Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles

Abstract

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane-derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including beta-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP-mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

Figure 1. OMV deliver multiple toxins to induce cytotoxicity in airway epithelial cells.

(A) Purified P. aeruginosa OMV deliver multiple virulence factors (Cif, alkaline phosphatase, β-lactamase, and PlcH) into the cytoplasm of airway epithelial cells. Western blot analysis of cell lysates of airway epithelial cells treated with purified OMV. CTRL, no OMV added. Δcif OMV applied to airway epithelial cells, cells lysed, and probed by Western blot analysis for Cif did not detect Cif in airway cell lysates (data not shown). (B) OMV elicit time-dependent cytotoxicity in host airway cells as determined by the CellTiter 96 AQ_ueous One cytotoxicity assay. (C) OMV elicit time-dependent cytotoxicity in host airway cells that is not dependent on the presence of Cif, as determined by the CellTiter 96 AQ_ueous One cytotoxicity assay. Δcif OMV, OMV purified from a strain of P. aeruginosa lacking the cif gene. Black bars, wild-type OMV (WT OMV); white bars, Δcif OMV. (D) Airway epithelial cells treated with lysed OMV components demonstrated a reduction in cellular cytotoxicity as determined by the CellTiter 96 AQ_ueous One cytotoxicity assay. Data are presented as mean+/−SEM. n = 3, * p<0.05, OMV versus Control, intact OMV versus lysed OMV.

Figure 2. OMV deliver functional Cif virulence factor to airway epithelial cells, across a mucus layer.

(A) Purified OMV applied to the apical surface of airway epithelial cells reduce CFTR in the plasma membrane in a time-dependent manner, compared to the buffer control (Control), as measured by Western blot analysis. Overnight supernatant (PA14 O/N Sup), previously reported to decrease apical membrane CFTR, serves as a positive control . (B) OMV purified from a P. aeruginosa Δcif mutant strain did not reduce apical membrane expression of CFTR, as measured by Western blot analysis. Black bar, wild-type OMV; striped bar, Δcif OMV. (C) Cif-containing OMV decrease apical membrane CFTR in airway epithelial cells (CFBE) and mucous-producing airway epithelial cells (Calu-3), as measured by Western blot analysis. Black bar, CFBE cells; striped bar, Calu-3 cells. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control.

Figure 3. Cif-containing OMV fuse with the epithelial cell lipid raft microdomains.

(A) Cif virulence factor and Omp85 localize to the lipid raft fraction. Omp85, a documented OMV protein, and Cif virulence factor co-fractionate with the lipid raft marker, flotillin-1, when raft and non-raft fractions are isolated from airway epithelial cells treated with OMV for 5 min, as measured by Western blot analysis. Right blot represents raft and non-raft fractions from the left blot, which are combined and the proteins precipitated to concentrate Cif signal to allow antibody detection. (B) Rhodamine R18-labeled OMV, which only fluoresce red upon fusion with the host plasma membrane, co-localize with the FITC-conjugated lipid raft marker cholera toxin B subunit (CtxB). All OMV co-localize with lipid rafts with the exception of five OMV that are indicated with small white arrows (see lower right panel). Scale bar equals 10 µm. (C) Cif virulence factor interacts with GPIp137 in the lipid raft fraction. Immunoprecipitation (IP) experiments demonstrate that the GPI-anchored protein p137 (GPIp137), a lipid raft marker, interacts with Cif in the lipid raft fractions of airway epithelial cells treated with wild-type OMV for 5 min. Serving as a negative control, cif mutant OMV (Δcif OMV)–treated airway epithelial cells cannot immunoprecipitate Cif and therefore do not demonstrate immunoprecipitation of GPIp137. 5% of total lysate run in Lysate lane. IB, immunoblot. Experiments repeated three times; representative blots/images are shown.

Figure 4. Disruption of lipid rafts blocks virulence factor delivery and function in airway epithelial cells.

(A) Rhodamine-R18 labeled OMV were applied to the apical side of polarized airway epithelial cells, and fluorescence was measured over time as readout for OMV fusion. Rhodamine-R18 signal is quenched at high concentration in OMV, but fluorescence increases as the rhodamine probe is diluted by fusion of the OMV with the host plasma membrane. OMV and cells alone serve as negative controls where the fluorescence does not change over time (background fluorescence). Black square, OMV+cells; black triangle, OMV alone; black inverted triangle, cells alone. Data are presented as mean fluorescence intensity. (B,C) Visualization and quantitative assay demonstrating that disruption of lipid rafts with the cholesterol-sequestering reagent, filipin III complex (10 µg/ml), inhibits OMV fusion with airway epithelial cells. Rhodamine-R18 labeled OMV were applied to the apical side of polarized airway epithelial cells in the presence or absence of filipin III, and fluorescence was measured over time. Wheat germ agglutin (WGA-blue) is a marker of host epithelial cell membranes. Scale bars equal 10 µm. Black square, control; black triangle, filipin III. (D) Filipin III prevents delivery of Cif to host cell endosomes, as measured by cytoplasmic and endosomal fractionation and Western blot analysis. Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. Cyto, cytoplasm; Endo, endosome. (E) Disruption of lipid rafts blocks Cif-mediated reduction in apical membrane CFTR expression, compared to buffer control (Control), as measured by Western blot analysis. “Centrifuge Sup” refers to the overnight culture supernatant of P. aeruginosa PA14 depleted of OMV, which serves as a negative control in this experiment. (F) Filipin III complex disruption of host cell lipid raft microdomains reduces the cytotoxic effect of OMV on host airway cells with 8 h OMV treatment, as measured with the CellTiter 96 AQ_ueous One cytotoxicity assay. Data are presented as mean+/−SEM. n = 3, * p<0.05, OMV versus Control; # p<0.05, OMV versus OMV+Filipin III.

Figure 5. Intact N-WASP–induced actin cytoskeleton is required for OMV fusion and virulence factor delivery.

(A) Wiskostatin and cytochalasin D disrupt actin cytoskeleton in airway cells. Alexa 647–conjugated phalloidin was used to label actin to monitor disruption of the actin cytoskeleton by two actin-disrupting agents, wiskostatin (Wisko, 10 µM for 30 min) and cytochalasin D (Cyto D, 2 µM for 45 min), by immunofluorescence microscopy. Scale bar equal to 20 µm. (B,C) Visualization and quantitative assay demonstrating that disruption of the actin cytoskeleton with either of the two actin-disrupting agents, wiskostatin (Wisko, 10 µM) or cytochalasin D (Cyto D, 2 µM), inhibits OMV fusion with airway epithelial cells. Rhodamine-R18 labeled OMV were applied to the apical side of polarized airway epithelial cells in the presence or absence of wiskostatin or cytochalasin D, and fluorescence was measured over time. Scale bar equals 10 µm (B). Representative images of three experiments revealing that OMV do not fuse with cells in which actin has been disrupted. WGA, wheat germ agglutin (blue) to label the cell surface. Black square, control; black triangle, Cytochalasin D; open circle, Wiskostatin. * p<0.05 (Control versus Cytochalasin D and Wiskostatin). (D) Wiskostatin or cytochalasin D treatment of airway epithelial cells prevents delivery of Cif virulence factor to endosomal fraction, as measured by cytoplasm and endosome fractionation and Western blot analysis. Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. Cyto, cytoplasm; Endo, endosome. (E) Disruption of the actin cytoskeleton with wiskostatin blocks the Cif virulence factor–mediated reduction in CFTR apical membrane expression in airway epithelial cells, as measured by Western blot analysis. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control.

Figure 6. Cif virulence factor is delivered to the cytoplasm and localizes to the cytoplasmic face of early endosomes.

(A) Cif localizes to the early endosomal (Rab5 GTPase, early endosomal antigen (EEA)-1 labeled) compartment after entry into airway epithelial cells. Airway epithelial cells were treated with OMV for 10 min, cells lysed, and endosomes were purified. Cif was immunoprecipitated from the endosomal fraction, and Western blot analysis was performed for Rab5 GTPase and EEA-1, early endosomal markers. IgG IP is a non-immune control immunoprecipitation experiment. (B) The ability of proteinase K (PK) to degrade Cif from the early endosomal fraction reveals that the Cif virulence factor localizes to the cytoplasmic face of early endosomes, as measured by Western blot analysis. The transferrin receptor serves as a control for a luminal endosomal protein marker, which is only exposed to proteinase K after treatment with Triton X-100 (TX). (C) Cif entry into airway epithelial cells is not altered by disruption of endosomal acidification by NH₄Cl (5 mM). Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. (D) Entry of Cif virulence factor into airway cells is unaffected by inhibition of retrograde transport with Brefeldin A (1 µM), as measured by Western blot analysis. Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. (E) Retrograde transport to the endoplasmic reticulum is not required for Cif to reduce plasma membrane CFTR in airway cells. Airway epithelial cells pretreated with Brefeldin A (1 µM), which inhibits retrograde transport, were treated with OMV. Brefeldin A had no effect on the ability of Cif to reduce plasma membrane CFTR, as measured by Western blot analysis. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control.

Figure 7. OMV required for virulence factor delivery to host airway cells.

(A) OMV delivery enhances the ability of Cif to reduce plasma membrane CFTR in airway cells by 17,000-fold. Cif was applied to airway epithelial cells as 1 ng, 10 ng, 50 µg recombinant protein or 3 ng in OMV, and its effect on CFTR expression at the apical plasma membrane was measured by Western blot analysis. (B) Cif was detected in airway cells treated with 50 µg Cif or 3 ng Cif packaged in OMV. Airway epithelial cells were treated with vehicle (Control-CTRL), 1 ng, 10 ng, or 50 µg of recombinant Cif protein, or with 3 ng of Cif packaged in OMV (OMV). Cif was detected in the airway cell cytoplasm by Western blot analysis. The presence of Cif protein in the airway cell cytoplasm correlated with the ability of Cif to reduce plasma membrane CFTR (A). (C) Intact OMV required for reduction of apical membrane CFTR expression (i.e., Cif virulence factor function). Airway epithelial cells were treated with OMV or lysed OMV components, and airway epithelial cells were assayed for the Cif-mediated reduction in apical membrane CFTR, as measured by Western blot analysis. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control.

Figure 8. Proposed model for P. aeruginosa OMV fusion with airway epithelial cells.

OMV are released from P. aeruginosa, diffuse to the host cell plasma membrane, and subsequently fuse with host cell lipid raft microdomains. Virulence factors are subsequently released into the cytoplasm of the airway epithelial cell, through an actin-dependent pathway.