Fig 1.
Akt phosphorylation occurs in two distinct phases in Salmonella-infected epithelial cells.
HeLa (A-D) or C2BBe1 (E) cells were infected with Salmonella Typhimurium. (A) Akt phosphorylation in infected HeLa cells peaks within the first 2 h following invasion but persists at a low level for 6–7 hpi. Western blot for phosphorylated Akt (pAkt) and total Akt, and band densitometry, normalized to total Akt (A) and uninfected (D and E), for phosphorylated Akt. Densitometry data show means ± SD for 3 independent experiments. Western blot is from one representative experiment. (B) Cytosolic replication of Salmonella is pronounced at 6–8 hpi and is characterized by the appearance of cells containing > 50 bacteria. Intracellular CFU were assessed by gentamicin protection assay, with (cytosolic bacteria, dotted red line) or without (total intracellular bacteria, solid red line) the addition of chloroquine. For analysis of intracellular bacteria in individual cells (Y2), cells were fixed and processed for immunostaining (bacteria) and intracellular bacteria counted by immunofluorescence microscopy. (C-E) Akt phosphorylation in individual cells. Salmonella-infected cells were fixed and processed for immunostaining (bacteria) and proximity ligation assay (PLA, pAkt). DNA was stained with DAPI. (C) Representative confocal images of infected HeLa cells. Scale bars: 10 μm. Analysis of pAkt levels in HeLa (D) or C2BBe1 (E) cells containing 1–20 bacteria/cell or >20 bacteria/cell. The pAkt spots/cell were counted and normalized to uninfected cells. Means ± SD of 3 independent experiments. ** P ≤ 0.01, *** P ≤ 0.001.
Fig 2.
Sustained delivery of SopB into epithelial cells by Salmonella.
Time course of SopB delivery in HeLa cells. Monolayers infected with Salmonella sopB3xFLAG were fixed and immunostained for bacteria (αCSA) and effector (αFLAG). (A) The percentage of effector-positive infected cells was quantified. Means ± SD from 3 independent experiments. (B) Representative confocal images of Salmonella-infected HeLa cells with SopB3xFLAG staining. Scale bars: 10 μm; inset 2 μm. (C) Analysis of intracellular Salmonella populations in SopB3xFLAG-positive cells. Effector-positive cells were classified according to the presence or absence of cytosolic bacteria. Means from 3 independent experiments. Statistical significance was analyzed by unpaired Student’s t-test. *** P ≤ 0.001, ns = not-significant. (D) Representative confocal images of HeLa cells infected with Salmonella sopB3xFLAG subjected to differential permeabilization. Cytosolic (Cyto.) bacteria (red) and the cytosolic tail of calnexin (yellow, permeabilization control) were labeled after digitonin permeabilization. Total bacteria (grey) and SopB3xFLAG (green) were detected post saponin permeabilization. Scale bars: 10 μm; inset 2 μm. See also S1 Fig.
Fig 3.
T3SS1 effectors are detected in murine gallbladder epithelial cells harboring hyper-replicating Salmonella.
Representative confocal images of gallbladders from mice infected with Salmonella expressing SopB3xFLAG (A), SipA3xFLAG (B), or no FLAG-tagged effector (C). Cryosections were immunostained for effector (αFLAG, grey) and bacteria (αCSA, green). DNA was stained with DAPI (cyan). Arrows indicate effector labeling. Whole gallbladder image (Scale bar: 200 μm) was compiled from tiled images; boxed areas are shown as enlarged overlay (scale bar: 20 μm) and single channel images (Scale bar: 5 μm). See also S2 Fig.
Fig 4.
Expression of SopB in cytosolic bacteria is sufficient to induce post-invasion Akt phosphorylation.
HeLa cells were infected with ΔsopB Salmonella complemented with plasmid borne sopB under the control of a promoter that is induced in the presence of glucose-6-phospate (PuhpT), or the arabinose inducible reporter (PBAD). (A) Induction of SopB expression in cytosolic bacteria stimulates Akt phosphorylation. Cells were fixed, and processed for PLA of phospho-Akt (pAkt) and immunostaining of SopB-2xHA (αHA). DNA was stained with DAPI. Representative confocal images are shown. Scale bars: 10 μm. (B, C) Western blot for total Akt and phosphorylated Akt (pAkt), and band densitometry, normalized to uninfected, for phosphorylated Akt, Densitometry data show means ± SD for 3 independent experiments. Western blot is from one representative experiment. Statistical significance was analyzed by 2-way ANOVA with Bonferroni’s post-test and compared to corresponding time points of ΔsopB (B) or WT (C) infected cells. * P ≤ 0.05, *** P ≤ 0.001, ns = not-significant.
Fig 5.
SopB promotes survival of cells containing cytosolic Salmonella.
(A, B) Cell death (A, LDH assay) and intracellular bacterial loads (B, gentamicin protection assay) at 6 hpi in HeLa cells infected with Salmonella WT, the ΔsopB strain, or plasmid complemented ΔsopB strain. Means ± SD of 3 independent experiments. Statistical significance was analyzed in comparison to WT infected cells. (C, D) Comparison of total and cytosolic Salmonella WT and ΔsopB in HeLa cells determined by gentamicin protection assay from untreated (total bacteria) or choloroquine treated cells (cytosolic bacteria). Means ± SD of 3 independent experiments. (E-G) Quantification of cytosolic replication and host cell death by live cell imaging of HeLa cells containing cytosolic WT and ΔsopB strains. To positively identify cytosolic bacteria the PuhpT-gfp reporter was used. The total green fluorescent signal per HeLa cell was measured. To detect cell death, propidium iodide was added to the culture media at the beginning of imaging. The mean time to PI staining of nuclei is indicated for cells containing cytosolic WT and ΔsopB strains. Statistical significance in (F, G) was analyzed by 1-way ANOVA with Bonferroni’s post-test and unpaired Student’s t-test, respectively. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
Fig 6.
Cytosolic replication of Salmonella correlates with widespread SPI1 expression.
HeLa cells were infected with WT Salmonella bearing a plasmid borne transcriptional reporter for the SPI1 gene prgH (PprgH-gfp[LVA], A-C). Cells were fixed with PFA (A, Non-amplified), or methanol (B and C, Amplified), and immunostained for bacteria (αCSA, red); methanol fixed cells were also stained for GFP (αGFP, green). Representative confocal images of cells containing cytosolic hyper-replicating (A, B) or vacuolar (C, <20 bacteria/cell) bacteria are shown. Magnified insets show single-channel images of boxed area. Nuclei and cell boundaries are outlined. Scale bars: 10 μm; inset 2 μm.
Fig 7.
T3SS1 is required for delivery of SopB and SipA by cytosolic Salmonella.
(A) Model of T3SS1. (B) Key to Salmonella strains used to determine requirement for T3SS1 and T3SS2 in effector delivery by cytosolic bacteria. Representative confocal images of infected HeLa (C) and C2BBe1 (D) cells stained for SopB3xFLAG. Infected monolayers were PFA fixed at 6 hpi and stained for effector (αFLAG), bacteria (αCSA) and LAMP1 (αLAMP1). Scale bars: 10 μm; inset 2 μm. The percentage of HeLa (E) or C2BBe1 (F) cells, containing >50 bacteria, with SopB or SipA staining. Means ± SD of 3 independent experiments. ** P ≤ 0.01, *** P ≤ 0.001. See also S4A–S4E Fig.
Fig 8.
SipB is dispensable for T3SS1 effector delivery by cytosolic Salmonella.
(A) Key to Salmonella strains used to determine requirement for the T3SS1 translocon in effector delivery by cytosolic bacteria. (B-E) Representative confocal images of infected HeLa (B) and C2BBe1 (D) cells, with quantification of effector delivery shown in (C) and (E), respectively. The percentage of infected cells containing >50 bacteria with effector staining was scored. Monolayers were infected, fixed at 6 hpi and immunostained as described in Fig 7. Means ± SD of 3 independent experiments. ns = not-significant. Scale bars: 10 μm; inset 2 μm. See also S4F–S4I Fig.
Fig 9.
Schematic of the distinct subpopulations of intracellular Salmonella during infection of epithelial cells.
SPI1-induced Salmonella invade non-phagocytic cells by translocating T3SS1 effectors into host cells. The majority of internalized bacteria remain within SCVs, although some escape into the cytosol. The mechanism of escape is not well understood but may be the result of damage by T3SS1 needles during invasion [30]. Subsequent adaptation to these two microenvironments (vacuolar vs cytosol) ultimately leads to physiologically and transcriptionally distinct intracellular bacterial populations. In particular, hyper-replicating cytosolic bacteria are SPI1 induced and secrete at least two T3SS1 effectors (SopB and SipA) into the cytosol where they can potentially target host cell pathways. Here we demonstrated that SopB-dependent Akt phosphorylation extends the lifespan of HeLa cells containing cytosolic bacteria. Findings from this study are highlighted in yellow.