Bacterial strains and plasmids
The P. pneumotropica reference and E. coli strains and plasmids used in this study are listed in Table 1.
pnxIIIA was first amplified using the primer pair pnx3A-pcr-f and pnx3A-pcr-r (Additional file 5 lists the oligonucleotide primers), and subsequently, the purified PCR product was used for a second amplification of pnxIIIA by using the primer pair pnx3A-protein-f and pnx3A-protein-r. The amplicon was cloned into an entry vector, pENTR/SD/D-TOPO vector (Invitrogen, Carlsbad, CA, USA), and subsequently recombined with the destination vector pBAD-DEST49 (Invitrogen), yielding pBAD-Pnx3A. Mutant PnxIIIA expression vectors, pBAD-Pnx3A209, pBAD-Pnx3A197, and pBAD-Pnx3A151, were also constructed as described below.
Bacterial and cell cultures and growth conditions
All P. pneumotropica strains were maintained in a brain-heart infusion medium (BD, Cockeysville, MD, USA) at 37°C and incubated for 48 h. Transformed E. coli bacteria were grown at 37°C for 16 h in Luria-Bertani medium supplemented with 100 μg/ml ampicillin, 50 μg/ml kanamycin, 125 μM 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and 1 mM isopropyl-β-D-thiogalactopyranoside, if required, to select and maintain recombinant E. coli. To induce gene expression in the recombinant E. coli, the cells were incubated at 37°C for 2-3 h until the optical density (OD, 600 nm) reached 1.0. Subsequently, 0.1% L-arabinose was added to the culture. During the induction of gene expression, the cell culture was incubated at room temperature (RT) for 16 h.
J774A.1 mouse macrophage cells (JCRB9108) were provided by Health Science Research Resources Bank (Osaka, Japan). The J774A.1 cells were cultivated at 37°C in 5% CO2 in Dulbecco's modified Eagle medium (DMEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum, 100 U penicillin, and 100 μg/ml streptomycin sulfate.
Nucleic acid extraction and purification
Plasmid and genomic DNA were extracted according to the method described in a previous study .
TA cloning, inverse PCR, and DNA sequencing
A fragment of pnxIIIA was amplified with the primer pair pnx2A-f and pnx2A-r by using Ex Taq (Takara Bio, Shiga, Japan), and the amplified product was purified using SUPREC-PCR (Takara Bio). The purified PCR amplicons were ligated with the pTAC-1 vector (Biodynamics Laboratory, Tokyo, Japan), and E. coli DH5α was transformed with the resultant vectors. The clones were screened via blue-white selection and direct colony PCR by using the M13 primer pair. For inverse PCR, the genomic DNA of P. pneumotropica ATCC 35149 was digested with various restriction enzymes that recognized a 6-nucleotide sequence, and subsequently, the digestion product was self-ligated with T4 ligase (Takara Bio) and then used as an inverse PCR template. Inverse PCR was performed using gradient PCR to determine the optimum annealing temperature for a model DNA Engine PTC-200 (Bio-Rad Laboratories, Hercules, CA, USA). The PCR products were ligated with the pTAC-1 vector and screened to ensure the accuracy of sequencing. Cycle sequencing was performed using the BigDye terminator premix (Applied Biosystems, Foster City, CA, USA). The products of the sequencing reaction were analyzed using an ABI 310 or ABI 3730XL DNA analyzer (Applied Biosystems).
Purification of recombinant Pnx proteins
rPnxIIIA was extracted and purified from the cell culture of E. coli strain TMU0812 harboring pBAD-Pnx3A. The cultured cells were suspended in 20 mM Tris-HCl, 150 mM NaCl, 5 mM imidazole, and 1 mM 2-mercaptoethanol (pH 8.0, binding buffer); they were then broken by sonication. The sonicate was centrifuged at 7,000 × g for 10 min and filtered using a 0.45-μm filter unit (Millipore, Billerica, MA, USA). The supernatant was loaded onto a 1-ml His-trap HP affinity column (GE Healthcare, Amersham, UK) mounted on an ÁKTAprime plus fast protein liquid chromatography device (FPLC device; GE Healthcare), and chromatography was performed by running a program for histidine-tagged protein purification according to the manufacturer's instructions. The collected sample was then eluted as a fraction in a buffer containing 20 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, and 1 mM 2-mercaptoethanol (pH 8.0; elution buffer). Fractions that mainly contained rPnxIIIA were monitored and confirmed by SDS-PAGE. For purification of rPnxIIIE, E. coli BL21-AI cultures harboring pET-Pnx3E were extracted in a binding buffer containing 6 M guanidine hydrochloride, and the extracts were purified with an elution buffer containing 6 M urea, similar to the method used to purify rPnxIIIA. The solvent of rPnxIIIA and rPnxIIIE was exchanged to a buffer containing 20 mM Tris-HCl and 150 mM NaCl by using FPLC and dialysis, respectively.
Purification of native rPnxIA and rPnxIIA was performed briefly according to previous described methods .
Generation of deletion mutants of rPnxIIIA variants
To compare the function of the unique repeat sequences in the rPnxIIIA variants, deletion mutant rPnxIIIA expression vectors were constructed. In brief, deletion mutant expression vectors pBAD-Pnx3A209, which lacked amino acid residues of a repeat sequence at position 287-735 (Figure 1B; Repeat 1), and pBAD-Pnx3A197, which lacked amino acid residues of a repeat sequence at position 1097-1666, (Figure 1B; Repeats 2 and 3) were directly constructed using the wild-type protein expression vector pBAD-Pnx3A as the template with primer pairs pnx3A-209-f and pnx3A-209-r and pnx3A-197-f and pnx3A-197-r, respectively. A PrimeSTAR Mutagenesis Basal Kit (Takara Bio) was used to create these deletion mutant expression vectors. Finally, pBAD-Pnx3A151, which lacked both repeat sequences, was constructed with the primer pair pnx3A-197-f and pnx3A-197-r with pBAD-Pnx3A209 as the PCR template. All the constructs were confirmed with DNA sequencing. The expression and purification of rPnxIIIA variants were performed in the same manner as that used for the wild-type rPnxIIIA.
The cytotoxicity of the recombinant Pnx proteins toward J774A.1 cells was determined via a LDH release assay that was performed according to the methods of Basler et al.  with minor modifications. Prior to incubation, the concentration of J774A.1 cells in a 96-well plate was adjusted 1 × 105 cells per well. The cells were grown in fresh DMEM supplemented with 20 mM CaCl2 and appropriate antibiotics. rPnxIIIA was added to the wells such that its concentrations were 0.1, 0.5, and 1.0 μg/ml of the final concentrations. The plate was incubated at 37°C in 5% CO2 for up to 24 h. LDH release from the J774A.1 cells was measured at 1, 2, 4, 6, 12, and 24 h by using the supernatant from the treated cells; a cytotoxicity detection kit (Roche Diagnostics, Mannheim, Germany) was used for this purpose. For the comparison of cytotoxicity among rPnxIA, rPnxIIA, and rPnxIIIA, 1.0 μg/ml of each recombinant protein was incubated with the J774A.1 cells for 4 h. Thereafter, LDH release from the J774A.1 cells was measured. Furthermore, to assess the effect of existence of CD11a on inhibition of rPnxIIIA-induced cytolysis, LDH release from the J774A.1 cells with the addition of 1.0 μg/ml rPnxIIIA with or without 1.0 μg/ml anti-CD11a rat MAb (Abcam, Cambridge, UK), which cross-reacts with the α subunit of mouse CD11 as a neutralizing antibody, was measured after 2, 6, 12, and 24 h of incubation as described above. To assess the cytotoxicity of P. pneumotropica reference strains toward J774A.1 cells, a whole bacterial cell cytotoxicity assay was performed briefly according to the methods of Kehl-Fie et al. . The results were reported as the percentage of LDH released from all the lysed cells. The experiments were repeated in triplicate.
ELISA was used to determine the binding of rPnxIIIA variants to components of rodent ECMs. In brief, a 96-well microtiter plate coated with rat collagen type I (BD BioCoat, BD) was used for the binding assay of rat collagen type I, and rat collagen type II (Chondrex, Redmond, WA, USA), mouse collagen type IV (BD), and mouse laminin (BD) were differently coated on a 96-well microplate (As one, Osaka, Japan) according to the manufacturer's instructions. ELISA was performed with a protein detector AP microwell kit (KPL, Gaithersburg, MD, USA), anti-6× Histidine tag monoclonal antibody (Biodynamics laboratory), and rPnxIIIA variants, the concentrations of which were adjusted to 0.5-50 μg/ml of the final concentration.
For the whole-cell binding assay of P. pneumotropica reference strains, precultured cells were adjusted the OD reached 1.0 and incubated on a 96-well microtiter plate coated with rat collagen type I (BD) at 37°C for 24 h. Thereafter, the plate was briefly washed and stained with 0.1% safranin, according to the method of Davey and Duncan . The quantification of adherent cells was determined by measuring the A490 with a plate reader.
Hemagglutination and hemolytic assay
Defibrinated sheep blood was obtained from Nippon Bio-Test Laboratories (Tokyo, Japan) and washed 3 times with sterilized phosphate-buffered saline (PBS) prior to conducting the assays. Hemagglutination activity was determined in V-cut bottom 96-well microtiter plates (Corning, Horseheads, NY, USA). Fifty micro milliliters of diluted rPnxIIIA variants or overnight cultures of P. pneumotropica reference strains were added to wells containing 2% sheep erythrocytes. The plate was incubated at RT for 1 h, and thereafter, the plate was imaged and visualized with a GeneGenius Bio Imaging System (Syngene, MD, USA). A hemolysis assay was performed according to a previously described method  that used 2% sheep erythrocytes.
Generation and purification of rabbit antisera
In brief, crude rabbit antisera against the entire rPnxIIIA and rPnxIIIE proteins were prepared using the methods of Schaller et al. . The crude antisera were further purified on an HiTrap rProtein A FF column (GE Healthcare) mounted on an FPLC device, and rabbit IgG was prepared for immunological experiments. The Institutional Animal Care and Use Committee of Tokyo Medical University approved all of the animal experimental procedures described here.
Fractionation of bacterial cell culture
Fractionation of the OM fraction, IM fraction, and soluble cell (SC) components was performed according to the methods of Valle et al. . P. pneumotropica ATCC 35149 cells in the mid-log phase were harvested, resuspended in 10 mM HEPES (pH 7.5) with 50 mM NaCl and 0.1 mg/ml lysozyme, and disrupted by sonication. The sonicate was centrifuged at 7,000 × g for 10 min, and subsequently, the supernatant was centrifuged at 100,000 × g for 1 h by using a Beckman Optima TL Tabletop Centrifuge (Beckman Coulter, Brea, CA, USA). The supernatant was used as the SC fraction, and the pellet containing the bacterial membrane was resuspended in a buffer containing 0.5% sarkosyl (N-laurylsarcosine) and allowed to stand for 30 min at RT. The sarkosyl-soluble fraction was centrifuged at 100,000 × g for 1 h. The supernatant was used as the IM fraction, and the pellet was resuspended in a 500 μl of 10 mM HEPES (pH 7.5) with 50 mM NaCl, 1% sarkosyl, and 10 mM EDTA and used as the OM fraction. To prepare a cell-free supernatant, the P. pneumotropica ATCC 35149 culture in the mid-log phase was centrifuged at 7,000 × g for 10 min, and the supernatant was filtered through a 0.22-μm pore size filter (Millipore) followed by a 0.45-μm pore size filter (Millipore). The filtrate was ultrafiltrated at 1000 × g for 20 min by using AmiconUltra-15 (Millipore). The resultant solution was used as the ultrafiltrated culture supernatant (UC) fraction. For SDS-PAGE analysis, the concentration of the SC, IM, OM, and UC samples were adjusted to 0.2 mg/ml, and 10 μl of each sample were subjected to 10% SDS-PAGE.
Cross-linking and pull-down assay
To determine the in vitro interaction of rPnxIIIA and rPnxIIIE, chemical cross-linking and IP were performed. A cross-linker for soluble proteins, bis[sulfosuccinimidyl] suberate-d0 (BS3-d0; Thermo Fisher Scientific, Waltham, MA, USA), was used for the cross-linking reaction of rPnxIIIA and rPnxIIIE according to the manufacturer's instructions. To terminate the cross-linking reaction, 20 mM NH4HCO3 was added. Thereafter, a mixed solution was subjected to IP by using an IP kit, Dynabeads Protein G (Invitrogen), and rabbit IgG against rPnxIIIA according to the manufacturer's instructions. The resultant solution was subjected to SDS-PAGE, and the interaction of rPnxIIIA with rPnxIIIA or rPnxIIIE was detected by Western blotting as described below.
Western blotting and Southern hybridization
Fractions of the P. pneumotropica cell culture, IP-treated sample, and cell lysates of P. pneumotropica reference strains were analyzed by Western blotting by using anti-rPnxIIIA IgG (1:20,000) or anti-rPnxIIIE IgG (1:20,000), followed by SDS-PAGE separation. Anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP; Thermo Fisher Scientific) for anti-rPnxIIIA IgG was used as secondary antibodies at a dilution of 1:50,000. To detect hybridization signals, the Femto Western chemiluminescence reagent (Thermo Fisher Scientific) was used as a substrate for HRP.
For Southern hybridization, digoxigenin-11-dUTP-labeled pnxIIIA probes were generated using the primer-pair pnx3A-probe-f and pnx3A-probe-r and the genomic DNA of P. pneumotropica ATCC 35149. The genomic DNAs of the reference strains were digested with HindIII and loaded on agarose gels. The hybridization and detection protocol used has been described previously .
Bacterial cells were fixed with 4% (w/v) paraformaldehyde, 0.25% (v/v) glutaraldehyde, and 5% sucrose in 1.5 ml of 0.1 M phosphate buffer (pH 7.4) for 2 h at 4°C. The cells were harvested at 1000 × g for 10 min. The pellets were then rinsed with the same buffer and dehydrated by passing them through an ethanol series. Samples were embedded in LR-white resin. Thin sections were placed in PBS with 5% bovine serum albumin (BSA) for 30 min at RT and then incubated with rabbit anti-PnxIIIA IgG diluted to 1:100 with 1% BSA in PBS for 4 h at RT. The sections were washed 3 times in PBS and incubated with goat anti-rabbit IgG conjugated with 10-nm immunogold particles (BBInternational, Cardiff, UK) diluted to 1:50 with 5% BSA in PBS for 1 h. The sections were subsequently stained with uranyl acetate and lead citrate and viewed under a JEOL JEM-1200EX electron microscope (JEOL, Tokyo, Japan) at 80 kV.
Nucleic acid accession numbers
The nucleotide sequences of pnxIIIE, pnxIIIA, pnxIIIB, pnxIIID, and tolC were deposited in GenBank through DNA Data Bank of Japan, and the assigned accession numbers were AB568084, AB568085, AB568086, AB568087, and AB568088, respectively.