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Originally published In Press as doi:10.1074/jbc.M413544200 on January 4, 2005

J. Biol. Chem., Vol. 280, Issue 10, 8668-8677, March 11, 2005
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Identification of a New Membrane-associated Protein That Influences Transport/Maturation of Gingipains and Adhesins of Porphyromonas gingivalis*{boxs}

Keiko Sato{ddagger}, Eiko Sakai§, Paul D. Veith¶, Mikio Shoji{ddagger}, Yuichiro Kikuchi{ddagger}, Hideharu Yukitake{ddagger}, Naoya Ohara{ddagger}, Mariko Naito{ddagger}, Kuniaki Okamoto§, Eric C. Reynolds¶, and Koji Nakayama{ddagger}||

From the Divisions of {ddagger}Microbiology and Oral Infection and §Oral Molecular Pharmacology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan and the Cooperative Research Centre for Oral Health Science, School of Dental Science, The University of Melbourne, Victoria, 3010, Australia

Received for publication, December 2, 2004 , and in revised form, December 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Periodontal disease, the major cause of tooth loss in the general population of industrial nations (1, 2), is a chronic inflammatory disease of the periodontium that leads to erosion of the attachment apparatus and supporting bone for the teeth (3) and is one of the most common infectious diseases of humans (4). The obligately anaerobic Gram-negative bacterium Porphyromonas gingivalis has become recognized as a major pathogen for adult periodontitis (5). The microorganism possesses several potential virulence factors for periodontopathogenicity (6). Among these factors the proteolytic enzymes are of special importance, since some of them have the abilities to destroy periodontal tissue directly or indirectly (7, 8). P. gingivalis produces large amounts of lysine-specific (Lys-gingipain, Kgp) and arginine-specific (Arg-gingipain, Rgp) cysteine proteinase on the cell surface and in the extracellular milieu (9–12). The Kgp and Rgp proteinases have the ability to specifically cleave substrates on the carboxyl side of lysine and arginine, respectively. Kgp and Rgp are encoded by one gene (kgp) and two separate genes (rgpA and rgpB) on the P. gingivalis chromosome, respectively and are widely implicated as important virulence factors in the pathogenesis of periodontal disease (13, 14).

The kgp and rgpA genes having 5,193- and 5,118-bp open reading frames (ORFs)1 encode proteins that consist of four domains: signal sequence, propeptide, mature proteinase, and C-terminal adhesin domains. The C-terminal adhesin domain region that is thought to be involved in hemagglutination comprises four subdomains. The rgpB gene has a 2,208-bp ORF, the amino acid sequence of which is similar to that of rgpA, but lacks most of the adhesin domain. These proteinases are synthesized as pre-proenzymes that are processed and secreted into the extracellular milieu as the mature proteinases or located on the cell surface as complexes non-covalently associated with the adhesin domain proteins; however, the precise mechanism of the transport/maturation is still unknown.

Previous studies have shown a link between colonial pigmentation on blood agar plates, hemagglutination and Kgp/Rgp activity in P. gingivalis cells (15, 16). P. gingivalis wild-type strains form black-pigmented colonies resulting from accumulation of the oxidized form of heme on the cell surface (17, 18), but the Kgp-null mutants exhibit reduced pigmentation and the Kgp/Rgp-null mutants show no pigmentation (15, 19). It suggests that these proteinases play an important role in acquisition of heme from erythrocytes (20, 21).

Transposon mutagenesis has been applied to the isolation of pigment-less mutants of P. gingivalis by several researchers (16, 22–24). Chen et al. (16) isolated non-pigmented mutants that had the transposon Tn4351 DNA within kgp. In addition, Simpson et al. (24) found that a non-pigmented mutant has the insertion sequence element IS1126 at the promoter locus of kgp. These results confirmed the involvement of kgp in pigmentation. Recently, non-kgp mutations causing no pigmentation have been found (16, 25). Chen et al. (16) found that Tn4351 was inserted into a putative glycosyl (rhamnosyl) transferase-encoding gene in several non-pigmented mutants and Abaibou et al. (25) found that the gene vimA located downstream of recA has a role in pigmentation. Recently, we have found that the gene porR, which is located at a gene cluster for glycan biosynthesis is involved in the biosynthesis of cell surface polysaccharide that may function as the anchor for Rgp and Kgp, via attachment to the C-terminal adhesins (26).

In this study, we isolated a non-pigmented mutant that has an insertion mutation within the new gene porT and found that the unprocessed gene products of kgp, rgpA, and rgpB are accumulated in the periplasmic space of the porT mutant cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Culture Conditions—All P. gingivalis strains used are shown in Table I. P. gingivalis cells were grown anaerobically (10% CO2, 10% H2, 80% N2) in enriched brain heart infusion (BHI) medium, and on enriched tryptic soy agar (28). For blood agar plates, defibrinated laked sheep blood was added to enriched tryptic soy agar at 5%. As a defined minimal medium, we used {alpha}-ketoglutarate/bovine serum albumin ({alpha}-KG/BSA) medium for the growth of P. gingivalis (29). For selection and maintenance of the antibiotic-resistant strains, antibiotics were added to the medium at the following concentrations: ampicillin, 50 µg/ml; kanamycin, 50 µg/ml; chloramphenicol (Cm), 20 µg/ml; erythromycin (Em), 10 µg/ml; and tetracycline (Tc), 0.7 µg/ml.


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  		TABLE I
P. gingivalis strains used in this study

 
Transposon Mutagenesis and Gene-directed Mutagenesis—Transposon mutagenesis of P. gingivalis strain 33277 (ATCC) with Tn4351 and gene-directed mutagenesis of P. gingivalis strains with electroporation were described previously (26).

Construction of Plasmids and Bacterial Strains—A PvuII DNA fragment (8 kb) containing Tn4351 DNA in the chromosomal DNA of P. gingivalis KDP106 was cloned into the HincII region of pACYC184 (30). The resulting plasmid was digested with AvaI and the larger AvaI fragment self-ligated to yield pKD263. The kanamycin-resistance gene (kan) block (1.3 kb) of pUC4K (31) was inserted into a unique SalI site within the porT gene of pKD263, resulting in pKD304. The NcoI-EcoRI fragment of pKD263 that contained the porT gene disrupted with the kan DNA block was then inserted into the PvuII site of pKD283, an EcoRI-fragment-deleted derivative of pMJF-2 (32), resulting in pKD305. P. gingivalis 33277 was transformed to be Em-resistant (Emr) by electroporation with pKD305, resulting in KDP117 (porT1::kan ermF) and KDP118 (porT+ ermF). A porT region DNA (1 kb) was PCR-amplified from the chromosomal DNA of 33277 using a pair of oligonucleotides 5'-TATTGTTGTGAGGTAGGTTATGC-3' and 5'-GCTCTAGAAATATCCAAAAAGCTTAGGCGTCG-3', digested with EcoRI and inserted into a unique EcoRI site of pKD713, a derivative of pKD703 (32) containing the tetQ DNA block of pKD375 (15) at the BamHI site, resulting in pKD850 (fimA::[porT+ tetQ]). KDP117 was then transformed with the NotI-linearized DNA of pKD850 to yield KDP350 (porT1::kan ermF fimA::[porT+ tetQ]). For construction of a plasmid containing the erm DNA block between a porT-upstream DNA and a porT-downstream DNA, the porT-upstream DNA region encoding PG0750 was PCR-amplified from the chromosomal DNA of 33277 using a pair of oligonucleotides, 5'-TAGGATCCTAGTTGTCACGCTCTTTTCGAC-3' and 5'-TAGGTCGACAGCGCTTGCGGCGGAAAAAGAAG-3', cloned into the pGEM-T Easy vector (Promega) and digested with SpeI and BamHI. The resulting DNA fragment was then inserted into the corresponding region of pKD355, a derivative of pBluescript II SK(-) carrying the erm DNA block (15) between the BamHI and EcoRI sites, resulting in pKD356. The porT-downstream DNA region encoding PG0752 was PCR-amplified using a pair of oligonucleotides, 5'-GTGAATTCCCTGAGAGAATAATCTTCAATTCT-3' and 5'-GCGGCCGCTATACAGGATCGTATTGAGTGCT-3', cloned into the pGEM-T Easy vector and digested with EcoRI. The resulting DNA fragment was inserted into the corresponding region of pKD356 to yield pKD357 (porT2::[ermF ermAM]). P. gingivalis KDP129 (kgp::cat) was then transformed with the NotI-linearized pKD357 DNA to yield KDP351 (kgp::cat porT2::[ermF ermAM]). For construction of a kgp'-'rgpB chimera gene, the DNA (0.6 kb) of the rgpB gene encoding the C-terminal domain was PCR-amplified from the chromosomal DNA of 33277 using a pair of oligonucleotides, 5'-GCTCTAGAAGAAACGAACTTGACGCTCACCGTA-3' and 5'-CATAAACGACTGCAATGCAACGGCGGCCGCA-5'. The amplified DNA was digested with EagI and XbaI, and inserted into the corresponding region of pKD851, a derivative of pBluescript II SK(-) that contained the His (X6)-tag DNA between BamHI and XbaI, resulting in pKD852. The middle region of the kgp gene was PCR-amplified from the chromosomal DNA of 33277 using a pair of oligonucleotides, 5'-TACTCGAGCTTATCGTGCAATGCCTAAGACC-3' and 5'-CGGATCCAATACATCGTTTGCAGGTTCGATCG-3', digested with XhoI and BamHI. The resulting XhoI-BamHI fragment was then inserted into the corresponding region of pKD852 to yield pKD853. The 2.6-kb AvaI DNA fragment encoding the signal peptide, propeptide, and mature proteinase portions of the kgp gene was isolated from the pNKV (33) and inserted into the AvaI site of pKD853 to yield pKD854 (kgp'-'rgpB). The KpnI-NotI DNA fragment of pKD854 containing the kgp'-'rgpB chimera gene DNA was inserted into the corresponding region of pKD713, resulting in pKD855 (fimA::[kgp'-'rgpB tetQ]). For construction of P. gingivalis strains possessing the kgp'-'rgpB chimera gene, KDP129 (kgp::cat) and KDP351 (kgp::cat porT2::[ermF ermAM]) were transformed with the NotI-linearized pKD855 DNA to yield KDP352 (kgp::cat fimA::[kgp'-'rgpB tetQ]) and KDP353 (kgp::cat porT2::[ermF ermAM] fimA::[kgp'-'rgpB tetQ]), respectively.

Hemagglutination Assay—Overnight cultures of P. gingivalis strains in enriched BHI medium were centrifuged, washed with PBS, and resuspended in PBS. The bacterial suspensions were then diluted in a 2-fold series with PBS. A 100-µl aliquot of each suspension was mixed with an equal volume of human erythrocyte suspension (1% in PBS) and incubated in a round bottom microtiter plate at room temperature for 3 h.

Enzymatic Assays—Kgp and Rgp activities were determined using the synthetic substrates t-butyl-oxycarbonyl-L-valyl-L-leucyl-L-lysine-4-methyl-7-coumarylamide (Boc-Val-Leu-Lys-MCA) (final concentration 20 µM) and carbobenzoxy-L-phenyl-L-arginine-4-methyl-7-coumarylamide (Z-Phe-Arg-MCA) in 20 mM sodium phosphate buffer (pH 7.5) containing 5 mM cysteine in a total volume of 1 ml. After incubation at 40 °C for 10 min, the reaction was terminated by adding 1 ml of 10 mM iodoacetamide (pH 5.0), and the released 7-amino-4-methylcoumarin was measured at 460 nm (excitation at 380 nm). One unit of enzyme activity was defined as the amount of enzyme required to release 1 nmol of 7-amino-4-methylcoumarin/ml under these conditions.

Northern Blot Analysis—Total RNA was extracted from P. gingivalis cells grown to mid-exponential phase (OD600, 0.3) using an RNA purification kit (RNeasy Protect minikit, Qiagen). 5 µg of RNA were electrophoresed in 1.2% agarose gel and then transferred to a nylon membrane (Hybond-N, Amersham Biosciences) according to the method described by Sambrook et al. (34). The antisense mRNA probes specific for the 0.5-kb BstXI(T325)-SphI(M482) region of rgpB and the 0.5-kb AccI(T346)-EcoRI(E510) region of kgp were constructed by using the pSPUTK plasmid (Stratagene). The RNA probes were labeled with digoxigenin using the DIG RNA labeling kit (Roche Applied Science). Northern blot hybridization and detection were carried out according to the manufacturer's recommendation.

Subcellular Fractionation—Subcellular fractionation of P. gingivalis cells was performed essentially according to Murakami et al. (35). Briefly, P. gingivalis cells from a 3,000-ml culture were harvested by centrifugation at 10,000 x g for 30 min at 4 °C, and resuspended with 100 ml of PBS containing 0.1 mM N{alpha}-p-tosyl-L-lysine chloromethyl ketone (TLCK), 0.1 mM leupeptin, and 0.5 mM EDTA. The cells were disrupted in a French pressure cell at 100 MPa by two passes. The remaining intact bacterial cells were removed by centrifugation at 2,400 x g for 10 min, and the supernatant was subjected to ultracentrifugation at 100,000 x g for 60 min. The pellets were treated with 1% Triton X-100 in PBS containing 20 mM MgCl2 for 30 min at 20 °C. The outer membrane fraction was recovered as a precipitate by ultracentrifugation at 100,000 x g for 60 min at 4 °C. The supernatant was obtained as the inner membrane fraction.

DEAE-Sepharose Chromatography and Affinity Chromatography— Cells of P. gingivalis KDP117 (porT) from a 500-ml culture were harvested by centrifugation at 10,000 x g for 30 min at 4 °C and resuspended with 20 mM phosphate buffer (pH 7.0) containing 0.1 mM TLCK, 0.1 mM leupeptin and 0.5 mM EDTA, and sonicated. Unbroken cells and large debris were removed by centrifugation (1,000 x g, 30 min, 4 °C), and the cloudy supernatant was applied to a column (2.6 x 40 cm) of DEAE Sepharose (Sepharose CL-6B, Amersham Biosciences), which had been equilibrated with 20 mM phosphate buffer (pH 7.0). After being washed thoroughly with the same buffer, proteins were eluted stepwise with 100 ml of the same buffer containing 100, 200, 300, 400, 500, and 700 mM NaCl at a flow rate of 0.5 ml/min. Proteins with high molecular masses (>150 kDa) were detected only in the 200 mM NaCl eluent. The 200 mM NaCl eluent, which immunoreacted with anti-Hgp44, was dialyzed against 0.1 M NaHCO3 (pH 8.3) and applied to a column of BrCN-activated Sepharose 4B (Amersham Biosciences) conjugated with anti-Hgp44 IgG, which had been equilibrated with the same buffer. The column was then eluted with 0.1 M Gly-HCl (pH 2.8), 0.5 M NaCl. The eluent was immediately equilibrated with 1 M Tris-HCl (pH 9.0).

The kgp'-'rgpB chimera gene product was purified using a resin precharged with Ni2+ (ProBondTM resin, Invitrogen). Briefly, P. gingivalis KDP353 cells (50 ml culture) were resuspended in 8 ml of the guanidinium lysis buffer (6 M guanidine HCl, 20 mM sodium phosphate (pH 7.8), 500 mM NaCl) and slowly rocked for 10 min at room temperature. After centrifugation at 3,000 x g for 15 min, the supernatant was applied to the resin column, which had been equilibrated with the denaturing binding buffer (8 M urea, 20 mM sodium phosphate, pH 7.8, 500 mM NaCl). The column was washed with the denaturing wash buffer (8 M urea, 20 mM sodium phosphate, pH 6.0, 500 mM NaCl) and then eluted with the denaturing elution buffer (8 M urea, 20 mM sodium phosphate, pH 4.0, 500 mM NaCl). The resulting fractions were analyzed by SDS-PAGE, followed by staining with Coomassie Brilliant Blue (CBB). These proteins were transferred to a polyvinylidene difluoride membrane (Millipore) and stained with CBB. The protein band migrating to the position corresponding to a molecular mass of 78 kDa was cut out and subjected to N-terminal amino acid sequencing with an automatic protein sequencer (protein sequencing system LF3600D, Beckman).

Preparation of Anti-Hgp44, Anti-HbR, and Anti-PorT Antisera— Recombinant Hgp44 protein was obtained as described previously (36). A peptide derived from the amino acid sequence (Thr221 to Leu233) of PorT with an N-terminal cysteine residue, CTHERPDLLDDYKL, which was conjugated to keyhole limpet hemocyanin was purchased from Sigma Genosys. The recombinant Hgp44 protein and the conjugated PorT peptide were mixed with Freund's complete adjuvant and injected subcutaneously into rabbits (Japan White) with two booster shots of a mixture of these antigens and Freund's incomplete adjuvant, resulting in anti-Hgp44 and anti-PorT antisera, respectively. Animal care and experimental procedures were conducted in accordance with the Guidelines for Animal Experimentation of Nagasaki University with approval of the Institutional Animal Care and Use Committee. Preparation of anti-HbR antiserum has been described previously (20).

Mass Spectrometry—In-gel digestion was performed as described previously (37). For MALDI-TOF analysis, digests were acidified with 1% trifluoroacetic acid and analyzed using the {alpha}-cyano-4-hydroxycinnamic acid thin layer technique on a 600-µm anchorchip target (Ultraflex TOF/TOF Bruker Daltonics, Bremen, Germany). For LC-MS analysis, acidified digests were preconcentrated and desalted on a C18 Pepmap precolumn and separated on a 75-µm C18 Pepmap column using an Ultimate nanoLC system (LC Packings, Amsterdam) and analyzed on-line by Ion Trap MS (Esquire HCT, Bruker Daltonics). MS/MS spectra were acquired automatically.

Spheroplast Formation and Proteinase Treatment—-Spheroplast formation and proteinase treatment of P. gingivalis cells was essentially performed by the method described previously (38). After being suspended in 50 mM Tris acetate buffer (pH 7.8) containing 0.75 M sucrose, P. gingivalis cells were treated with lysozyme (final concentration, 0.1 mg/ml) on ice for 2 min. Conversion to spheroplasts was performed by slowly diluting the cell suspension over a period of 10 min with 2 volumes of cold 1.5 mM EDTA. After centrifugation at 10,000 x g for 10 min, the resulting precipitates were gently resuspended in 50 mM Tris acetate buffer (pH 7.8) containing 0.25 M sucrose and 10 mM MgSO4 (spheroplasts). The supernatants were used as the periplasm fraction and the proteins in this fraction were precipitated with trichloroacetic acid, and subjected to SDS-PAGE and immunoblot analysis. Formation of spheroplasts was examined by phase contrast microscopy. Spheroplasts were treated on ice with proteinase K (final concentration 1 mg/ml) in the presence or absence of 2% Triton X-100 for 1 h. After quenching proteinase K with phenylmethylsulfonyl fluoride (final concentration, 5 mM) for 5 min, the whole volume of the sample was mixed with 4 volumes of Laemmli sample buffer and subjected to SDS-PAGE and immunoblot analysis.

Registration of the Nucleotide Sequence Data—The GenBankTM/EMBL/DDBJ accession number for the sequence reported in this study is AB016085 [GenBank] .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of a Non-pigmented Mutant of P. gingivalis by Transposon Mutagenesis, and Identification of a Gene Disrupted by the Transposon Insertion—Several non-pigmented clones were isolated among Emr P. gingivalis transconjugants after mating between Escherichia coli HB101 containing R751::Tn4351{Omega}4 and P. gingivalis 33277. One of the non-pigmented strains named KDP106 was further characterized in this study. Southern blot hybridization analysis revealed that the KDP106 chromosome contained a single Tn4351 insertion. A PvuII fragment (8 kb) of KDP106 chromosomal DNA that contained the inserted Tn4351 DNA was cloned by using the method of marker (Tcr on Tn4351 DNA) rescue. Sequencing of the flanking regions revealed that there was one ORF truncated by the transposon insertion. The ORF coding for 244 amino acids was designated porT (Fig. 1A).



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  		FIG. 1.
Chromosomal structure around porT. A, P. gingivalis chromosomal structure in the vicinity of porT. The triangle indicates the Tn4351 insertion site of KDP106. B, schematic structures of the porT regions of the chromosomal DNA of the Emr transconjugant KDP117 (porT) and KDP118 (porT+). Dotted boxes, porT gene DNA fragments; empty box, pKD283 DNA; hatched box, the kan DNA block. The unit of the numbers is kilobase.

 
Construction of a PorT Mutant by Gene-directed Mutagenesis—To determine whether non-pigmentation of KDP106 was attributable to porT, we constructed a mutant with disruption of porT by gene-directed mutagenesis. We introduced the kan DNA block into the SalI region within porT and constructed a suicide vector plasmid containing the disrupted porT gene (pKD305). Introduction of pKD305 into P. gingivalis 33277 cells by electroporation produced a number of Emr transformants. Southern blot hybridization analysis with chromosomal DNA of 10 Emr transformants revealed that the chromosomal DNA from all of the transformants examined possessed full-length plasmid pKD305 DNA inserted at the porT region. The transformants were classified into two types with respect to location of the kan DNA block. Transformants exhibiting chromosomal structures I and II were designated KDP117 (porT) and KDP118 (porT+), respectively (Fig. 1B). KDP117 exhibited no pigmentation on the blood agar plates (Fig. 2A), whereas the pigmentation of KDP118 was the same as the wild-type parent strain, indicating that porT was essential for colonial pigmentation of P. gingivalis. To further confirm the relation between porT and colonial pigmentation, the wild-type porT gene was introduced into the fimA locus of KDP117. The resulting strain KDP350 (porT1::kan ermF fimA::[porT+ tetQ]) showed the same colonial pigmentation as the wild-type strain (Fig. 2A).



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  		FIG. 2.
Properties of the porT mutant. A, colonical pigmentation. P. gingivalis cells were anaerobically grown on blood agar plates at 37 °C for 4 days. Panels: a, KDP117 (porT); b, KDP129 (kgp); c, KDP351 (KDP129 porT); d, KDP136 (kgp rgpA rgpB); e, KDP350 (KDP117 fimA::porT+); f, KDP352 (KDP129 fimA::[kgp'-'rgpB]); g, KDP353 (KDP129 porT fimA::[kgp'-'rgpB]);. B, hemagglutination. P. gingivalis cells were grown in enriched BHI broth, washed with PBS, and resuspended in PBS at an optical density at 0.4. The suspension and its dilutions in a 2-fold series were applied to the wells of a microtiter plate from left to right and mixed with sheep erythrocyte suspension. Rows: a, KDP117 (porT); b, KDP118 (porT+); c, 33277 (wild type). C, FimA fimbrilin. The whole cell lysates of 33277 (lane 1) and KDP117 (lane 2) were subjected to SDS-PAGE and immunoblot analysis with anti-FimA. D, growth in enriched BHI medium. An overnight culture was diluted 20-fold with enriched BHI medium and incubated anaerobically at 37 °C. Growth was monitored by measuring the optical density at 600 nm. E, no growth of the porT mutant on the {alpha}-KG/BSA-defined agar plate. P. gingivalis cells were heavily streaked on the {alpha}-KG/BSA-defined agar plate and incubated anaerobically at 37 °C for 7 days. Sectors: 1, KDP117 (porT); 2, KDP118 (porT+); 3, 33277 (wild type); 4, KDP129 (kgp); 5, KDP150 (fimA); 6, KDP107 (porR); 7, KDP108 (porR+); 8, KDP112 (rgpA rgpB).

 
Hemagglutination and Fimbriation of the porT Mutant— KDP117 (porT) showed no hemagglutination (Fig. 2B). Immunoblot analysis with anti-FimA fimbrilin showed that KDP117 produced precursor forms (45 and 43.5 kDa) of FimA fimbrilin, but not mature fimbrilin (43 kDa) (Fig. 2C), which was consistent with the electron microscopic observation that KDP117 cells showed no fimbriation (data not shown).

Kgp and Rgp Activities of the porT Mutant—The porT mutant failed to grow on {alpha}-KG/BSA-defined medium, whereas it could grow in enriched BHI medium as well as the wild-type parent strain (Fig. 2, D and E). These results indicated that the porT mutant might not produce Kgp or Rgp proteinase since the Kgp/Rgp-null mutant also shows no colonial pigmentation, hemagglutination or fimbriation, and fails to grow on {alpha}-KG/BSA-defined medium (15). Therefore we determined Kgp and Rgp activities of the porT mutant. KDP117 (porT) showed very weak activities of Kgp and Rgp in the cell lysates and culture supernatants, whereas KDP350 (porT fimA::porT+) exhibited almost the same Kgp and Rgp acitivities as 33277 (wild type) (Fig. 3).



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  		FIG. 3.
Rgp and Kgp activities of the porT mutant. A, P. gingivalis cells were anaerobically grown in enriched BHI medium at 37 °C for 36 h. Kgp (panels 1 and 3) and Rgp (panels 2 and 4) activities of the cell lysates (panels 1 and 2) and the culture supernatants (panels 3 and 4) were measured. B, immunoblot analysis with anti-PorT. Lanes: 1, KDP136 (kgp rgpA rgpB); 2, KDP117 (porT); 3, KDP350 (porT fimA::porT+); 4, 33277 (wild type).

 
Northern Blot Hybridization Analysis Using rgp and kgp Probes—Total RNA was purified from the early-exponential phase cultures of the porT, rgpA, rgpB, kgp, and wild-type strains. Northern blot hybridization analysis using kgp and common rgp probe RNA revealed that the kgp, rgpA and rgpB genes of the porT mutant were transcribed (data not shown), indicating that the porT mutation did not affect gene expression of rgpA, rgpB, or kgp at the level of transcription.

Immunoblot Analysis Using Anti-Kgp, Anti-Hgp44, and Anti-HbR—The kgp and rgpA genes comprising 5,193-bp and 5,118-bp ORFs, respectively, encode polyproteins that consist of four segments: signal peptide, propeptide, proteinase, and adhesin domains. The C-terminal adhesin domains comprise four subdomains (Hgp44/A1, Hgp15(HbR)/A2, Hgpl7/A3, and Hgp27/A4) that are involved in hemagglutination and hemoglobin binding. The rgpB gene comprises a 2,208-bp ORF and lacks most of the adhesin domain. Cells of the porT mutant (KDP117) and the wild-type parent strain (33277) were fractionated into the cytoplasm/periplasm, inner membrane and outer membrane fractions, and immunoblot analyses with anti-Kgp, anti-Hgp44 and anti-HbR were performed (Fig. 4). In the wild-type strain, 190- and 50-kDa proteins immunoreactive with anti-Kgp were found in the cytoplasm/periplasm fraction. The 50-kDa protein was also found in the total membrane fractions, especially in the outer membrane fraction, but the 190-kDa protein was not found in the total membrane fraction (Fig. 4A). In the porT mutant, on the other hand, the 190-kDa protein was found in the cytoplasm/periplasm fraction, whereas the 50-kDa protein could not be detected in any fraction. The membrane fractions of the porT mutant showed no proteins immunoreactive with anti-Kgp, anti-Hgp44 or anti-HbR (Fig. 4B). Because anti-Hgp44 and anti-HbR seemed to react to several protein bands with high molecular masses in addition to the 190-kDa protein in the cytoplasm/periplasm fraction of the porT mutant, the cytoplasm/periplasm fraction of the porT mutant was subjected to DEAE Sepharose chromatography and immunoblot analyses with anti-Kgp, anti-Hgp44 and anti-HbR, and affinity column chromatography using anti-Hgp44 antiserum (Fig. 5). The cytoplasm/periplasm fraction of the porT mutant was found to contain 210-kDa, 190-kDa, and 185-kDa proteins that could be purified by affinity column chromatography using anti-Hgp44 antiserum. All of the proteins reacted with anti-Hgp44 and anti-HbR antisera, whereas only the 190-kDa protein reacted with anti-Kgp, suggesting that the 210-kDa, 190-kDa, and 185-kDa proteins might be derived from hagA, kgp, and rgpA, respectively. To determine whether the 190-kDa anti-Kgp-reactive protein was located in the cytoplasm or periplasm, cells of the porT mutant were subjected to spheroplast formation and proteinase K treatment followed by immunoblot analysis with anti-Kgp (Fig. 6). The 190-kDa anti-Kgp-reactive protein in spheroplasts of the porT mutant cells was sensitive to the proteinase K treatment, whereas the P. gingivalis GroEL protein mainly located in the cytoplasm was insensitive to the treatment, suggesting that the 190-kDa protein was located on the surface of the porT spheroplasts. The 190-kDa protein was also found in the periplasm fraction. Taken together, the 190-kDa anti-Kgp-reactive protein appeared to be located in the periplasm.



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  		FIG. 4.
Accumulation of the precursor form of Kgp in the cytoplasm/periplasm fractions of the porT mutant. The whole cell (lane 1), cytoplasm/periplasm (lane 2), total membrane (lane 3), inner membrane (lane 4), and outer membrane (lane 5) fractions of 33277 (wild type) (A) and KDP117 (porT) (B) were analyzed with SDS-PAGE followed by immunoblot analyses with anti-Kgp, anti-Hgp44, and anti-HbR.

 



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  		FIG. 5.
Partial purification of anti-Hgp44-immunoreactive proteins with high molecular masses in the porT mutant. Whole cell lysates were fractionated on the DEAE Sepharose column as described in "Experimental Procedures." Fractions were analyzed by SDS-PAGE followed by staining with CBB (A) and immunoblot analyses with anti-Hgp44 (B), anti-Kgp (C), and anti-HbR (D). E, fractions no. 36–43 were pooled and applied to a column of BrCN-activated Sepharose 4B conjugated with the anti-Hgp44 IgG. Proteins were eluted from the column with 0.1 M Gly-HCl, pH 2.8, 0.5 M NaCl.

 



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  		FIG. 6.
Spheroplast formation and proteinase treatment. Spheroplast and periplasm fractions of P. gingivalis cells were formed as described in "Experimental Procedures." Spheroplasts were subjected to the proteinase K treatment in the presence or absence of 2% Triton X-100. Samples were subjected to SDS-PAGE (10% gel) followed by staining with CBB (A) and immunoblot analyses with anti-Kgp (B) and anti-GroEL (C). Same samples were also subjected to SDS-PAGE (15% gel) followed by immunoblot analysis with anti-PorT (D). Lanes: 1, KDP136 (kgp rgpA rgpB); 2, KDP117 (porT); 3, 33277 (wild type); 4, KDP118 (porT+). sph, spheroplasts; peri, periplasm fraction.

 
MS Analysis of Proteins in the porT Mutant—Several bands from the CBB-stained gel of cell lysates from the porT mutant were identified by MS analyses of in-gel tryptic digests. Protein bands 1, 2, and 3 with molecular masses of 190, 185, and 85 kDa were found to correspond to the gene products of kgp, rgpA, and rgpB, with MS/MS sequence coverage ranging from 30–40% (Fig. 7). Peptides were matched to all domains of the precursor proteins excepting the signal peptides (see Supplementary Data 1). The N-terminal peptide could not be identified. Several peptides were matched by MS/MS to the C-terminal 80 residue region of the proteins, which was interesting as peptides from this region are not recovered from the processed domains in the wild type.



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  		FIG. 7.
PMF analysis of proteins with high molecular masses in the porT mutant. A, SDS-PAGE gel with the whole cell lysates of KDP117 (porT). Protein bands 1, 2, and 3 were analyzed with PMF. B, schematic drawing of the regions indicated by the PMF data of the protein bands 1–3. PMF, peptide mass fingerprinting.

 
Analysis of the kgp'-'rgpB Chimera Gene—We were unsuccessful in our attempts to determine the N-terminal amino acid sequences of the 190-, 185-, and 85-kDa proteins, which was attributed to their high molecular masses and difficulty in purification. To overcome these perceived difficulties, we constructed a kgp'-'rgpB chimera gene that comprised the signal peptide, propeptide, and proteinase domain of kgp, His tag, and the C-terminal portion of rgpB (Fig. 8A and Supplementary Data 2). Using the kgp'-'rgpB chimera DNA, we obtained KDP352 (kgp::cat fimA::[kgp'-'rgpB tetQ]) and KDP353 (kgp::cat porT2::[ermF ermAM] fimA::[kgp'-'rgpB tetQ]) by introduction of the kgp'-'rgpB chimera DNA into the fimA loci of KDP129 (kgp) and KDP351 (kgp porT), respectively. KDP352 showed colonial pigmentation on the blood agar plate and Kgp activity in the cell lysates and culture supernatants that were equivalent to that of the wild-type kgp+ strain, whereas KDP353 showed no pigmentation and very weak Kgp activity (Figs. 2A and 8B). Immunoblot analysis with anti-Kgp and anti-His tag revealed that a precursor form (78 kDa) of the kgp'-'rgpB chimera gene product was accumulated in KDP353 (Fig. 8C). The precursor protein was purified with Ni2+ column chromatography and subjected to N-terminal amino acid sequencing. No result was obtained suggesting a blocked N terminus presumably through pyroglutamate formation.



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  		FIG. 8.
Proteinase activity and anti-Kgp-immunoreactive proteins of P. gingivalis strains possessing the kgp'-'rgpB chimera gene. A, structure of the kgp'-'rgpB chimera gene. B, Kgp and Rgp activities. P. gingivalis cells were anaerobically grown in enriched BHI medium at 37 °C for 36 h. Kgp and Rgp activities of the bacterial cells, and the culture supernatants of the cultures were determined. Columns: 1, KDP129 (kgp); 2, KDP351 (kgp porT); 3, KDP352 (kgp fimA::[kgp'-'rgpB]); 4, KDP353 (kgp porT fimA::[kgp'-'rgpB]); 5, 33277 (wild type). C, immunoblot analysis with anti-Kgp. An arrow indicates a precursor form of the kgp'-'rgpB gene product. Lanes: 1, 33277 (wild type); 2, KDP352 (kgp fimA::[kgp'-'rgpB]); 3, KDP353 (kgp porT fimA::[kgp'-'rgpB]); 4, KDP129 (kgp).

 
Subcellular Localization of PorT—-The wild-type strain (33277) was fractionated into the cytoplasm/periplasm, inner membrane and outer membrane fractions and then subjected to Tris-Tricine SDS-PAGE and immunoblot analysis with anti-PorT antiserum (Fig. 9). A 28-kDa anti-PorT-immunoreactive protein was found in the inner membrane fraction. From spheroplast formation and proteinase K treatment followed by immunoblot analysis with anti-PorT, anti-PorT immunoreactive protein was found to be in the spheroplast fraction of the porT+ strains, but not in the periplasmic fraction (Fig. 6D). The anti-PorT-immunoreactive protein band in the spheroplast fraction was significantly reduced by treatment with proteinase K and almost disappeared by treatment with Triton X-100 and proteinase K. These results suggest that PorT is an inner membrane-associated protein exposed to the periplasmic space.



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  		FIG. 9.
Subcellular localization of the PorT protein. The whole cell, cytoplasm/periplasm, total membrane, inner membrane and outer membrane fractions of 33277 were subjected to SDS-PAGE and immunoblot analysis using anti-PorT antiserum. Lanes: 1, whole cell lysates; 2, cytoplasm/periplasm fraction; 3, total membrane fraction; 4, inner membrane fraction; 5, outer membrane fraction.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The gingipain/adhesin-encoding genes (kgp and rgpA) code for polyproteins comprising the signal peptide, propeptide, proteinase, and adhesin domains (33, 39). rgpB encodes a protein comprising the signal peptide, propeptide, and proteinase domains (40, 41). Although the adhesin domain region is missing in the rgpB ORF, the C-terminal region of the rgpB primary gene product shares homology with the C-terminal regions of the kgp and rgpA primary gene products (14, 40). The kgp, rgpA, and rgpB primary gene products have typical Gram-negative signal peptides and are predicted to be transported by the type II secretory pathway (or so-called general secretory pathway) to the periplasm. The identification of P. gingivalis genes by the Institute for Genomic Research (www.tigr.org) and the annotation of the genes by the Los Alamos National Laboratory (www.oralgen.lanl.gov) have revealed that the P. gingivalis genome encodes the signal recognition particle (PG0996) that helps target secretory proteins to the cytoplasmic membrane, two subunits of the preprotein translocase, SecY (PG1673) and SecF (PG1536), which help form the channel for transport across the cytoplasmic membrane, and signal peptidase I (PG1746 and PG1747) and signal peptidase II (PG1397), responsible for cleaving standard signal peptides and lipoprotein signal peptides respectively, allowing the translocated protein to be released into the periplasm (42). Very recently, we have found that maturation of several cell-surface proteins such as a fimbrilin and a 75-kDa protein is affected by treatment with globomycin, a specific inhibitor of signal peptidase II. Furthermore, the maturation of one outer membrane protein, HmuR, that has a typical signal peptide cleaved by signal peptidase I is unaffected by the presence of globomycin, suggesting that both signal peptidases of P. gingivalis are functional (27).

In this study, we found a new gene porT from genetic analysis of the pigmentation-deficient mutant of P. gingivalis. porT encodes a 28-kDa precursor and appears to contain a signal peptide that would most likely be cleaved at 29AQ30, which has been reported to be the favored cleavage site for P. gingivalis signal peptidase I (43). The mature sequence of PorT does not contain any predicted trans-membrane helices, suggesting that it is not an integral inner membrane protein. The sequence of PorT therefore suggests a periplasmic, outer membrane or extracellular location. The finding in this study that PorT was associated with the inner membrane fraction can be reconciled by consideration of the fractionation technique. The membrane fraction isolated by French pressure disruption and centrifugation was further fractionated by selective solubilization of the inner membrane with detergent. The contents of the inner membrane fraction would therefore include cytoplasmic and periplasmic proteins that were firmly bound to either side of the inner membrane. In addition, proteins bound to either surface of the outer membrane but not integrated within the outer membrane may also be released into the inner membrane fraction by the detergent. Immunoblot analysis with the spheroplast fraction treated with or without proteinase K revealed that PorT is exposed to the periplasmic space. Taken together, the results suggest that PorT is an inner membrane-associated protein exposed to the periplasmic space.

A BLAST search for PorT homologues revealed that the genomes of Cytophaga hutchinsonii and Prevotella intermedia encode a putative PorT homologue. Both of the bacterial species are members of the Cytophaga-Flavobacteria-Bacteroides group; however, Bacteroides fragilis and Bacteroides thetaiotaomicron belonging to the same group do not possess a PorT homologue, suggesting that PorT homologues may exist in a limited group of bacterial species.

Deficiency in colonial pigmentation, hemagglutination, and fimbriation of the porT mutant can be explained by very weak activities of Rgp and Kgp in the porT mutant since the Rgp/Kgp-null mutants also show deficiency in pigmentation, hemagglutination, and fimbriation (15). Subcellular fractionation analysis revealed that precursor forms of the kgp, rgpA, and hagA gene products were accumulated in the periplasmic space of the porT mutant cells. MS analyses of these proteins showed that all domains from the pro domain to the conserved C-terminal domain were present. The signal peptides however were not identified, consistent with a periplasmic location. N-terminal sequencing of these proteins and the kgp'-'rgpB chimera gene product in the porT mutant produced no results, indicating a blocked N terminus, consistent with previous predictions that the pro domains of these proteins have N-terminal pyroglutamate (43).

Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized (44). They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. The FUP family consists of a group of large proteins present in the outer membranes of Gram-negative bacteria. They are believed to contain a large domain that spans the membrane 24 times as {beta}-strands, presumably forming a {beta}-barrel structure and a transmembrane pore (45, 46). They also possess N-terminal and C-terminal periplasmic domains, which may function in protein folding and subunit assembly (47, 48). The OMF family proteins probably form homotrimeric, 12-stranded, {beta}-barrel-type pores in the outer membrane through which the solutes pumped out of the cytoplasm or cytoplasmic membrane pass in response to the energy-coupled export process catalyzed by the cytoplasmic membrane permease. The P. gingivalis genome has at least four genes (PG0094, PG0538, PG0679, and PG1667) that are involved in the OMF family; however, it might be unlikely that the gingipains utilize this pathway since the OMF family proteins function in conjunction with a primary cytoplasmic membrane transporter of the major facilitator superfamily, the ABC superfamily, the RND superfamily and the PET family, whereas the gingipains appear to utilize the type II secretory pathway across the inner membrane. The AT family proteins have C-terminal 250–300 aminoacyl residues which fold and insert into the outer membrane to give rise to putative {beta}-barrel structures with 14 transmembrane {beta}-strands (49–52). This structure presumably forms a pore through which the N-terminal virulence factor (passenger domain) is transported to the extracellular milieu. A number of extracellular and cell-surface proteinases such as the IgA1 proteinase produced by Neisseria gonorrhoeae are transported to the outer membrane and the extracellular milieu by the AT pathway (53). However, there are several differences between the AT family proteins and gingipains. First, the kgp and rgpA gene products have relatively large C-terminal adhesin domains, whereas the AT family proteins have the C-terminal {beta}-domain consisting of 250–300 amino acid residues. Second, the kgp, rgpA, and rgpB gene products have the pro-peptide region between the signal peptide and the proteinase domain, whereas the AT family proteins generally have a different organization (44). Third, from a phylogenetic point of view the AT family proteins are restricted to the phylum Proteobacteria, including the {alpha}-, {beta}-, {gamma}-, and {epsilon}-proteobacteria classes, and to the phylum Chlamydiae (44). The TPS family pathway utilizes an outer membrane channel that is formed by a separate protein (54, 55). Exoproteins that utilize this pathway contain a conserved N-terminal secretion domain that targets them to the outer membrane channel. This pathway is a widespread mechanism for the secretion of large virulence factors across the outer membrane in nascent form. Folding occurs on the outside of the cells. Gingipains cannot use this system however because they lack an N-terminal secretion domain. The Secretin family consists of a group of Gram-negative bacterial outer membrane proteins that form multimeric pores through which macromolecules, usually proteins, can pass (47, 56, 57). These proteins form homomultimeric ring structures, 10–20 subunits per complex, with large central pores. The P. gingivalis genome seems not to have a homologue of the Secretin family protein as revealed by a BLAST search with the P. gingivalis genome data base. Taken together, transport of gingipains/adhesins from the periplasm to the outer membrane and the cell surface may not be classified using these known pathways. Involvement of the membrane-associated protein PorT in the transport/maturation of gingipains/adhesins may be indicative of a novel transport system. The C-terminal region of the proproteins of gingipains/adhesins has been suggested to be glycosylated, which may be a prerequisite for translocation of the proproteins across the outer membrane. PorT may be involved in that glycosylation of the proproteins in the periplasmic space. Alternatively, PorT may attach to the N-terminal prosequence or C-terminal region of the proproteins and assist them in translocation across the outer membrane.

In this study, we constructed the P. gingivalis strain possessing the kgp'-'rgpB chimera gene as the sole potential gene for Kgp activity. The strain exhibited a level of Kgp activity in the cell lysates and culture supernatants that was equivalent to the wild-type strain. These results suggest that the kgp- and rgpA-encoding adhesin domains (except the C-terminal region) do not influence the catalytic activity of the Kgp or Rgp proteinase.

In conclusion, we have identified a new membrane-associated protein PorT that influences transport and maturation of gingipains/adhesins encoded by kgp, rgpA, and rgpB of P. gingivalis. This finding may lead to the identification of a novel transport system across the outer membrane from the periplasm to the cell surface and the extracellular milieu.


   FOOTNOTES
 
* This work was supported by Grants-in-aid 14370585 and 16017282 for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplementary data.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB016085 [GenBank] . Back

|| To whom correspondence should be addressed. Tel.: 81-95-849-7648; Fax: 81-95-849-7650; E-mail: knak{at}net.nagasaki-u.ac.jp.

1 The abbreviations used are: ORFs, open reading frames; PBS, phosphate-buffered saline; CBB, Coomassie Brilliant Blue; BSA, bovine serum albumin; BHI, brain heart infusion; Cm, chloramphenicol; Em, erythromycin; Tc, tetracycline; OMF, outer membrane factor; {alpha}-KG, {alpha}-ketoglutarate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back


   ACKNOWLEDGMENTS
 
We thank Drs. M. Ehara and F. Yoshimura for subcellular fractionation using the French pressure cell and members of Division of Microbiology and Oral Infection for useful discussion.



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