Fig 1.
Purification of N. gonorrhoeae secreted OMVs.
(A) N. gonorrhoeae MS11-A was analyzed by transmission (top panels) and scanning (bottom panels) electron microscopy (EM). Arrows indicate OMVs. Scale Bar = 0.5, 0.2 and 0.1μm. (B) Crude OMV preparations from FA1090 and MS11-A cultures were analyzed by transmission electron microscopy (TEM) (right panels show higher magnification images) (C) N. gonorrhoeae MS11-A derived OMVs were fractionated by OptiPrep density gradient ultracentrifugation and 11 fractions and proteins analyzed by colloidal coomassie staining. Densitometry of the prominent band that corresponds to PorB (shown on the top). Immunoblot analysis of each of the fractions probed with anti-PorB (bottom). Molecular weight markers (kDa) are indicated on the left. (D) TEM images of the negatively stained pooled fractions from OptiPrep density gradient as indicated at the bottom. Scale Bar = 0.2 μm.
Fig 2.
OMVs secreted by N. gonorrhoeae contain distinct proteins.
(A) Total membranes of N. gonorrhoeae were separated by sucrose gradient ultracentrifugation and fractions were probed with anti-PorB and anti-BamA to detect fractions with outer membranes and anti-F1-β with inner membranes. Fractions 10 and 5 were used as outer and inner membrane fractions, respectively, in further analysis. (B) OMVs, outer membrane (OM), inner membrane (IM), total membrane (TM) and whole bacterial lysate (WBL) were probed for two outer membrane proteins (BamA and PorB) and inner membrane protein, F1-β. (C) Colloidal coomassie staining of proteins of N. gonorrhoeae secreted outer membrane vesicles (OMV), outer membrane (OM) and inner membrane (IM). The positions of molecular size markers (kDa) are shown on the left. PorB (36 kDa) is indicated on the right and over represented proteins in OMVs by asterisks.
Fig 3.
Porins are the major membrane proteins identified in purified OMVs.
(A) Proteome of purified N. gonorrhoeae MS11-A OMVs as identified by LC-MS/MS relative abundance based on peptide matched spectra. (B) Predicted subcellular localization of the number of identified proteins in purified and crude OMVs. (C) Predicted subcellular localization of identified proteins in purified and crude OMVs based on the relative abundance of identified peptides. (D) The relative percentage of proteins containing signal peptides in purified and crude OMVs, based on SignalP v.4.1 analysis. (E) All identified proteins in purified OMVs were functionally classified based on COG database using WebMGA server.
Fig 4.
PorB complex formation in OMVs.
(A) Super-resolution microscopy (dSTORM) of N. gonorrhoeae (left) and outer membrane vesicles (middle and zoomed in right) probed with anti-PorB serum. Panel on the right shows fluorescence intensity along the line across vesicles. Scale bar = 500 nm. (B) Transmission electron microscopy of N. gonorrhoeae (left panel) and OMVs (right panel) probed for PorB using 10 nm-gold conjugated antibodies. Gold particle distribution around the surface of bacteria and vesicles represented by arrow. Scale bar = 0.2 μm. (C) Boiled and unboiled purified OMVs were solubilized with increasing concentrations of detergent (sodium dodecyl sulfate, SDS) as indicated and analyzed by colloidal coomassie staining after semi-native gel electrophoresis. Monomeric and a higher order molecular weight PorB protein complex are indicated by arrows. (D) Immunoblot analysis of OMVs and bacterial total membranes isolated from N. gonorrhoeae with increasing concentrations of detergent (SDS) as indicated and probed for PorB after semi-native gel electrophoresis. Monomeric (ɵ) and higher order molecular weight PorB protein complex (*) are indicated.
Fig 5.
OMVs enable mitochondria targeting of PorB.
(A) Bone marrow-derived macrophages (BMDMs) were treated with purified N. gonorrhoeae OMVs and analyzed by confocal laser scanning microscopy for PorB (red) and Tom20 (green) localization at indicated times. Cells were stained with DAPI (blue) to visualize nuclei. Scale bar = 10 μm. Representative images of more than 200 cells from three biological samples. (B) BMDMs treated with purified OMVs for 12 hours were analyzed by RapidSTORM reconstructed dual colour super-resolution imaging for PorB (red) and Tom20 (green) localization. (C) OMV treated BMDMs (12 hours) were probed with anti-PorB (green) and Tom20 (red) antibodies and analyzed by 3D single-molecule localization super-resolution microscopy.
Fig 6.
OMVs associate with mitochondria.
(A, B and C) Bone marrow-derived macrophages (BMDMs) were treated with purified N. gonorrhoeae OMVs for 24 hours and imaged by transmission electron microscopy by negative staining and (C) 10 nm-gold conjugated antibodies against PorB. (D) Crude mitochondria (P) and cytoplasmic fractions (S) were obtained from OMV-treated BMDMs and analyzed for PorB and BamA and mitochondria (Tim23, Tom40 and F1β) and cytosolic (GAPDH) markers by immunoblot analysis after semi-native gel electrophoresis. (E) Mitochondria-enriched fractions were incubated with purified OMVs or PBS and subsequent supernatant (S) and pellet (P) fractions were analyzed by immunblotting with anti-PorB, BamA and Tim23 antibodies after SDS gel electrophoreses.
Fig 7.
N. gonorrhoeae depends on OMVs for PorB delivery to macrophages.
(A) Wild type (WT) and ΔNGFG_01788 deletion mutant were cultured in GC media and growth was monitored by optical density at 600 nm. Mean and standard deviation from triplicate experiments. (B) Transmission electron microscopy of ΔNGFG_01788 mutant showing diplococci and OMV secretion. (C) Purified OMVs and whole bacteria lysates were analyzed by coomassie staining after SDS gel electrophorese and the amount of PorB within OMVs relative to whole bacterial cells determined by densitometry. Relative OMV-mediated secretion of PorB in the mutant is based on wild type levels (100%). Mean and standard deviation from three independent experiments are shown. (D) BMDMs were incubated with wild type N. gonorrhoeae or (E) ΔNGFG_01788 mutant in transwells and analyzed for PorB (red) and Tom20 (green) localization by immunofluorescence analysis after 24 hours. DAPI (blue) indicates cell nucleus. (F) The number of PorB signals (puncta) was quantified from >100 cells and two independent experiments.
Fig 8.
OMVs trigger loss of mitochondrial health and activation of apoptotic caspases in macrophages.
(A) BMDMs treated with N. gonorrhoeae OMVs (20 and 40 μg/mL) for 48 hours were analyzed by the MTT assay to determine cell viability and mitochondrial activity relative to PBS treated cells. Mean and SD from 3 independent experiments are shown (asterisks indicate p<0.01). (B) BMDMs were labelled with TMRM and treated with purified OMVs (40 μg/mL) for 48 hours to determine the mitochondrial membrane potential (ΔΨm) compared to PBS treated cells. Mean and SD from three independent experiments, containing triplicate biological samples with more than 500 cells (asterisks indicate p<0.001). (C) BMDMs were treated with staurosporine (STS), OMVs and PBS for 24 hours and mitochondria and cytosolic fraction were analyzed by immunoblotting for cytochrome c and Tim23. A nonspecific (NS) band from anti cytochrome c antibody was used as a control for loading. (D) BMDMs were treated with PBS, OMVs, STS, ABT-737 and cycloheximide (CHX) for 24 hours and analyzed by immunoblotting for cleaved caspase-3 (p17 kDa). Anti-PorB and anti-tubulin were used as control for OMV treated cells and loading, respectively. Data representative of three independent experiments. (E) Quantification of cleaved caspase-3 (p17 kDa) from panel D by densitometry. Mean and SD from three independent experiments shown. (F) OMV treated BMDMs were incubated with Draq7 (blue) to stain nuclei of dead cells and analyzed by time lapse imaging. Time frames are shown. Arrows indicate blebbing cells. Scale bar = 100 μm.
Fig 9.
OMVs induce delayed macrophage death.
BMDMs treated with PBS, N. gonorrhoeae purified OMVs and the pan-caspase inhibitor, Q-VD-PH (QVD) were analyzed by live-cell imaging every 30 minutes for 48 hours to determine (A) cell death (Draq7 positive cells), (B) mitochondrial membrane potential (ΔΨm, TMRM positive cells) and (C) caspase-3/7 activity (caspase fluorogenic substrate positive cells). Mean and standard error of the mean (SEM) are shown for three independent experiments, containing triplicate biological samples.
Fig 10.
PorB delivered by E. coli OMVs induces macrophage apoptosis.
(A and B) N. gonorrhoeae PorB was expressed in E. coli and OMVs from E. coli (Ec OMV) and PorB expressing E. coli (Ec PorB OMV) analyzed by transmission electron microscopy and anti-PorB immunogold labelling. (C and D) BMDMs were treated with PBS and OMVs from N. gonorrhoeae (Ng OMV), PorB expressing E. coli (Ec PorB OMV) or control E. coli (Ec OMV) and (C) cell death (Draq7) and (D) caspase-3/7 activity (fluorogenic substrate) was determined every 30 minutes for 48 hours using live-cell imaging. Mean and standard deviation from two independent experiments with three biological repeats are shown.
Fig 11.
Schematic model of PorB delivery to mitochondria by OMVs.
PorB is expressed in the outer membrane of N. gonorrhoeae as a trimeric complex. PorB is also present in a similar complex in OMVs. Macrophages sense and take up OMVs, although the mechanism remains to be elucidated. Intact N. gonorrhoeae OMVs containing PorB are released into the cytosol, suggesting escape from endo-lysosomal compartments. Cytosolic OMVs in close proximity to mitochondria enable transport of PorB and other OMV proteins such as BamA to mitochondrial membranes, including outer and inner membranes. Mitochondria-associated PorB is largely present as monomeric protein. PorB targeting of mitochondria induces cytochrome c release, the activation of caspase-3, plasma membrane blebbing and macrophage cell death.