Bacterial strains and growth conditions
Neisseria gonorrhoeae F62 strain was grown overnight in gonococcus medium (GC) agar (Difco) or in liquid GC broth supplemented with 1% isovitalex (BBL) at 37°C in 5% CO2.
Cloning and construction of isogenic mutants
The pIII and ng1873 genes devoid of the sequence for the predicted leader peptide (sequences coding for amino acids 1–22) and the stop codon were amplified using the primers FOR-pIII-5′-cgcggatcccatatg GGCGAGGCGTCCGTT-3′ (NdeI site), REV-pIII-5′-cccgctcgagGTGTTGGTGATGATTGCG-3′ (XhoI site), FOR-ng1873-5′-cgcggatcccatatgGCAAATCTGGAGGTGCGCC-3′ (NdeI site), REV-ng1873-5′-cccgctcgagTTGGAAAGGGTCGGAATCG-3′ (XhoI site). The PCR products were inserted into the NdeI/XhoI sites of the pET21b expression vector in order to obtain the pET-pIII-His and pET-ng1873-His constructs.
Knockout mutants in F62 strain, in which the pIII and the ng1873 genes were truncated and replaced with an antibiotic cassette, were prepared as described in . The gene deletion was verified by PCR and lack of protein expression was confirmed by Western blot analysis.
Purification of recombinant His-tagged proteins and preparation of polyclonal antisera
PIII and NG1873 His-tagged proteins were expressed in E. coli as described  and proteins were purified on a metal-chelate affinity chromatography column (MCAC); proteins were eluted in a single step with 50 mM phosphate buffer pH 8.0, 300 mM NaCl, 250 mM imidazole and protease inhibitors.
To prepare antisera against PIII and NG1873 His-tagged proteins, 20 μg of each purified protein were used to immunize CD1 female mice. The recombinant proteins were given i.p. together with Al(OH)3 for three doses (day 0, day 21, day 35). Blood samples were taken on day 49 and pooled.
Confocal immunofluorescence microscopy
To visualize PIII protein on bacterial surface, F62 strain was grown in GC up to OD600 0.5 and washed in PBS. Bacterial pellet were fixed with 2% PFA for 20 min at room temperature and spotted on chamber slides coated with poly-lysine. Bacteria were then blocked with 2% BSA for 15 min and incubated with mouse polyclonal anti-PIII antibodies diluted in 2% BSA for 30 min at room temperature. Bacteria were then stained with goat anti-mouse Alexa Fluor 568 conjugated antibodies (Molecular Probes) for 20 min at room temperature. Labeled samples were mounted with ProLong® Gold antifade reagent with DAPI and analyzed with Zeiss LSM710 confocal microscope.
Negative staining and TEM
A drop of a 109 cfu/mL bacterial suspension in D-PBS was placed on Parafilm and bacteria were adsorbed for 15 min to formvar/carbon 200 mesh grids. Bacteria were fixed for 15 min with 2% p-formaldehyde and grids were then rinsed four times in PBS and air-dried. Grids were finally treated with uranyl acetate and examined by TEM GEOL 1200EX II transmission electron microscope.
Paper disk diffusion inhibiting assays
F62 and F62ΔpIII strains were grown overnight on GC agar, suspended in D-PBS and adjusted to OD600 = 0.1 (≅108 cfu/mL). An aliquot of 0.1 mL of the bacterial suspension was seeded on GC agar. 10 μL of the following detergents were applied to paper disk (Oxoid): SDS at 0.125, 0.25, 0.5, 1%, Triton X-100 at 0.03, 0.06, 0.125, 0.25% and deoxycolate at 0.8, 0.9, 1.2, 1.4%. Control disks with PBS were included in the assay. Disks were then placed on the GC agar inoculated with bacteria. All plates were incubated at 37°C in 5% CO2.
Cell fractionation and protein analysis
Total cell lysates (TL), inner membranes (IM) and outer membrane (OM) were prepared from bacteria at exponential growth phase. Total cell lysates were obtained by three freezing-thawing cycles.
For IM and OM preparation, bacteria were sonicated, unbroken cells were removed by centrifugation and the supernatant centrifuged at 50000 × g for 90 min at 4°C. The pellet containing the membranes was incubated in 2% Sarkosyl in 20 mM Tris–HCl, pH 7.5 at room tempertaure for 20 min to solubilize the inner membranes. The suspension was centrifuged at 10000 × g for 20 min at 4°C and the supernatant was centrifuged overnight at 75000 × g at 4°C. The supernatant was collected as IM fraction and the pellet, containing the OM, was resuspended in 20 mM Tris–HCl, pH 7.5.
SDS-PAGE electrophoresis with NuPage 4-12% Bis-Tris gels (Invitrogen) and Western blot analysis were performed according to standard procedures.
Opa proteins were detected by monoclonal antibody 4B12, kindly provided by M. Achtman. Pili were detected by monoclonal antibody SM1, kindly provided by M. Virji. OpaB protein was detected by polyclonal antisera against NG0070 His-tagged protein; purification of the protein and mice immunization were performed as described before. Bands were visualized with Super Signal Chemiluminescent Substrate (PIERCE).
Two-dimensional gel electrophoresis and image analysis
200 μg of proteins were precipitated with 0.015% sodium deoxycholate and 48% trichloroacetic acid and dissolved in 7 M urea, 2 M thiourea, 2% CHAPS, 2% ASB14, 1% DTT, 2 mM tributylphosphine, 20 mM Tris and 2% carrier ampholyte. Proteins were absorbed overnight onto Immobiline DryStrips (7 cm; pH-gradient 3–10 non linear) and the first dimension was run using a IPGphor Isoelectric Focusing Unit (Ge Healthcare), applying sequentially 150 V for 1 hour, 500 V for 35 min, 1000 V for 30 min, 2600 V for 10 min, 3500 V for 15 min, 4200 V for 15 min and finally 5000 V to reach 12kVh. For the second dimension, strips were equilibrated as described and proteins were separated on linear 4–12% polyacrylamide gels. Bidimensional gel was acquired with a Personal Densitometer SI (Molecular Dynamics) and images were analyzed with the software Image Master 2D v2003.02 (Ge Healthcare).
In-gel protein digestion and MALDI-TOF mass spectrometry analysis
Protein spots were excised from the gels, washed with 50 mM ammonium bicarbonate/acetonitrile 50/50 (v/v) and air-dried. Dried spots were digested for 2 hours at 37°C with sequencing grade modified trypsin in 5 mM ammonium bicarbonate, loaded on a matrix prespotted Anchorchip (PAC 384 HCCA, Bruker-Daltonics, Bremen, Germany), air-dried and washed with 70% ethanol, 0.1% trifluoracetic acid. Mass spectra were acquired on an ultraflex MALDI TOF mass spectrometer (Bruker-Daltonics). Spectra were externally calibrated by using the combination of standards present on the PAC (Bruker-Daltonics). Monoisotopic peptide matching and protein search were performed automatically by MASCOT software.
Ectocervical and Endocervical cells (Ect1/E6E7 and End1/E6E7 from ATCC) were maintained in keratinocyte serum-free medium (KSFM, Gibco) supplemented with 50 μg/mL bovine pituitary extract, 0.1 ng/mL epidermal growth factor, 0.4 mM CaCl2 and antibiotics at 37°C in 5% CO2.
Transformed urethral epithelial cells (kindly provided by M. Apicella, Department of Microbiology, University of Iowa) were maintained in Prostate Epithelial Cell Growth Medium (PrEGM, Clonetics) supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and growth factors included in PrEGM BulletKit at 37°C in 5% CO2.
Binding assay and FACS analysis
Cells were non-enzymatically detached using cell dissociation solution (CDS, Sigma), harvested and suspended in RPMI medium supplemented with 1% FBS. Approximately 105 cells were placed in 96-well microplates and mixed with different concentrations of purified PIII and NG0694 (negative control) proteins or medium alone for 1 hour at 37°C, mixing every 20 min to avoid the attachment of cells.
Excess unbound proteins were removed by two washings and centrifugations and cells were incubated for 1 hour at 4°C with anti-PIII and anti- NG0694 antisera followed by incubation with R-Phycoerythrin-conjugated anti-mouse IgG for 30 min at 4°C.
Cell-bound fluorescence was analysed with FACSCalibur flow cytometer (Becton Dickinson) by using the CellQuest software program. The mean fluorescence intensity (MFI) for each population was calculated.
Ectocervical, endocervical and tUEC cells were seeded in 96-well tissue culture plates and incubated overnight in the respective antibiotic-free media. Bacteria were grown overnight on GC agar plates, suspended in D-PBS at ≅108 cfu/mL in antibiotic-free medium. MOI (multiplicity of infection) was 100 bacteria per cell; aliquots of bacterial suspensions were diluted in D-PBS and plated at the time of infection for precise determination of bacterial starting inoculum. Cells were incubated with bacteria for 3 hours at 37°C in 5% CO2; to determine the number of intracellular bacteria, infected cells were washed four times with medium and treated with 200 μg/mL gentamicin for 1 hour at 37°C. After washing, cells were lysed by 1% saponin and plated. In parallel, to determine the growth rate of bacteria during the infection, bacteria without cells were incubated at 37°C in cell medium; after 3 hours the number of replicating bacteria was determined by serial dilution and plating. The bacterial colonies were monitored for piliation and Opa morphology by examination with a stereomicroscope.
For immunofluorescence analysis, ectocervical cells seeded on chamber slides were incubated with bacteria as described above. After incubation, wells were washed with PBS and fixed with 2% PFA for 20 min at room temperature. Subsequently, samples were blocked with 2% BSA for for 15 min and incubated with mouse polyclonal serum anti-OM (1:1000) for 1 h at room temperature. Wells were washed several times with PBS and incubated with goat anti-mouse Alexa Fluor 488 conjugated antibodies (Molecular Probes) and Alexa Fluor 568-conjugated phalloidin for 30 min at room temperature. Labeled samples were mounted with ProLong® Gold antifade reagent with DAPI and analyzed with Zeiss LSM710 confocal microscope.