Fig 1.
SopF is translocated by T3SS1.
(A) SopF translocation. J774A.1 macrophage-like cells were infected with S. Typhimurium wild type (WT), ΔprgI::FRT or ΔssaR strains harboring SopF-CyaA (SopF), SopB-CyaA (SopB) or SseK1-CyaA (SseK1) fusions. Lysates were collected at 1 h and 8 h p.i. and subject to ELISA quantification of cAMP (fmol cAMP/well). Results are the mean ± SD (n≥3 experiments). Asterisks indicate data that is significantly different from WT bacteria with no CyaA plasmid (-) (p<0.05, ANOVA with Dunnett’s post-hoc test). (B) Timecourse of intracellular SopF production. HeLa and HCT116 epithelial cells were infected with ΔsopF pSopF-3xFLAG bacteria; J774A.1 macrophage-like cells were infected with ΔsopF pSopF-2xHA bacteria. Cell lysates were collected at the indicated times p.i., then proteins were separated by SDS-PAGE and subject to immunoblotting with anti-FLAG or anti-HA antibodies. Loading was normalized to equivalent CFU for each timepoint. Molecular mass markers are indicated on the left. (-) indicates uninfected lysate. Results are representative of 2–3 independent experiments. (C) SopF is not required for bacterial entry into non-phagocytic cells. HeLa epithelial cells were infected with S. Typhimurium wild type (WT), ΔsopF, ΔsopB or ΔsopBΔsopF (ΔsopBF) bacteria and invasion efficiency (the percent of inoculum internalized at 1 h p.i.) was quantified by gentamicin resistance assay. The invasion efficiency of S. Typhimurium WT was set to 100% for each experiment. Data are the mean ± SD (n = 3 experiments). Asterisks represent data significantly different from WT bacteria (one-way ANOVA with Dunnett’s post-hoc test). (D) Translocated SopF localizes to Salmonella-induced plasma membrane ruffles and around bacteria. HeLa cells seeded on coverslips were infected with ΔsopF pSopF-3xFLAG bacteria (constitutively expressing mCherry) and fixed/permeabilized at 30 min or 1 h p.i. Translocated SopF-3xFLAG was detected using tyramide signal amplification. Representative confocal images of SopF accumulation (shown in green) in plasma membrane ruffles (left panel) or around bacteria, presumably on the SCV (right panel). mCherry bacteria (STm) are shown in red. Scale bars are 10 μm. Insets show enlargements of boxed areas. Pie charts show the mean percentage distribution of SopF localization patterns at 30 min and 1 h p.i. (n≥3 independent experiments). Categories are plasma membrane ruffles, bacteria, membrane tubule(s) originating from the SCV or a combination of these (bacteria plus ruffles, ruffles plus tubule(s) or bacteria plus tubules).
Fig 2.
SopF associates with mammalian cell membranes.
(A) HeLa cells were transfected with a plasmid encoding for EGFP-SopF for 18 h and then subject to confocal fluorescence microscopy or sequential detergent fractionation. Left panel shows a representative confocal microscopy image. EGFP-SopF (greyscale), DNA (blue). Scale bar is 10 μm. Right panel shows immunoblotting analysis. Cells were collected and subject to sequential detergent fractionation. Equal volumes of saponin-soluble, TX-100-soluble, and SDS-soluble fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against GFP, Hsp27 (cytosol), calnexin (membranes) and lamin A/C (nucleus). Molecular mass markers are indicated on the left. Results are representative of two independent experiments. (B) As for (A) except HeLa cells were transfected with a plasmid encoding for FLAG-SopF. FLAG-SopF was detected by immunostaining (left panel) or immunoblotting (right panel) with anti-FLAG antibodies. (C) SopF partially colocalizes with actin-binding proteins found at cell adhesion sites. HeLa cells were transfected with pFLAG-SopF for 18 h, then fixed and immunostained with anti-FLAG, anti-moesin, anti-lamellipodin and anti-vasodilator-stimulated phosphoprotein (VASP) antibodies. Representative confocal microscopy images show FLAG-SopF in green and moesin, lamellipodin or VASP in red. Scale bars are 10 μm. Insets show enlargements of boxed areas.
Fig 3.
(A) Saccharomyces cerevisiae wild type and six mutant PI kinase strains were transformed with yEGFP-SopF. Tet-off strains were repressed for 24 h with doxycycline before the expression of SopF was induced with galactose. The subcellular localization of SopF was monitored in live cells by widefield fluorescence microscopy. SopF relocalizes from internal membrane sites to the plasma membrane in the mss4tet-off strain, which has decreased PI(4,5)P2 and increased PI(4)P levels at the plasma membrane. yEGFP-2xPH-Osh2, which localizes to the Golgi and plasma membrane in yeast via PI(4)P binding, was used as a control for each strain. Representative fluorescence images are shown. Scale bars are 2 μm. (B) SopF colocalizes with multiple phosphoinositide pools present on the plasma membrane in mammalian cells. HeLa cells were co-transfected with mCherry-SopF and EGFP or EGFP-PH domain chimeras for 15 h and fixed. Representative confocal microscopy images show phosphoinositide-binding probes in green and mCherry-SopF in red. PI(4,5)P2, PI(4)P, and PI(3,4,5)P3 pools at the plasma membrane are bound by PH-PLCδ1, 2xPH-Osh2 and PH-Btk, respectively. Scale bars are 10 μm. Insets show enlargements of boxed areas. (C) SopF binds to multiple phosphoinositides in vitro. Recombinant glutathione S-transferase (GST) and GST-SopF were purified by affinity chromatography and incubated with PIP Strips (Echelon Biosciences) at 1 μg/ml. Bound GST and GST-SopF were detected using anti-GST antibodies followed by chemiluminescence detection. Compounds spotted on the membrane (100 pmol lipid per spot) are: lysophosphatidic acid (LPA); lysophosphatidylcholine (LPC); phosphatidylinositol phosphate (PI); PI(3)P; PI(4)P; PI(5)P; phosphatidyl ethanolamine (PE); phosphatidyl choline (PC); sphingosine-1-phosphate (S1P); PI(3,4)P2; PI(3,5)P2; PI(4,5)P2; PI(3,4,5)P3; phosphatidic acid (PA); phosphatidylserine (PS).
Fig 4.
SopF promotes nascent SCV membrane integrity.
(A) HeLa epithelial cells (left panel), HCT116 epithelial cells (middle panel) and J774A.1 mouse macrophage-like cells (right panel) were infected with S. Typhimurium wild type (WT), ΔsopF or ΔsopF pSopF-3xFLAG (comp) bacteria. The chloroquine resistance assay was used to quantify the proportion of cytosolic bacteria at 90 min p.i. Data represent the mean ± SD (n≥3 independent experiments). Asterisks indicate data significantly different from WT infection (one-way ANOVA with Dunnett’s post-hoc test). (B) Fluorescence detection of cytosolic S. Typhimurium. Wild type (WT) and ΔsopF bacteria constitutively expressing mCherry and harboring a PuhpT-gfpova reporter were used to infect HeLa cells. The unstable GFP variant (GFP-OVA) is under the control of the S. Typhimurium uhpT promoter, which is induced by glucose-6-phosphate, a metabolite found exclusively in the mammalian cytosol. GFP fluorescence is therefore indicative of S. Typhimurium that are in damaged vacuoles and/or free in the cytosol. The number of GFP-positive bacteria was scored by fluorescence microscopy. Data represent mean ± SD (n≥5 independent experiments). Asterisks indicate data significantly different from WT bacteria (Student’s t-test). (C) Timecourse of galectin-8 (GAL8) association. HeLa cells were infected with S. Typhimurium wild type (WT), ΔsopF or ΔsopF pSopF-3xFLAG (comp) bacteria (all strains are constitutively expressing mCherry) and at the indicated times, infected monolayers were fixed and immunostained for GAL8, which decorates damaged SCVs. The number of GAL8-positive bacteria was quantified by fluorescence microscopy. Data represent mean ± SD (total of >450 bacteria per strain from 3 independent experiments). Asterisks indicate data significantly different from WT bacteria (one-way ANOVA with Dunnett’s post-hoc test). (D) Timecourse of LC3 association. HeLa cells were infected as in (C) and at the indicated times cells were fixed and immunostained for microtubule-associated protein 1A/1B-light chain 3 (LC3), a marker of autophagy. The number of bacteria associated with LC3 was quantified by fluorescence microscopy. Data are the mean ± SD (n≥3 experiments). Asterisks represent data significantly different from WT bacteria (one-way ANOVA with Dunnett’s post-hoc test). Representative confocal images show LC3 association (green) with bacteria (red, STm) at 1 h p.i. and 2 h p.i. Scale bars are 10 μm. Insets are enlargements of the boxed areas.
Fig 5.
The C-terminus of SopF is required for its membrane association in eukaryotic cells.
(A) HeLa epithelial cells were infected with S. Typhimurium wild type (WT), ΔsopF, ΔsopF pSopF (comp), ΔsopF pSopF(1–345), ΔsopF pSopF(1–367) or ΔsopF pACYC177 (empty vector) bacteria. The proportion of cytosolic bacteria was determined by CHQ resistance assay at 90 min p.i. (upper panel) or GAL8 recruitment at 1 h p.i. (lower panel, all bacteria are constitutively expressing mCherry for fluorescence detection). Upper panel: data represent the mean ± SD (n≥3 independent experiments). Lower panel: Data represent the mean ± SD (total of >600 bacteria per strain from n≥3 independent experiments). Asterisks indicate data significantly different from WT infection (one-way ANOVA with Dunnett’s post-hoc test). (B) HeLa cells were transfected with plasmids encoding for FLAG-SopF(1–367) or FLAG-SopF(1–345) for 18 h. Cells were fixed and immunostained with anti-FLAG antibodies. DNA was stained with Hoechst 33342. Representative confocal microscopy images show FLAG-SopF in greyscale and DNA in blue. Scale bars are 10 μm. (C) Subcellular fractionation of transfected cells. HeLa cells were transfected with plasmids encoding for FLAG-SopF, FLAG-SopF(1–367) or FLAG-SopF(1–345) for 18 h, then collected and subjected to sequential detergent fractionation. Equal volumes of saponin-soluble, TX-100-soluble, and SDS-soluble fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against the FLAG epitope, Hsp27 (cytosol), calnexin (membranes) and lamin A/C (nucleus). Molecular mass markers are indicated on the left. Results are representative of two independent experiments. (D) C-terminal truncations of SopF lose plasma membrane association in the S. cerevisiae mss4tet-off strain. Wild type (WT) and mss4tet-off yeast strains were transformed with plasmids encoding for yEGFP-SopF, yEGFP-SopF C370S, yEGFP-SopF(1–367) or yEGFP-SopF(1–345) and the subcellular localization of SopF in live cells was visualized by widefield fluorescence microscopy. Representative fluorescence images are shown. Scale bars are 2 μm. The role of a potential lipidation site in SopF localization was assessed by site-directed mutagenesis of the Cys370 residue (C370S). The grey box depicts a domain of unknown function (DUF), DUF3626, spanning amino acid residues 178–338 of SopF. (E) Quantification of SopF localization in WT and mss4tet-off yeast strains that were transformed and visualized as described in (D). Subcellular localization was categorized as cytosol, internal membrane sites (IMS), plasma membrane (PM), or IMS and PM. Results are expressed as the mean percentage of total yeast transformants (n = 300 cells from three independent transformations).
Fig 6.
SopF and SopB have opposing effects on vacuole stability.
(A) HeLa cells were infected with the following mCherry-expressing S. Typhimurium strains–wild type (WT), ΔsopF, ΔsopB, ΔsopB pWKSDE (in trans complementation with SopB/SigD and its cognate chaperone SigE, ΔsopB DE), ΔsopB pWSKDE C460S (in trans complementation with SopB/SigD C460S and its cognate chaperone SigE, ΔsopB C460S), ΔsopBΔsopF (ΔsopBF), ΔsopBΔsopF pWSKDE (ΔsopBF DE) or ΔsopBΔsopF pWSKDE C460S (ΔsopBF C460S). Monolayers were fixed at 1 h and 2 h p.i. and immunostained for GAL8 (upper panel), a marker of damaged SCVs, or the autophagy-associated protein, LC3 (lower panel). The number of GAL8- or LC3-positive bacteria was scored by fluorescence microscopy. Data represent the mean ± SD (n≥3 independent experiments). Asterisks indicate data significantly different from WT infection for each timepoint (one-way ANOVA with Dunnett’s post-hoc test). (B) Translocated SopF fractionates to host cell membranes independent of SopB. HeLa cells were infected with S. Typhimurium ΔsopF pSopF-3xFLAG or ΔsopBΔsopF (ΔsopBF) pSopF-3xFLAG bacteria. At 1 h p.i., cells were mechanically disrupted and subject to differential centrifugation to obtain three fractions–P (unbroken host cells, host cell nuclei, intact bacteria), M (host cell membranes) and C (host cell cytosol). Equal volumes were separated by SDS-PAGE and subject to immunoblotting with antibodies directed against FLAG, LAMP-1 (lysosomal membranes), Hsp27 (cytosol) and DnaK (bacteria). Molecular mass markers are indicated on the left. Results are representative of two independent experiments. (C) HeLa cells were infected with ΔsopBΔsopF pSopF-3xFLAG bacteria and the localization of translocated SopF was determined by tyramide signal amplification followed by fluorescence microscopy. Pie charts show the mean percentage distribution of SopF localization patterns at 30 min and 1 h p.i. (n = 3 independent experiments). Categories are plasma membrane ruffles, bacteria, membrane tubule(s) originating from the SCV or a combination of these (bacteria plus ruffles, ruffles plus tubule(s) or bacteria plus tubules).