Fig 1.
PIK3C3 promotes Shigella flexneri cell-to-cell spread in HT-29 cells.
(A-C) Representative images showing infection foci formed by CFP-expressing S. flexneri in HT-29 cells 8 hours post-infection in presence of the specific PIK3C3 inhibitor VPS34-IN1 (A) or SAR405 (B), or upon treatment with siRNA duplexes targeting PIK3C3 or N-WASP (as positive control for no spread) (C). Scale bar, 50 μm. (D-F) Quantification of foci size (area) in arbitrary units 8 hours post-infection in presence of VPS34-IN1 (D), or SAR405 (E) or upon treatment with siRNA duplexes targeting PIK3C3 (F). Each dot represents the average of one biological replicate. At least 50 infection foci were analyzed in each of three independent biological replicates. Error bars represent standard error of the mean. Statistical analysis, one-way ANOVA with Dunnett’s multiple comparisons test; ns, not significant; *, p < 0.05; **, p < 0.005; ****, p < 0.0001. (G) Western blot showing knockdown efficiency of two siRNA duplexes targeting PIK3C3. (H) Quantification of the knockdown efficiency as shown in (G) for three biological replicates. PIK3C3 was normalized to loading control (Actin) and knockdown efficiency was calculated relative to mock treated cells. Error bars represent standard error of the mean. Statistical analysis, one-way ANOVA with Dunnett’s multiple comparisons test; ns, not significant; ****, p < 0.0001. (I) Percentage of bacteria displaying an actin tail per infection focus 4 hours post-infection in presence of VPS34-IN1. Eight foci were analyzed per replicate and three biological replicates were performed. Error bars represent standard error of the mean. Statistical analysis, one-way ANOVA with Dunnett’s multiple comparisons test; ns, not significant.
Fig 2.
PIK3C3 promotes Shigella flexneri cell-to-cell spread through resolution of VLPs into DMVs.
(A) Schematic representation of S. flexneri cell-to-cell spread. The bacterium (grey) displays actin-based motility (red filaments) and projects into adjacent cells at cell-cell contacts through formation of membrane protrusions (light blue). The collapse of the actin cytoskeleton in the protrusion neck results in the formation of an intermediate structure termed Vacuole-Like Protrusions (VLPs, purple). The resolution of the membrane tether linking VLPs to primarily infected cells lead to the formation of Double Membrane Vacuoles (DMVs, yellow). DMV escape grants the bacteria access to the cytosol of secondary infected cell where they resume actin-based motility (red). Created in BioRender. Rolland, S. (2023) BioRender.com/z03v009. (B) Representative tracking analysis using live-fluorescence confocal microscopy of HT-29 cells infected with CFP-expressing S. flexneri in absence (top) or presence (bottom) of VPS34-IN1 at 500 nM. Each bar represents the tracking of a single bacterium over 4h30. At least 30 bacteria were tracked for each condition per biological replicate. Color code (bottom right): dark blue, primarily infected cells; light blue, protrusion; purple, vacuole-like protrusion (VLP); yellow, double membrane vacuole (DMV) and red, escape and actin-based motility in the cytosol of adjacent cells. (C) Pie chart showing the proportion of bacteria failing at a given stage in three biological replicates. Means and standard error of the means are indicated. Statistical analysis, two-way ANOVA with Sidak’s multiple comparisons; ns, not significant; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. (D-F) Time spent in protrusions (D), VLPs (E), or DMVs (F) for bacteria that successfully transitioned to the next stage of the dissemination process. Red dots correspond to bacteria that did not escape DMVs by the end of tracking. Error bars represent the standard error of the means. Statistical analysis, unpaired t-tests; ns, not significant; *, p < 0.05; ****, p < 0.0001. (G) Time spent in protrusions for all bacteria that failed to spread from cell-to-cell and did not form VLPs. Error bars represent the standard error of the means. Unpaired t-tests were performed, ns, not significant.
Fig 3.
PIK3C3 promotes Listeria monocytogenes cell-to-cell spread in HT-29 cells through DMV escape.
(A-B) Representative images showing infection foci formed 8 hours post-infection in HT-29 cells infected with GFP-expressing L. monocytogenes in presence of VPS34-IN1 (A) or SAR405 (B). Scale bar, 50 μm. (C-D) Quantification of foci size (area) in arbitrary units at 8 h post-infection in presence of VPS34-IN1 (C) or SAR405 (D). Each dot represents the average of one biological replicate. Three independent biological replicates were performed, each containing at least 50 foci. Error bars represent the standard error of the mean. Statistical analysis, one-way ANOVA with Dunnett’s multiple comparisons test; ns, not significant; **, p < 0.005; ****, p < 0.0001. (E) Representative tracking analysis using live-fluorescence confocal microscopy of HT-29 cells infected with CFP-expressing L. monocytogenes in absence (top panel) or presence of VPS34-IN1 at 500 nM (bottom). Each bar represents the tracking of a single bacterium over 4 hours. Color code (bottom right): dark blue, primarily infected cells; light blue, protrusion; yellow, double membrane vacuole (DMV) and red, escape and mobility in the cytosol of adjacent cells. (F) Pie chart showing the proportion of bacteria failing at a given stage in three biological replicates. Means and standard error of the means are indicated. Statistical analysis, two-way ANOVA with Sidak’s multiple comparisons; ns, not significant; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. (G-H) Time spent in protrusions (G), or DMVs (H) for bacteria that transition to the next stage of the dissemination process. Each dot represents the time spent of a single bacterium in the corresponding phase. Red dots correspond to bacteria that did not escape DMVs by the end of tracking. Error bars represent the standard error of the means. Statistical analysis, unpaired t-tests; ns, not significant; *, p < 0.05; ****, p < 0.0001.
Fig 4.
PIK3C3 promotes the resolution of VLPs into DMVs through formation of PtdIns(3)P at the protrusion/VLP membrane.
(A) Representative images of membrane YFP-expressing cells infected with CFP-expressing S. flexneri dissemination from a PX-mCherry non-expressing cell to a PX-mCherry expressing cell. PX-mCherry specifically binds PtdIns(3)P. Scale bar, 2 μm. (B) Representative tracking analysis in HT-29 cells infected with CFP-expressing S. flexneri in absence (top panel) or presence of VPS34-IN1 at 500 nM (bottom panel). Each bar represents the tracking of a single bacterium spreading from a non-expressing cell into a PX-mCherry expressing cell over 4 hours. At least 30 bacteria were tracked for each condition. Color code: dark blue, primarily infected cells; light blue, protrusions; purple, vacuole-like protrusions (VLPs); yellow, double membrane vacuoles (DMVs) and red, cytosol of adjacent cells; black bar, recruitment of PX-mCherry. (C) Representative example of relative fluorescence intensity calculation. (D) Relative fluorescence of mCherry-PX at the VLP membrane the time frame before DMV formation. Error bars indicate standard deviation of the mean. Statistical analysis, unpaired t-tests were performed; ns, not significant; ****, p < 0.0001. (E) Graphs representing the relative fluorescence intensity at the membrane of protrusion/VLP/DMV for an example of successful cell-to-cell spread. (F-G) Graphs representing the relative fluorescence intensity at the membrane of protrusions and VLPs for an example of failure in protrusion (F), and failure in VLP (G), in presence of VPS34-IN1(500 nM).
Fig 5.
DNM2 promotes Shigella flexneri cell-to-cell spread through transient recruitment at the neck of VLPs, leading to DMV formation.
(A) Representative images of membrane YFP-expressing cells infected with CFP-expressing S. flexneri showing the recruitment of DNM2-mCherry during cell-to-cell spread. Scale bar, 2 µm. (B) Representative tracking analysis in HT-29 cells infected with CFP-expressing S. flexneri in absence (Mock, top panel) or presence of VPS34-IN1 at 500 nM (VPS34-IN1, bottom panel). Each bar represents the tracking of a single bacterium spreading from a non-expressing cell into a DNM2-mCherry expressing cell. At least 30 bacteria were tracked for each condition. Color code: dark blue, primarily infected cells; light blue, protrusions; purple, vacuole-like protrusions (VLPs); yellow, double membrane vacuoles (DMVs) and red, cytosol of adjacent cells; black bar, recruitment of DNM2-mCherry. (C) Representative example of relative fluorescence intensity calculation. (D-E) Graphs representing the relative fluorescence intensity at the membrane of protrusion/VLP/DMV for an example of successful cell-to-cell spread (D), and failure in VLP (E).
Fig 6.
PIK3C3 is required for efficient cell-to-cell spread in vivo.
(A) Representative images of infant rabbit colon sections showing infection foci formed by S. flexneri (red) in E-cadherin-positive epithelial cells (green) 6 hours post-infection. Scale bar, 50 μm. (B) Foci size average (area in arbitrary units) formed by Shigella in infant rabbit colon sections in absence and in presence of SAR405. Each dot represents the average foci size per animal. Statistical analysis, unpaired t-tests; ns, not significant; ****, p < 0.0001. (C) Percentage of bacteria displaying an actin tail per infection focus 6 hours post-infection in presence of SAR405 in HT-29 cells. Eight foci were analyzed per replicate in three independent biological replicates. Statistical analysis, two-way ANOVA with Sidak’s multiple comparisons; ns, not significant.
Fig 7.
Model of Shigella cell-to-cell spread supported PIK3C3 and DNM2.
After escape from primary vacuoles and acquisition of actin-based mobility, S. flexneri projects into adjacent cells through formation of membrane protrusions. Collapse of the actin cytoskeleton in protrusions leads to the formation of VLPs. PIK3C3-dependent formation of PtdIns(3)P at the protrusion/VLP membrane is required for the recruitment at the VLP neck of the membrane scission machinery, DNM2, leading to the formation of DMVs. S. flexneri escape from DMVs does not rely on PIK3C3-dependent formation of PtdIns(3)P. After escaping DMVs, S. flexneri resumes actin-based motility in the cytosol of adjacent cells. Created in BioRender. Rolland, S. (2023) BioRender.com/r81i566.