PorA, a conserved C-terminal domain-containing protein, impacts the PorXY-SigP signaling of the type IX secretion system

Porphyromonas gingivalis, a periodontal pathogen, translocates many virulence factors including the cysteine proteases referred to as gingipains to the cell surface via the type IX secretion system (T9SS). Expression of the T9SS component proteins is regulated by the tandem signaling of the PorXY two-component system and the ECF sigma factor SigP. However, the details of this regulatory pathway are still unknown. We found that one of the T9SS conserved C-terminal domain-containing proteins, PGN_0123, which we have designated PorA, is involved in regulating expression of genes encoding T9SS structural proteins and that PorA can be translocated onto the cell surface without the T9SS translocation machinery. X-ray crystallography revealed that PorA has a domain similar to the mannose-binding domain of Escherichia coli FimH, the tip protein of Type 1 pilus. Mutations in the cytoplasmic domain of the sensor kinase PorY conferred phenotypic recovery on the ΔporA mutant. The SigP sigma factor, which is activated by the PorXY two-component system, markedly decreased in the ΔporA mutant. These results strongly support a potential role for PorA in relaying a signal from the cell surface to the PorXY-SigP signaling pathway.


Construction of a complemented strain of the ∆porA mutant
The promoter region of Porphyromonas gulae catalase gene (accession no. AB083039 in GenBank/EMBL/DDBJ databases) was PCR amplified from P. gulae VPB3492 chromosomal DNA using the pair of primers PRO-U and PRO-R-B, digested with KpnI plus BamHI, and inserted into the KpnI-BamHI site of pBSSK to yield pKD1304. The entire porA gene containing its downstream region was PCR amplified from P. gingivalis ATCC 33277 chromosomal DNA using the pair of primers N0123-coF-B/N0123-coR-(NotI), the amplified DNA was cloned into the pGEM-T Easy vector (Promega) to yield pKD1305. pKD1305 DNA was digested with BglII plus NotI (restriction site within the pGEM-T Easy vector), and then ligated with the larger BamHI-NotI fragment of pKD1304 to construct pKD1306. The smaller KpnI-NotI fragment of pKD1306 was then ligated with the larger KpnI-NotI fragment of pTCB to construct pKD1307. After mating of Escherichia coli S17-1 47 containing pKD1307 with the P. gingivalis ∆porA mutant KDP1041 or KDP1045, an Em r Tc r transconjugant (KDP1042) or an Ap r Tc r transconjugant (KDP1051) were obtained.

Immunoprecipitation with α-PorA antibody
Cells were grown in BHI broth (200 ml) until late exponential phase (overnight culture).
Cells were harvested by centrifugation at 10,000 × g for 20 min and washed with HEPES buffer (10 mM HEPES, pH 8.0, 0.15 M NaCl) two times and concentrated eight-fold by suspension in 25 ml of 10 mM HEPES pH8.0. Cells were cross-linked using 0.25 mM dimethyl 3,3'-dithiobispropionimidate (DTBP) (easily reversed with dithiothreitol (DTT)) for 1 h at room temperature. The reactions were quenched for 10 min with 1.25 ml (50 mM final concentration) of 1 M Tris-HCl (pH 8.0). Cells were centrifuged at 10,000 × g for 10 min and suspended in 20 ml of Buffer A (10 mM HEPES pH 8.0 with 1 mM TLCK, 1 mM Leupeptin) and then mixed with 25 µg/ml DNase I and 25 µg/ml RNase A. After gentle shaking for 15 min at 4 °C, the suspension was disrupted by three passes at 100 MPa in a French pressure cell press. Remaining intact bacteria cells were removed by centrifugation at 2,400 × g for 10 min.
Supernatant was centrifuged at 100,000 × g for 60 min at 4 °C. The pellets were suspended in Buffer A and then mixed with 1% DDM (vol/vol). After gentle shaking for 30 min at RT, the suspension was centrifuged at 8,000 × g for 10 min at RT. The supernatant was stored at 4 °C as the membrane fraction. The membrane fraction was immunoprecipitated by use of Protein G agarose beads with 5 µl of α-PorA polyclonal antibody. Before the immunoprecipitation analysis, the α-PorA polyclonal antibody was cross-linked to the Protein G beads by dimethyl pimelimidate dihydrochloride to prevent co-elution of the antibody with the target protein. In Figs. 4A and S8A, the resulting precipitate was dissolved with the same volume of the sample buffer and loaded on an SDS (12%) gel. In Fig. S8B, the resulting precipitate was dissolved with the elution buffer (0.1 M glycine-HCl, pH 2.7), and then supplemented with neutralization buffer (1 M Tris-HCl, pH 9.0) and loaded on a Native gel. Immunoblot analysis was performed with α-PorA and α-PorV.

Hemagglutinating activity
Overnight cultures of P. gingivalis strains grown in enriched BHI medium were centrifuged, washed once with PBS, and suspended in PBS at an optical density of 0.5 at 595 nm. The bacterial suspensions were then diluted in a twofold series with PBS. A 100-μl aliquot of each suspension was mixed with an equal volume of defibrinated sheep erythrocyte suspension (1% in PBS) and incubated in a round-bottom microtiter plate at room temperature (RT) for 3 h.

Enzymatic assay
When we measured Kgp and Rgp activities in cells or in culture supernatants in Figs.
1C and 2D, cultures of P. gingivalis strains at OD600 of 1.0 were centrifuged to separate the culture supernatants and cells. Precipitated cells were resuspended with an original volume of phosphate-buffered saline (PBS). Each volume (2 µl for Kgp and 5 µl for Rgp) of bacterial cell suspensions and of culture supernatants were added to the reaction mixture (1.0 ml) containing 5 mM cysteine, 20 mM sodium phosphate buffer, pH 7.5, and 10 mM benzyloxycarbonyl-L-histidyl-L-glutamyl-L-lysine 4-methylcoumaryl-7amide (Peptide Institute,Osaka, Japan) (for Kgp) or benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methylcoumaryl-7-amide (Peptide Institute) (for Rgp). After 10 min incubation at 40 o C, the reaction was terminated by adding 100 mM sodium acetate buffer, pH 5.0, containing 10 mM iodoacetic acid (1.0 ml). The released 7-amino-4methyl-coumarine was measured at 465 nm (excitation at 365 nm) by fluorescence spectrophotometer Beckman Coulter DTX 800 (Brea, CA). Enzymatic activities are expressed as ∆E/min/µl. Whole cell cultures without separation to cells and culture supernatants were used in the experiments shown in Fig. S6.

Preparation of the outer membrane vesicle fraction
P. gingivalis strains were mono-or co-cultured in 60 ml of enriched BHI medium at 37 °C for 48 h. P. gingivalis cells were harvested by centrifugation at 6,000 × g for 20 min at 4 °C. The supernatants were ultracentrifuged at 100,000 × g for 60 min at 4 °C. The pellets were washed once with PBS, and then dissolved in PBS containing 0.1 mM TLCK and 0.1 mM Leupeptin.

MS analysis
A gel plug containing proteins was subjected to the following procedures: washing with 50% (v/v) acetonitrile, washing with 100% acetonitrile, reduction with 10 mM DTT, alkylation with 55 mM iodoacetamide, washing/dehydration with 50% (v/v) acetonitrile, and digestion for 10 h with 10 µg/ml trypsin. The resulting peptides were extracted from the gel plug with 0.1% (v/v) trifluoroacetic acid/50% (v/v) acetonitrile.
Digests were spotted on a MALDI target using α-cyano-4-hydroxycinnamic acid as a matrix. Spectra were acquired on a 4800 MALDI TOF/TOF analyzer (Applied Biosystems). Data analysis and MS database searching were performed using GPS ExplorerTM and MASCOT software.

qRT-PCR analysis
Total RNA was isolated from P. gingivalis cells grown to mid-exponential phase (OD600  Table S2. qRT-PCR conditions were as follows: 1 cycle at 95 °C for 10 min, and 35 cycles of 95 °C for 30 s, and 60 °C for 1 min. At each cycle, accumulation of PCR products was detected by the reporter dye from the dsDNA-binding SYBR Green. To confirm that a single PCR product was amplified, after the PCR, a dissociation curve (melting curve) was constructed in the range of 55 °C to 95 °C. All data were analyzed using Mx3005P software. The expression level of each targeted gene was normalized to that of the 16S rRNA gene, which was used as a reference. All PCR reactions were carried out in triplicate. The efficiency of primers binding was determined by linear regression by plotting the cycle threshold (CT) value versus the log of the cDNA dilution. Relative quantification of transcript was determined using the comparative CT method (2 −ΔΔCT ) calibrated to 16S rRNA. qRT-PCR experiments were performed multiple times independently with comparable results.

Construction of Halo-CTD chimera proteins
The signal sequence region coding for amino acids (M1-A19) of the kgp gene containing its promoter region was PCR amplified from P. gingivalis ATCC 33277 chromosomal DNA using the pair of primers kgp-U400-F-KpnI/kgp-sigR-SalI, digested with KpnI plus SalI, and inserted into the KpnI-SalI site of pBSSK to yield pKD1312.
The entire HaloTag gene region was PCR amplified from pFC15A containing Halo-tag (Promega) using the pair of primers Halo-F-SalI/ Halo-stopR-BglII or Halo-R-BglII.

Isolation of Halo-CTD chimera protein fraction with DTBP-mediated crosslinking from partially purified samples
Isolation of Halo-CTD(PorA) chimera protein fraction with DTBP-mediated crosslinking was performed as described previously 59 . Partially purified samples (PP) were prepared as follows. Cells were grown in BHI broth (200 ml) until late exponential phase (overnight culture). Cells were harvested by centrifugation at 10,000 × g for 20 min and washed twice with HEPES buffer (10 mM HEPES pH 8.0, 0.15 M NaCl) and concentrated eight-folds by suspension in 25 ml of HEPES buffer. Samples were crosslinked using 0.25 mM DTBP (easily reversed with DTT) for 1 h at room temperature.
Samples were centrifuged at 8,000 × g, 4 °C for 25 min. Pellets were resuspended in 40 ml ice-cold solution (step-2: 5 mM MgCl2, PIC) and gently mixing for 10 min. Samples were centrifuged at 8,000 × g, 4 °C for 25 min and resulting supernatants were concentrated by Amicon Ultra-15, 10K (Merck KGaA). One ml of concentrated supernatants was mixed with 12.5 µl of settled HaloLink Resin and incubated for 1 h at room temperature. After centrifugation at 4 °C for 5 min, the supernatants were removed and the resin was washed three times with 1 ml of HaloTag purification buffer (1×TBS [prepared from 10×TBS; 1 M Tris, 1.5 M NaCl, pH 7.6] and 0.05% IGEPAL-CA630). After centrifugation, the resin was dissolved with 50 μl of 2×SDS sample buffer containing DTT and heat denatured at 85 °C for 3 min.

Purification of PorA-N
His-PorA (Q28-K246) was expressed and purified with Ni-NTA affinity chromatography as described above. The His-tag and CTD of His-PorA (Q28-K246) were removed by the addition of trypsin (Sigma) for 1 h at 27 °C, and the reaction was  Table   S3. The diffraction data were processed with MOSFLM 61 and were scaled with Aimless 62 . The experimental phase was calculated using the SAD data of the Se-Met derivative and the initial model was automatically constructed using the program Phenix 63 . The atomic model was manually modified with Coot 64 and refined to 1.3 Å with Phenix against the native crystal data that showed the best resolution limit. The final refinement R factor and the free R factor were converged to 17.8% and 19.7%, respectively. The Ramachandran plot indicated that 99.3% and 0.7% residues were in the most favored and allowed region, respectively. Structural refinement statistics are summarized in Supplemental Table S3.

Ethics statement
Animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University with approval from the Institutional Animal Care and Use Committee (approval number 1205010983).