Quantitative assessment of cytosolic Salmonella in epithelial cells - PubMed (original) (raw)
Quantitative assessment of cytosolic Salmonella in epithelial cells
Leigh A Knodler et al. PLoS One. 2014.
Abstract
Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Hyper-replicating invasion-primed Salmonella occur in numerous epithelial cell lines.
Epithelial cells were infected with mCherry S. Typhimurium (left and middle panels) or S. Typhimurium harboring a reporter plasmid, P_prgH_-GFP[LVA] (right panels). Left panel; cells were fixed at 1 h and 8 h p.i. and the number of internalized bacteria per cell was scored by fluorescence microscopy. Each dot represents one infected cell (≥50 infected cells were scored for each timepoint). Data are from one experiment representative of at least three independent experiments. Middle and right panels; representative confocal images of hyper-replicating, cytosolic Salmonella. Cells were fixed at 8 h p.i., permeabilized and immunostained for the vacuolar membrane marker, LAMP1 (middle panels), and flagellin, FliC (right panels). DNA was stained with Hoechst 33342. Scale bars are 20 µm.
Figure 2. Vacuolar lysis is not dependent upon bacterial load.
HeLa cells were infected with mCherry S. Typhimurium. At 1 h p.i., the plasma membrane was permeabilized with digitonin, followed by delivery of anti-Salmonella LPS antibodies to the cytosol. Monolayers were visualized by fluorescence microscopy. (A) In epithelial cells containing at least one cytosolic bacterium (LPS-positive after digitonin permeabilization), the total number of internalized bacteria was scored. Combined data from three independent experiments (n = 201 cells). (B) In cells containing at least one cytosolic bacterium, the proportion of cytosolic bacteria in the total bacterial load was calculated. Data was binned into three categories: cells containing 1–5 total bacteria, 6–10 total bacteria and >10 total bacteria. Each dot represents one cell. Combined data from two independent experiments (n = 147 cells).
Figure 3. Vacuolar, but not cytosolic, Salmonella are susceptible to chloroquine treatment.
HeLa epithelial cells were seeded on Thermanox® plastic coverslips and infected with wild type S. Typhimurium. At 7 h p.i., cells were left untreated or treated with 400 µM CHQ for 1 h. Untreated and CHQ-treated cells were then fixed at 8 h p.i. and processed for transmission electron microscopy. (A) Vacuolar bacteria in untreated cells. (B) Vacuolar bacteria in CHQ-treated cells. (C) Hyper-replicating, cytosolic bacteria in untreated cells. (D) Hyper-replicating, cytosolic bacteria in CHQ-treated cells. (E) Inset of (C). (F) Inset of (D). Arrowheads indicate bacteria enclosed within vacuoles. N, nucleus. Scale bars are 0.5 µm for (A), (B), (E) and (F), 2 µm for (C) and (D).
Figure 4. Vacuolar bacteria are transcriptionally inactive and non-replicative after CHQ treatment.
(A, B) HeLa epithelial cells were seeded on glass coverslips and infected with wild type S. Typhimurium harboring plasmid-borne GFPmut3 under the control of the tetRA promoter. At 5 h p.i., cells were treated with 400 µM CHQ for 1 h. CHQ was then washed out and cells were further incubated with 300 ng/ml ATc for 3 h to allow gfp transcription. Cells were fixed, permeabilized and immunostained for Salmonella LPS (shown in red) and LAMP1 (blue). (A) Confocal image of cells treated with ATc only. Both vacuolar and cytosolic bacteria are GFP-positive. Scale bar is 10 µm. (B) Confocal image of cells treated with CHQ, washed out, then incubated with ATc. Only hyper-replicating, LAMP1-negative bacteria are fluorescent. Scale bars are 10 µm. (C) HeLa cells were infected with mCherry S. Typhimurium. Cells were left untreated or incubated with 400 µM CHQ from 30–90 min p.i., then washed out and incubated in CHQ-free growth medium until 8 h p.i. Cells were fixed at 90 min and 8 h p.i. and the number of bacteria per cell scored by fluorescence microscopy. Each dot represents one infected cell. Results are from a representative experiment. The proportion of infected cells containing ≥100 bacteria from three independent experiments (mean ± SD) is shown for each condition at the top of the graph.
Figure 5. Wortmannin increases the proportion of cytosolic Salmonella early after bacterial internalization.
(A) Digitonin permeabilization assay. HeLa cells were infected with wild type or Δ_sifA_ bacteria. Additionally, cells were pretreated with 100 nM WTM for 45 min prior to infection with wild type bacteria, and inhibitor treatment continued until 90 min p.i., then washed out. The proportion of intracellular bacteria accessible to anti-LPS antibodies delivered to the cytosol was determined at each time point. (B) Cells were infected as in (A) and the proportion of cytosolic bacteria was quantified at 1.5 h p.i. by the chloroquine resistance assay. Data (mean ± SD) are from three independent experiments. Asterisks indicate data significantly different from wild type bacteria, analysis of variance (ANOVA) with Dunnett’s post-hoc analysis, p<0.05.
Figure 6. Access to the cytosol induces SPI-1 gene expression.
(A) Gentamicin protection assay in HeLa cells. HeLa cells were infected with wild type or Δ_sifA_ bacteria. Additionally, cells were pretreated with 100 nM WTM for 45 min prior to infection with wild type bacteria, and inhibitor treatment continued until 90 min p.i., then washed out. Infected cells were solubilized at 1 h and 10 h p.i. and viable bacteria enumerated by plating on LB agar. Fold-replication represents CFU at 10 h p.i. divided by CFU at 1 h p.i. Values (mean ± SD) are from three independent experiments. (B) Cells were infected as described above with mCherry wild type or mCherry Δ_sifA_ mutant bacteria. At 10 h p.i., cells were fixed and the number of bacteria per cell scored by fluorescence microscopy. Each dot represents one infected cell. Data are from a representative experiment. The proportion of infected cells containing ≥100 bacteria from three independent experiments (mean ± SD) is shown for each condition at the top of the graph. (C) Cells were infected as described above with wild type or Δ_sifA_ bacteria harboring a GFP reporter plasmid for SPI-1 activity, pMPMA3ΔPlac-P_prgH_-GFP[LVA]. At 10 h p.i., cells were fixed and bacteria immunostained with anti-LPS antibodies. The frequency of infected cells containing at least one GFP-positive bacterium was scored by fluorescence microscopy. Data are from three independent experiments (mean ± SD). wt, wild type bacteria; Δ_sifA_, Δ_sifA_ mutant bacteria; WTM, wild type bacteria plus WTM treatment. Asterisks indicate data significantly different from wild type bacteria, analysis of variance (ANOVA) with Dunnett’s post-hoc analysis, p<0.05.
Figure 7. Time course of cytosolic replication in epithelial cell lines.
Epithelial cells (HeLa, Caco-2 C2Bbe1, HuTu 80 and HCT 116) were seeded in 24-well plates and infected with wild type S. Typhimurium. One hour prior to each time point, two wells were treated with CHQ. At the indicated time, duplicate untreated (total CFU, black dots) and duplicate CHQ-treated cells (cytosolic CFU, red dots) were solubilized and serial dilutions plated on LB agar for CFU enumeration. Results are representative of at least three independent experiments.
Figure 8. SPI-1 is required for vacuole lysis and proliferation in the cytosol.
Caco-2 C2Bbe1 cells were infected with the following S. Typhimurium strains; SL1344 wild type, Δ_ssaR_ (defective for T3SS2 assembly), pSLT- (virulence-plasmid cured), Δ_flgB_ (defective for flagellar assembly), Δ_sptP_Δ_sopE_Δ_sopE2_Δ_sopB_Δ_avrA_Δ_sopA_Δ_sipA_ (“effectorless” mutant), Δ_prgI_ (defective for T3SS1 assembly). CHQ was added for 1 h prior to each time point. At the indicated times, untreated and CHQ-treated monolayers were solubilized and plated on LB agar for CFU enumeration. Total bacteria are shown by black dots, CHQ-resistant bacteria (cytosolic) by red dots. Results are representative of at least three independent experiments.
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