragments that were generated was inserted into the appropriate ltx promoter at the site of IS1301 integration. The entire orfA/ltx promoter region containing the desired PCR fragments was then released by digestion with KpnI and BamHI and ligated into the E. coli/A. actinomycetemcomitans shuttle vector pYGKlacZ (Brogan et al., 1996). Recombinant reporter plasmids were transformed into E. coli DH5 and verified by restriction analysis. Reporter constructs were subsequently electroporated into competent A. actinomycetemcomitans strain 652 by pulsing for 2.5 ms at 2.5 kV. Plasmid uptake was confirmed by the presence of blue colonies on BHI agar containing X-Gal and by recovery of the appropriate plasmid from the recombinant strains. Promoter activity was assessed by determining β-galactosidase activity using the colorimetric substrate ONPG assays as described below. A similar approach was used to insert IS1301 into the ltx promoter of strain JP2.
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Construction of lacZ reporter constructs containing frameshift mutations of orfA.
Three frameshift mutants of orfA were constructed and were designated FS270, FS400 and FS500. FS400 was constructed by cleaving orfA at its unique SphI site and filling the overhanging nucleotides with T4 DNA polymerase in the presence of all four dNTPs for 30 min at 37 °C. The blunted ends were then religated with T4 DNA ligase. This mutant was capable of translating the first 80 amino acids of OrfA before prematurely terminating (full-length OrfA contains 150 residues). FS270 and FS500 were each constructed in pBluescript by assembling two promoter fragments that were amplified from strain 652 genomic DNA. The primers for these reactions are shown in Table 2
. Fragments FS270(1) and FS500(1) encoded 24 and 98 residues of OrfA respectively and were ligated to pBluescript cleaved with KpnI and XbaI. Inclusion of the XbaI site in these fragments generated the frameshift in orfA. Subsequently, amplified fragments FS270(2) and FS500(2) were ligated into the constructs described above cleaved with XbaI and BamHI to regenerate the full-length orfA containing the frameshift mutation. Clone FS270 was capable of expressing only the first 24 residues of OrfA. Clone FS500 possessed a frameshift mutation at the codon encoding amino acid 98, but encoded an additional 38 residues from an alternative reading frame before terminating. FS500 was therefore capable of producing a polypeptide of 136 amino acids. All three frameshift mutants were excised from pBluescript by cleavage with KpnI and BamHI and cloned into pYGKlacZ as described above. The resulting reporter constructs were introduced into an A. actinomycetemcomitans background that was deficient in OrfA (see below).
Insertional inactivation of orfA in A. actinomycetemcomitans.
The orfA gene was inactivated by insertion of a spectinomycin resistance marker obtained from pVT1461 (kindly provided by Dr K. Mintz, University of Vermont) by cleavage with SphI. This fragment was ligated into the unique SphI site of orfA in the ltx promoter region of A. actinomycetemcomitans 652 contained in pBluescript. The SphI site is located at nucleotide 396 in the 1000 bp ltx promoter region of this strain and is shown in Fig. 1. After selection for spectinomycin-resistant clones, the promoter fragment was removed by cleavage with KpnI and BamHI and cloned into pGEM-T containing the sacB gene under the control of the inducible tac promoter. The resulting plasmid was introduced into A. actinomycetemcomitans 652 by electroporation and recombinants were selected on agar plates containing 50 µg spectinomycin ml–1. Resistant clones were then grown in broth culture (BHI containing 50 µg spectinomycin ml–1) to mid-exponential phase and were induced for 2 h with 1 mM IPTG before plating on BHI agar plates containing 50 µg spectinomycin ml–1, 5 % sucrose and 1 mM IPTG. SpecR SucR recombinants were selected and confirmed by two additional passages on the above selective agar medium. Integration of orfA containing the SpecR determinant was then confirmed by PCR amplification using primers FS270(1)F and FS270(2)R (see Table 2). Loss of orfA transcripts in the recombinant organisms was then confirmed by RT-PCR using primers 16 (5'-TAGGAATTTATCCGGTCAAAG-3') and 20 (5'-TTTTAGGTTTAGGGCGATG-3') of Mitchell et al. (2003), which flank the SphI site used to insert the spectinomycin-resistance marker. The resulting strain was used for analysing the orfA frameshift reporter plasmids described above.
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0.5. Enzymic activity was determined in the following reaction: 5 µl bacteria were suspended in 226 µl 0.1 M sodium phosphate buffer, pH 7.5, containing 0.0003 % SDS and 10 µl chloroform. Samples were incubated at room temperature for 10 min and then 3 µl magnesium buffer (0.1 M MgCl2, 4.5 M β-mercaptoethanol) and 66 µl ONPG (4 mg ml–1) were added. Samples were incubated for 10 min at 37 °C and reactions were stopped by the addition of 500 µl 1 M Na2CO3. Units of β-galactosidase activity were calculated using the method of Miller (1972).
Real-time PCR.
Real-time PCRs were performed using Ready-to-Go reverse transcriptase beads (Amersham Biosciences) in a reaction volume of 25 µl. Reverse transcription was carried out according to the manufacturer's instructions. Each reaction mixture contained 7.5 pmol of the appropriate antisense primer and 50 ng bacterial RNA. After completion of cDNA synthesis, serial dilutions of cDNA were amplified by addition of 7.5 pmol sense primer and 0.5x SYBR-Green dye (Roche Applied Science). Amplifications were carried out using the Smart Cycler system (Cephied). The amplification conditions for real-time PCR were as follows: denaturation at 95 °C for 270 s for a single cycle, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and elongation at 72 °C for 60 s. The threshold cycle for each reaction was determined from a second-derivative plot of total fluorescence as a function of cycle number using the software package supplied with the Smart Cycler system. All reactions were normalized against real-time PCR reactions using primers specific for the A. actinomycetemcomitans 5S RNA gene. The threshold cycle for each reaction was determined from a second-derivative plot of total fluorescence as a function of cycle number using the software package supplied with the Smart Cycler system. All reactions were carried out at least twice using independently obtained template samples with consistent results.
Expression of His6-tagged OrfA.
The orfA open reading frame was amplified by PCR using the RTS E. coli Linear Template Generation set for His6 tags (Roche Applied Sciences). In this two-step protocol, the initial PCR was performed using A. actinomycetemcomitans 652 genomic DNA as the template. This reaction used the forward primer 5'-CTTTAAGAAGGAGATATACCATGTCCGGTACAGAATATGCTCC-3',which contains an orfA-specific sequence (underlined) and additional nucleotides that are required for priming of the secondary PCR reaction. The reverse primer (5'-TGATGATGAGAACCCCCCCCCCATGGCAACGGTAGAAGG-3') also possesses an orfA-specific region (underlined) along with additional sequence encoding a portion of the His6 tail and necessary for priming the secondary PCR reaction. The PCR reaction profile was 94 °C for 30 s, 55 °C for 1 min, 72 °C for 2 min, for 30 cycles. The secondary PCR was performed with the orfA amplicon as the template, using primers provided by the manufacturer. This reaction added a ribosome-binding sequence and a T7 polymerase promoter to the 5' end of the orfA amplicon, and the hexahistidine tag and a translational termination sequence to the 3' end of the fragment. The product of the secondary PCR was cloned directly into the pGEM-T Easy vector using the TA cloning sites provided (Promega). Ligated plasmids were transformed into competent E. coli DH5 and the appropriate plasmid construct was confirmed by digestion of purified plasmids with EcoRI. Plasmid purification was carried out using the Wizard Plus Miniprep DNA purification system (Promega). For expression of OrfA, 15 µg purified plasmid DNA was used in the RTS500 in vitro coupled transcription–translation system (Roche) according to the manufacturer's instructions. Expression of OrfA was confirmed by PAGE analysis and Western blotting using monoclonal anti-polyhistidine antibodies and goat anti-mouse peroxidase-conjugated antibody.
His6-tagged OrfA was purified from the in vitro expression mixture by immunoprecipitation. Monoclonal anti-polyhistidine antibody, clone His-1 (Sigma), was added to samples to a final concentration of 10 µg ml–1 and incubated at 4 °C for 2 h. Following antibody binding, 100 µl of a 50 % (w/v) slurry of Protein A-Sepharose 4 Fast Flow beads (GE Healthcare) were added and samples were incubated for an additional 2 h at 4 °C with gentle rotation. Beads were then pelleted by centrifugation for 5 min at 300 g and washed three times in 500 µl 50 mM Tris, pH 7.6 and bound OrfA was eluted by five 1 min incubations in 200 µl each of 0.1 M glycine, pH 2.0 followed by centrifugation as above. Samples were neutralized by the addition of an equivalent volume of 1 M Tris, pH 8.0 and then lyophilized. Dried fractions were suspended in 200 µl sterile MilliQ H2O and 20 µl volumes were analysed by PAGE followed by Coomassie staining and Western blotting. The sample was subjected to two additional rounds of immunoprecipitation and the final OrfA protein concentration was determined using a BCA microplate assay (Pierce Biotechnology). Samples were then separated into 50 ng aliquots, lyophilized and stored at –80 °C until used.
Electrophoretic mobility shift assays (EMSAs).
Probes corresponding to various segments of the lyx promoter of strain 652 (Fig. 1) were generated by PCR using the biotinylated primers (Biosynthesis) shown in Table 3. PCRs were carried out in a volume of 100 µl for 40 cycles using an annealing temperature of 60 °C for 1 min and a 2 min extension reaction at 72 °C followed by melting at 94 °C for 1 min. The resulting products were precipitated with 2-propanol, dried and resuspended in 50 µl TE buffer. All products were confirmed by agarose gel electrophoresis. Biotinylated probes were diluted 1 : 100 or 1 : 50 as needed in MilliQ H2O and 20 µl binding reactions were set up containing 4 µl 5x binding buffer (50 mM Tris/HCl, pH 8.0, 750 mM KCl, 2.5 mM EDTA, 0.5 % Triton X-100, 62.5 %, v/v, glycerol and 1 mM DTT) and 1 µg poly(dI.dC). Where indicated, 2 µl diluted biotinylated probe and 5 ng His6-OrfA were added to the reactions. For competition reactions, 2 µl undiluted and unlabelled probe was added. All binding reactions were incubated for 20 min at room temperature. After the addition of 5 µl loading dye (15 % Ficoll, 0.25 % xylene cyanol, 0.25 % bromophenol blue), samples were separated by electrophoresis on 10 % TBE gels (Invitrogen) and transferred to Hybond Immobilon N+ nylon membranes (GE Healthcare) at a constant current of 380 mA for 1.5 h. Membranes were immediately UV cross-linked at 1200 µJ cm–2 and then probed using the LightShift nucleic acid detection kit (Pierce), following the manufacturer's instructions. Developed membranes were visualized on a Kodak 1D ImageStation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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previously suggested that upstream displacement of a cis-acting negative regulator in the ltx promoter (CNR in Fig. 1b) by the integration of IS1301 may contribute to increased expression of the leukotoxin operon. To test this hypothesis, we generated lacZ reporter constructs in which progressively larger fragments of random sequence derived from the coding region of ltxA were inserted into the native ltx promoter at the site of integration of IS1301. As shown in Fig. 2, the transcriptional activity of these constructs was similar to that of the control reporter in which lacZ was driven by the ltx promoter from the minimally leukotoxic parent strain A. actinomycetemcomitans 652, even when a fragment of approximately the same size (900 bp) as IS1301 was inserted. This suggests that displacement of CNR does not lead to increased transcriptional activity of the ltx promoter.
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). As shown in Fig. 3(b), deleting 100 bp from the 5' end of IS1301 (construct IS800) reduced promoter activity by 5.7x104 Miller units, which represents 60 % of the difference in expression between the IS promoter and the parent 652 promoter constructs. Further deletions in IS1301 resulted in slight additional reductions in β-galactosidase activity. This suggests that sequences within 100 bp of the 5'-end of IS1301 are primarily responsible for elevated transcriptional activity of the ltx promoter of strain IS. The sequence of this region of IS1301 is shown in Fig. 3(c); it contains the 5' imperfect terminal repeat (underlined) and a consensus E. coli –10 element (in bold) upstream of the start codon of the putative transposase gene. To determine if the –10 element contributes to increased promoter activity, a lacZ reporter was constructed in which the TATAAT sequence was altered to GGGAAT by site-specific mutagenesis. As shown in Fig. 4(a, b), this construct showed β-galactosidase activity similar to the parent A. actinomycetemcomitans 652 reporter construct. A second lacZ reporter in which the unaltered IS1301 sequence was inverted in orientation relative to orfA and lacZ also exhibited β-galactosidase activity similar to the parent strain promoter construct. Finally, real-time RT-PCRs using primers complementary to the transposase ORF of IS1301 showed a 5–6-fold decrease in the appropriate transcripts in the strain containing the site-specific mutation (see Fig. 4c). However, a transcript spanning the transposase ORF and orfA was not detected, consistent with the results of Mitchell et al. (2003). Together, these results suggest that the –10 element and its orientation relative to orfA contribute either directly or indirectly to increased transcription of the ltx operon.
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). This suggests that an intact orfA gene may be required for IS1301 to exert its effect. To address this, the activity of a JP2 promoter–lacZ reporter was examined in both 652 and JP2 backgrounds. As shown in Fig. 4(b), lacZ expression was significantly higher in the 652 background. This suggests that OrfA may be a positive regulator of ltx expression.
Frameshift mutations in orfA alter ltx promoter activity
To determine if orfA regulates ltx promoter activity, three independent reporter plasmids containing frameshift mutations in orfA were analysed in an OrfA-deficient A. actinomycetemcomitans strain that was constructed by insertional inactivation of orfA (see Methods). As shown in Fig. 5, each of the frameshift mutations resulted in significantly lower β-galactosidase activity than the parental construct. Frameshift mutations at nucleotides 270 and 400 of the orfA sequence lead to 93 % and 96 % reductions of ltx promoter activity respectively. These constructs are capable of expressing OrfA polypeptides of 24 and 80 residues whereas the full-length ORF encodes a putative protein of 150 residues. The frameshift mutation at nucleotide 500 directed higher levels of β-galactosidase expression than the reporters containing mutations at nucleotides 270 and 400 but still had only 30 % of the β-galactosidase activity expressed by the control reporter plasmid. Thus, the introduction of premature translational stop codons into orfA dramatically affected ltx promoter activity.
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). As shown in Fig. 6(b), a biotinylated fragment corresponding to the intergenic region between orfA and the upstream glyA gene exhibited reduced mobility when incubated with His6-OrfA and the interaction of OrfA with this fragment was inhibited by the addition of excess unlabelled promoter DNA. To localize the putative OrfA binding site, a series of nested 5' deletions of the target DNA fragment were amplified and analysed. As shown in Fig. 6(c, d), OrfA retarded the mobility of all of the promoter fragments that contained the region between the –35 element and the start codon of orfA. The mobility of fragments lacking this region, such as probe CNR (see Fig. 6e), was unaffected by OrfA. These results suggest that OrfA interacts with sequences residing downstream from the –35 element in the ltx promoter.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Mitchell et al., 2003), and hyperleukotoxic strains of the organism are known to possess genetic alterations in the promoter region that lead to increased transcription of the ltx operon. Indeed, He et al. (1999) showed that integration of IS1301 occurred near the 5'-end of an AT-rich regulatory sequence that resembles an UP element (Estrem et al., 1999; Gourse et al., 2000; Ross et al., 1993) in the hyperleukotoxic strain IS, but IS1301 does not appear to functionally disrupt the putative UP element (Mitchell et al., 2003). Instead, Mitchell et al. (2003) suggested that the acquisition of IS1301 may uncouple negative regulation of the ltx operon by displacing a cis-acting negative regulatory sequence 800 bp upstream. Our current results, however, show that displacement of this negative regulator by random sequences derived from ltxA did not result in increased transcription of downstream genes, suggesting that specific sequences within IS1301 are required to stimulate toxin expression.
Analysis of lacZ reporters containing serial deletions in IS1301 indicated that a 100 bp region at the 5' end of IS1301 was required for increased transcriptional activity. This region contains a consensus –10 element upstream from the transposase gene, and several lines of evidence suggest that this putative –10 sequence functions to modulate the activity of the ltx promoter. First, inverting the orientation of the intact IS1301 relative to orfA and second, site-specific mutagenesis of the putative –10 sequence both reduced ltx promoter activity to that of the parental minimally leukotoxic strain which lacks IS1301. In addition, real-time PCR indicated a 5–6-fold reduction in the steady-state levels of transposase transcripts in the strain possessing the –10-site-specific mutation. However, consistent with Mitchell et al. (2003), no transcript was detected that spanned the transposase and orfA ORFs. Thus, alteration of the putative transposase –10 sequence may have an indirect effect on orfA expression or, alternatively, the primary transcript encompassing both genes may undergo rapid post-transcriptional processing. Indeed, the primary transcript from the ltx promoter in both minimally leukotoxic and hyperleukotoxic A. actinomycetemcomitans strains is quickly processed to generate independent orfA and ltxCA (or ltxCABD) transcripts (Kolodrubetz et al., 1996). Together, these data suggest that the TATAAT sequence upstream of the IS1301 transposase gene contributes to increased expression of leukotoxin.
Interestingly, recombinant insertion of IS1301 into a lacZ reporter plasmid containing the ltx promoter of A. actinomycetemcomitans JP2 did not increase β-galactosidase expression. A. actinomycetemcomitans JP2 contains a 530 bp deletion that truncates orfA and removes much of the orfA–ltxC intergenic region (see Fig. 1a). This led us to examine a potential regulatory role for the putative OrfA polypeptide. Reporter plasmids containing three independent frameshift mutations in orfA resulting in premature termination of translation each produced significantly decreased β-galactosidase activity compared to the unaltered ORF. Furthermore, recombinantly expressed OrfA protein bound to a region of the ltx promoter at or near the –35 sequence, suggesting that OrfA may function to regulate ltx promoter activity. Thus, it is possible that increased transcription of orfA mediated by the –10 element in IS1301 results in overproduction of the OrfA protein, which in turn stimulates ltx operon expression leading to the hyperleukotoxic phenotype. However, the OrfA binding site near the –35 sequence suggests that it may not function as a typical trans-regulatory protein. One possibility is that OrfA is a DNA-binding protein that alters the structure of the ltx promoter, which in turn facilitates increased transcription. Interestingly, the C-terminus of OrfA is highly basic (11 of 35 residues are positively charged), consistent with a potential DNA-binding activity, and this basic region is encoded by a sequence that is deleted in strain JP2. OrfA does not possess a predicted signal sequence or transmembrane domain, suggesting that it is a cytoplasmic protein. However, it exhibits no significant similarity to other polypeptides in the protein database and does not possess any conserved structural domains when analysed by PFAM. Thus, OrfA may represent a novel regulatory protein that controls the expression of the leukotoxin in A. actinomycetemcomitans; studies are currently under way to further examine its DNA-binding activity.
While our results suggest a potential role for OrfA in stimulating ltx expression in A. actinomycetemcomitans 652, they do not fully explain the hyperleukotoxic phenotype of strain JP2, where truncation of orfA is associated with increased ltx expression. The expression of lacZ from the JP2 promoter in a 652 background was significantly greater than that in a JP2 background. This is consistent with a role for OrfA as a positive regulator of ltx expression. However, the JP2 promoter still directed higher overall expression of lacZ in the 652 background than did the 652 promoter, suggesting that other factors may also contribute to the elevated expression observed from the JP2 promoter. One possible explanation is that the closer proximity of the ltx promoter to ltxCABD in strain JP2 (as a result of the 530 bp deletion) partially compensates for the loss of OrfA induction in JP2. In addition, the primary ltx transcript appears to be rapidly cleaved between orfA and ltxC (Kolodrubetz et al., 1996; D. R. Demuth, unpublished). Thus, it is also possible that the 530 bp deletion spanning the orfA–ltxC intergenic region alters processing of the ltx primary transcript in JP2, resulting in increased expression of the ltx operon.
In summary, hyperleukotoxic strains of A. actinomycetemcomitans are associated with localized aggressive periodontitis but the molecular mechanisms leading to the hyperleukotoxic phenotype are poorly understood. Our results suggest that the acquisition of IS1301 upstream of orfA introduces a new promoter-like sequence that may increase transcription of orfA and the downstream ltx genes. We also show that the OrfA protein acts as a positive regulator of ltx expression, which may further amplify toxin expression in strain IS. However, the mechanism of OrfA trans-activation may be unique and will require further study to define.
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ACKNOWLEDGEMENTS |
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Edited by: M. A. Curtis
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REFERENCES |
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Received 1 August 2007;
revised 5 October 2007;
accepted 1 November 2007.
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0.0001) decrease in activity relative to the parental strain, 652.
CNR alone; 2,