Modulation of autophagy affects bacterial proliferation in the epithelial cell cytosol

We hypothesized that the modulation of autophagy in epithelial cells would selectively perturb the cytosolic, and not vacuolar, proliferation of S. Typhimurium. Autophagy levels can be manipulated by pharmacological or genetic means (reviewed in [30]). To test our hypothesis, we used nutrient starvation to upregulate autophagy and the class III phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin (WTM), to inhibit autophagy (Fig 1A). To enumerate cytosolic bacteria after autophagy activation/inhibition, we used the digitonin permeabilization assay [12,13] to label the bacteria accessible to cytosol-delivered anti-S. Typhimurium lipopolysaccharide (LPS) antibodies at the early stages of infection (15 min– 3 h p.i., Fig 1B). Wild-type bacteria were constitutively expressing mCherry from a plasmid, pFPV-mCherry. In untreated cells (basal levels of autophagy), 6.8% of bacteria were accessible to the cytosol as early as 15 min p.i. This proportion increased to ~20% by 45 min p.i. and remained at a steady-state thereafter (Fig 1B). Treatment with Earle’s balanced salt solution (EBSS), i.e. starvation-induced autophagy, reduced the fraction of bacteria that were accessible to the cytosol at 45 min, 90 min and 180 min p.i. (Fig 1B), consistent with enhanced autophagic capture of bacteria in, or repair of, damaged vacuoles. In contrast, inhibition of autophagy (WTM) increased the proportion of cytosolic bacteria at all timepoints examined (Fig 1B), in agreement with previous findings [14,31].

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Fig 1. EBSS and wortmannin inversely affect the cytosolic population.

(A) Schematic of the experimental design employed to differentially modulate host cell autophagy. (B) Epithelial cells seeded on glass coverslips were pretreated with EBSS or 100 nM WTM as depicted in (A) and infected with S. Typhimurium wild-type bacteria harboring pFPV-mCherry for plasmid-borne, constitutive expression of mCherry. The proportion of bacteria accessible to anti-Salmonella LPS antibodies delivered to the mammalian cell cytosol was determined by digitonin permeabilization assay and fluorescence microscopy. n≥3 independent experiments. (C) Epithelial cells seeded on coverslips were pretreated as in (A) and infected with mCherry-S. Typhimurium (wild-type glmS::Ptrc-mCherryST::FRT bacteria constitutively expressing a chromosomal copy of mCherry) harboring the fluorescent reporter plasmid, PuhpT-gfpova (pNF101). At the indicated times, cells were fixed and the proportion of infected cells containing cytosolic bacteria (GFP-positive) was scored by fluorescence microscopy. The mean from each experiment is represented as a large dot (n = 3). (D) Cells seeded on coverslips were pretreated as in (A) and infected with wild-type bacteria harboring pFPV-mCherry. The number of bacteria in each infected cell was scored by fluorescence microscopy. Cells with ≥100 bacteria contain cytosolic S. Typhimurium. Each small dot represents one infected cell. n = 2 (1 h) or 3 (8 h) experiments. (E) Epithelial cells were infected with wild-type bacteria and the number of intracellular bacteria at 1 h and 8 h p.i. was determined by gentamicin protection assay. Fold-replication is CFUs at 8 h/1 h. The mean from each experiment is represented as a large dot (n = 3). For all panels, control = untreated cells; EBSS = Earle’s balanced salt solution treatment; WTM = wortmannin treatment. Asterisks indicate data significantly different from control (ANOVA with Dunnett’s post-hoc test, p<0.05).

https://doi.org/10.1371/journal.ppat.1009280.g001

To verify cytosolic localization, we used a transcriptional reporter plasmid, pNF101, that only expresses gfp-ova in the sub-population of S. Typhimurium that are in damaged vacuoles and/or free in the cytosol. Epithelial cells were infected with mCherry-S. Typhimurium (S. Typhimurium wild-type bacteria constitutively expressing mCherry on the chromosome) harboring pNF101. In untreated cells, 35% and 13% of infected cells harbored GFP-positive (cytosolic) bacteria at 2 h and 8 h p.i., respectively (Fig 1C). EBSS treatment lowered this proportion at both timepoints to 23% and 8%, respectively. Conversely, WTM treatment increased the level of infected cells with cytosolic bacteria at both 2 h and 8 h p.i. (79% and 48%).

To quantify the niche-specific effects of EBSS and WTM treatments on bacterial replication, epithelial cells were infected with S. Typhimurium harboring pFPV-mCherry and the number of bacteria per cell was scored. Cytosolic S. Typhimurium that evade autophagic clearance in epithelial cells replicate at a much faster rate than vacuolar bacteria [12,13,32], eventually occupying the entire cytosolic space (≥100 bacteria/cell). By contrast, vacuolar bacteria display low to moderate replication (2–40 bacteria/cell). Comparing the three treatment conditions, the incidence of infected cells with ≥100 bacteria at 8 h p.i. (8.2±1.6% in control, n = 3 experiments) was reduced by EBSS treatment (1.2±2.0%) and promoted by WTM treatment (16±5.4%) (Fig 1D). A similar trend was seen with the chloroquine (CHQ) resistance assay, which quantifies the proportion of cytosolic bacteria in the total population—51±8.7%, 28±14% and 65±10% (n = 4 experiments) of intracellular bacteria were cytosolic at 8 h p.i. in untreated, EBSS-treated and WTM-treated cells, respectively. Consequently, total bacterial proliferation was restricted and promoted by EBSS and WTM treatment, respectively, compared to untreated cells (Fig 1E). Importantly, neither bacterial invasion at 1 h p.i. nor vacuolar replication at 8 h p.i. was overtly affected (Fig 1D). Collectively, these data confirm that modulation of autophagy selectively affects the proliferation of S. Typhimurium in the cytosol of epithelial cells.

Identification of cytosol- and vacuole-induced genes by RNA-seq analysis

To define the transcriptional signature of cytosolic S. Typhimurium, we isolated bacterial RNA from epithelial cells that had been treated with EBSS or WTM, conditions that depleted or enriched the cytosolic population, respectively. Treated cells were infected with wild-type bacteria (Fig 1A), samples were collected and processed at 8 h p.i. and bacterial RNA was extracted using an enrichment protocol described previously ([33]; S1 Fig). Importantly, EBSS and WTM treatments were restricted to the early stages of infection (≤90 min p.i.) (Fig 1A) to limit any effects on bacterial gene expression at the time of sample collection (8 h p.i.). Four cDNA libraries were derived from two biological replicates and analyzed by RNA-seq (see Materials and Methods; [34]). The entire dataset is available in S1 Dataset and includes the expression profiles of the >5,000 coding and non-coding genes identified previously in S. Typhimurium [34,35]. Genes for which the adjusted p-value was 0.05 or less (DeSeq2 analysis) were deemed as preferentially induced in the cytosol or vacuole, resulting in a shortlist of 216 “up cytosol” genes and 443 “up vacuole” S. Typhimurium genes (Fig 2A and S1 Dataset).

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Fig 2. Summary of RNA-seq results.

(A) RNA-seq results displayed as a volcano plot. Statistical significance is represented on the y-axis and magnitude of change on the x-axis. Statistically significant mRNAs and sRNAs are shown in blue dots for “up vacuole” and red dots for “up cytosol” categories. mRNAs/sRNAs that did not pass statistical significance are indicated by grey dots. Select genes of interest are indicated. (B) Pathway enrichment analysis. Top panels indicate regulons generated from transcriptional profiling of key regulatory mutants, while bottom panels display enriched gene sets generated from KEGG, GO, or from RNA-seq profiling of S. Typhimurium under 22 infection-relevant in vitro growth conditions. All displayed enrichments are significant (adjusted p-value <0.05) according to Fisher’s exact test with Benjamini-Hochberg correction for multiple comparisons. The number of genes in each category is shown in log₂ scale. Only a selection of non-redundant pathways is presented; the complete list is available as S2 Dataset.

https://doi.org/10.1371/journal.ppat.1009280.g002

Of the twelve chromosomally-encoded pathogenicity islands (PAIs) in S. Typhimurium, only four were up-regulated in the cytosolic environment (S2 Fig). Most obvious was the abundance of SPI1 genes that were up-regulated, consistent with the reported induction of prgH in the cytosolic population at later times in epithelial cells [12,36]. SPI4, which encodes a giant non-fimbrial adhesin and its cognate type I secretion system and is co-regulated with SPI1 [37,38], was also up-regulated in the cytosol. Lastly, genes encoding for two T3SS1 effectors within SPI5 and SPI11, SopB [39,40] and SopF [24,25,41], respectively, were up-regulated in cytosolic bacteria. The remaining SPI5 and SPI11 genes were either up-regulated in the vacuolar population, or had unchanged expression, highlighting the mosaic nature of PAIs. Most SPI2 genes were up-regulated in the vacuolar population, as were genes encoding two type III effectors translocated by T3SS2 (pipB in SPI5 and sspH2 in SPI12). SPI3 genes were up-regulated in the SCV of epithelial cells, like in macrophages [42,43]. Similarly, most genes within SPI11, a PAI that is important for macrophage survival [44], and SPI12 were up-regulated in the epithelial SCV. The majority of SPI6 genes (which encode a type VI secretion system; [45]), all SPI9 genes (encode a type I secretion system; [46]) and SPI16 genes were not expressed in bacteria residing within either niche in epithelial cells. Overall, the transcriptional signatures of cytosolic and vacuolar bacteria at the PAI level (S2 Fig) validates the basis of our RNA-seq analysis.

To put the observed gene expression changes into the context of the extensive S. enterica literature, we used an innovative approach that involved the curation of 80 custom pathways from previously published regulons that had been defined by RNA-seq, ChIP-seq or microarray-based analysis. We combined these pathways with KEGG and GO gene sets to annotate the subsets of “up cytosol” and “up vacuole” S. Typhimurium genes (Fig 2B and S2 Dataset). The list of “up vacuole” genes included an abundance (e.g. SPI2, pipB, pgtE, PinT, InvS, steB) that are positively regulated by the two-component systems that govern SPI2 induction in the SCV, namely PhoPQ, SsrAB and OmpRZ. Conversely, genes that are negatively regulated by these two component systems were in the “up cytosol” category. The SPI1-encoded transcription factors, HilA, HilD, and InvF, are required for the expression of SPI1 genes [47], and accordingly many of the “up cytosol” genes/sRNAs (e.g. sipBCDA, siiCD, sopF, sopB) grouped with these regulons. Our analysis also revealed several specific gene signatures (Fig 2B). For example, genes associated with iron transport were solely up-regulated in the cytosol. Up-regulation of the tricarboxylic acid (TCA) cycle, lysine degradation and molybdate transport was evident in vacuolar bacteria, suggesting increased reliance of Salmonella on these energy sources in the SCV. Interestingly, genes/sRNAs that are repressed by Ferric uptake regulator (Fur) in iron-replete conditions and activated by Hfq, BarA/SirA and FliZ were up-regulated in both the vacuole and cytosol.

We used the expression profiles of S. Typhimurium grown in 22 distinct infection-relevant in vitro growth conditions [34] to predict the environmental conditions experienced by S. Typhimurium when in the cytosol and vacuole. For the 216 genes up-regulated in the cytosol, three main patterns emerged: (i) up-regulation by bile shock (3% bile for 10 min) and/or low Fe²⁺ shock (200 μM 2,2-dipyridyl (DPI) for 10 min) (e.g. mntH, sitABCD, entCEBA), (ii) up-regulation by NaCl shock (0.3 M NaCl for 10 min) (e.g. proVW, ilvX), (iii) up-regulation during late exponential/early stationary phase of growth (e.g. sopE2, sipBCDA, siiABCDEF) (S1 Dataset). This suggests that cytosolic bacteria are growing fast and exposed to a low iron, high osmolarity physicochemical environment. The rapid growth of cytosolic S. Typhimurium has been previously observed [12,13,32], validating the former prediction. A common theme observed for the 443 genes up-regulated in the vacuole was induction by: (i) in vitro SPI2 growth conditions and/or (ii) internalization into macrophages (S1 Dataset).

Cytosolic iron-limitation is a cue for S. Typhimurium gene induction

To independently assess the cytosolic expression of genes predicted by RNA-seq analysis, we used green fluorescent protein (GFP)-based transcriptional fusions. This fluorophore is an ideal reporter to study differential gene expression in individual bacteria in infected host cells and tissues [12,18,48,49]. Previously gfpmut3 transcriptional fusions have demonstrated the vacuole-specific induction of SPI2 genes [50], for example. Due to the low abundance of many transcripts in the “up cytosol” shortlist (according to TPM; S1 Dataset), we also chose to use gfpmut3, which encodes for a stable GFP variant, to increase the likelihood of detecting fluorescent bacteria in epithelial cells. We initially focused on those genes that are induced when Salmonella are exposed to low Fe²⁺ and/or bile shock in broth [34] (S1 Dataset). Epithelial cells were infected with mCherry-S. Typhimurium carrying individual gfpmut3 reporters and bacterial GFP fluorescence intensity was assessed qualitatively and quantitatively at 8 h p.i. The following genes were up-regulated in the cytosol: entC (entCEBA operon), fepA (fepA-entD operon), fepB, fhuA (fhuACBD operon), fhuE, iroB (iroBCDE operon), iroN, mntH, nrdH (nrdHIEF operon), sitA (sitABCD operon), SL1802, SL3990 (SL3990-SL3989 operon), sufA (sufABCDSE operon), yjjZ (SL1344_4483), STnc3080 and STnc3250 (Figs 3 and S3). Up-regulation of STnc4000, ilvC and SL1344_2715 was not observed by fluorescence microscopy (S4 Fig). Quantification using ImageJ software of the mean fluorescence intensity (MFI) of the GFP signal associated with individual bacteria confirmed the qualitative analysis for iroB (11-fold increase in average MFI for cytosolic bacteria/vacuolar bacteria, n = 2 experiments), nrdH (11-fold), sitA (13-fold), sufA (7-fold), yjjZ (23-fold) and STnc3080 (11-fold) (Fig 3, lower panel). Bacteria harboring these reporters and grown to late log-phase in LB-Miller broth, i.e. t₀ conditions, showed only background levels of fluorescence (Fig 3, lower panel).

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Fig 3. Iron-associated genes are induced in the epithelial cytosol.

(A) Epithelial cells seeded on coverslips were infected wild-type mCherry-S. Typhimurium harboring gfpmut3 transcriptional reporters. At 8 h p.i., cells were fixed and stained with Hoechst 33342 to detect DNA. Representative confocal microscopy images show induction of iroB, nrdH, sitA, sufA, yjjZ and STnc3080 promoters in cytosolic bacteria. Green = transcriptional reporter, red = S. Typhimurium, blue = DNA. Scale bars are 10 μm. (B) Quantification of the mean fluorescence intensity (MFI) of GFP signal by fluorescence microscopy and ImageJ. Bacteria were designated as being cytosolic (Cyto) or vacuolar (Vac) if residing within cells with ≥100 bacteria or 2–40 bacteria, respectively. t₀ represents the infection inoculum i.e. bacteria grown to late log phase in LB-Miller broth. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of two experiments. Acquisition parameters (exposure time and gain) were set up using PsitA-gfpmut3 (the highest GFP intensity) and these same parameters were applied throughout. Dashed lines indicate the range of background fluorescence in the GFP channel measured for mCherry-S. Typhimurium (no reporter plasmid).

https://doi.org/10.1371/journal.ppat.1009280.g003

All of the confirmed S. Typhimurium genes are up-regulated by “low Fe2+ shock” in broth (addition of 200 μM DPI for 10 min [34]). DPI chelation is not specific to iron, however [51,52], raising the possibility that other metals also regulate the expression of these genes. We used iroN, sitA, yjjZ, sufA, fepA and STnc3250 promoters to determine metal cation specificity in vitro. The effect of increasing concentrations (0.1–100 μM) of Co²⁺, Fe³⁺, Mn²⁺, Ni²⁺ or Zn²⁺ on GFP fluorescence for bacteria grown in defined minimal media (M9) was measured (S5 Fig). For all reporters, maximal expression was observed in the absence of any added metal. Upon addition of Fe³⁺ and Co²⁺, GFP expression of S. Typhimurium carrying PiroN-gfpmut3, PyjjZ-gfpmut3 and PfepA-gfpmut3 reporters decreased in a dose-dependent manner. Notably, the promoter responses were 10- to 100-fold more sensitive to Fe³⁺ than Co²⁺. PsitA-gfpmut3 expression was modulated by Fe³⁺, Mn²⁺ and Co²⁺, although Fe³⁺ and Mn²⁺ were clearly the most potent repressors by an order of magnitude, in agreement with a previous study using a sitA::lacZ reporter [53]. Lastly, expression of PSTnc3250-gfpmut3 and PsufA-gfpmut3 were only reduced by increased concentrations of Fe³⁺. PiroN-gfpmut3 was the most sensitive iron reporter, with a >50% reduction in fluorescence at 0.1 μM Fe³⁺. Collectively, the in vitro repression of these genes/sRNAs by 0.1–1 μM Fe³⁺ hinted that the induction cue in the cytosol was iron limitation (<0.1 μM).

To test the response of reporters to iron concentrations during infection, we used ferric ammonium citrate (FAC) to increase free iron levels in mammalian cells [54,55]. Epithelial cells were incubated overnight in growth media containing increasing concentrations (10–300 μM) of FAC. Untreated and FAC-treated cells were infected with mCherry-S. Typhimurium carrying transcriptional reporters, fixed at 8 h p.i. and the MFI of GFP signal for cytosolic and vacuolar bacteria quantified by fluorescence microscopy and ImageJ (Fig 4). The fluorescence intensity of cytosolic bacteria harboring PiroN-gfpmut3, PsitA-gfpmut3 or PyjjZ-gfpmut3 reporters was reduced in a dose-dependent manner over the FAC concentration range. Specifically, the average MFI was reduced by 57-, 36- and 15-fold, respectively, upon the addition of 300 μM FAC to growth media (Fig 4A), indicating repression of iroN, sitA and yjjZ expression in the cytosol under iron-replete conditions. The low MFI for vacuolar bacteria in untreated cells limited our analysis of the iron-dependent repression of iroN, sitA and yjjZ in the SCV.

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Fig 4. Manipulation of cellular iron levels primarily affects cytosolic bacteria.

(A) Epithelial cells seeded on coverslips were left untreated (control) or treated overnight in growth media supplemented with 10 μM, 30 μM, 100 μM or 300 μM ferric ammonium citrate (FAC). Cells were infected with wild-type mCherry-S. Typhimurium harboring PiroN-gfpmut3, PsitA-gfpmut3 or PyjjZ-gfpmut3 reporters, fixed at 8 h p.i. and the MFI of bacterial GFP signal was quantified by fluorescence microscopy and Image J. Bacteria were designated as being cytosolic (Cyto) or vacuolar (Vac) if residing within cells with ≥100 bacteria or 2–40 bacteria, respectively. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of two experiments. Acquisition parameters (exposure time and gain) were set-up using PiroN-gfpmut3 (the highest GFP intensity) and these same parameters were applied throughout. Dashed lines indicate the range of background fluorescence in the GFP channel measured for mCherry-S. Typhimurium (no reporter plasmid). Asterisks indicate data significantly different from control (Kruskal-Wallis test). (B) Epithelial cells seeded on coverslips were left untreated (control) or treated overnight with growth media containing 200 μM 2,2’-dipyridyl (DPI), a metal-chelating compound, 200 μM DPI and 200 μM ferric ammonium citrate (DPI+FAC), or 200 μM FAC alone (FAC). Infection strains and quantification of GFP signal were as described for (A). Asterisks indicate data significantly different from control (Kruskal-Wallis test). (C) Cells were treated as in (B) and infected with wild-type bacteria. The number of intracellular bacteria was quantified by gentamicin protection assay at 1 h and 8 h p.i. Fold-replication is CFUs at 8 h/1 h. The mean from each experiment is represented as a large dot (n = 3). Asterisks indicate data significantly different from control (ANOVA with Dunnett’s post-hoc test). (D) Cells were treated as in (B) and infected with wild-type bacteria. The proportion of cytosolic bacteria was quantified by CHQ resistance assay at 7 h p.i. The mean from each experiment is represented as a large dot (n≥3). Asterisk indicates data significantly different from control (ANOVA with Dunnett’s post-hoc test). (E) Cells were treated as in (B) and infected with mCherry-S. Typhimurium harboring a plasmid-borne reporter of cytosolic access, pNF101, and fixed at 8 h p.i. Left panel: the proportion of infected cells containing only cytosolic (all bacteria are GFP+, mCherry+), only vacuolar (all bacteria are GFP-, mCherry+) or a mixed population (Cyto&Vac) of bacteria was blindly scored by fluorescence microscopy. n≥5 independent experiments. Right panel: the number of cytosolic or vacuolar bacteria in each cell was blindly scored. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of two experiments.

https://doi.org/10.1371/journal.ppat.1009280.g004

We used the lipophilic, cell-permeable chelator, DPI, as an independent way to assess the effects of metals on S. Typhimurium gene expression in the mammalian cytosol. Because DPI has a high affinity for various metal cations, we assessed the specificity for iron by means of an “add-back” experimental design [51,56,57] whereby an equimolar amount of Fe³⁺ was added to the DPI-treated media. Epithelial cells were treated overnight with 200 μM DPI, 200 μM DPI plus 200 μM FAC, or 200 μM FAC alone and infected with mCherry-S. Typhimurium harboring PiroN-gfpmut3, PsitA-gfpmut3 or PyjjZ-gfpmut3 reporters. Cells were fixed at 8 h p.i. and the intensity of GFP signal for cytosolic and vacuolar bacteria quantified via fluorescence microscopy. Addition of DPI increased the expression of iroN, sitA and yjjZ for both cytosolic and vacuolar bacteria (Fig 4B), indicating that DPI accesses both cellular compartments. However, the effects of divalent cation chelation were more pronounced for the vacuolar population (13- to 43-fold increase in average MFI) than the cytosolic population (1.7- to 2.2-fold increase in average MFI). Add-back of iron restored the MFI to that of untreated cells for cytosolic bacteria harboring PiroN-gfpmut3 and PsitA-gfpmut3 reporters (Fig 4B), demonstrating that iron limitation is the primary stimulus for the up-regulation of these genes in the cytosol. Add-back of iron had a lesser effect on the average MFI of bacteria harboring the PyjjZ-gfpmut3 reporter, regardless of their intracellular niche (Fig 4B), possibly due to a lower sensitivity of yjjZ to changes in free iron levels (Fig 4A) or a responsiveness to deprivation of metals other than iron in the intracellular environment (S5 Fig). Add-back of iron only partially restored the expression of sitA and iroN in vacuolar bacteria (Fig 4B) suggesting that sitA and iroN respond to multiple divalent cations in the SCV or FAC treatment is less effective at increasing free iron levels in endocytic-derived compartments compared to the cytosol. Taken together, our data show that iron limitation in the mammalian cytosol is a major cue sensed by S. Typhimurium.

Using the same treatment conditions, we determined the role of iron limitation on bacterial replication in epithelial cells. As assessed by gentamicin protection assay (Fig 4C), total bacterial replication was limited by treatment with DPI (4.0-fold replication over 8 h) and DPI plus FAC (5.6-fold), but not FAC alone (8.8-fold), compared to untreated cells (10.0-fold). The CHQ resistance assay showed a similar profile (Fig 4D). Compared to untreated cells (50±3.4% cytosolic bacteria), treatment with DPI reduced the levels of cytosolic bacteria to 28±6.7% at 7 h p.i. The proportion of cytosolic bacteria was increased to 42±11% upon add-back of iron (DPI+FAC), whereas it was indistinguishable from untreated cells for FAC alone (52±5.5%). Notably, DPI treatment (200 μM) also retards the growth of S. Typhimurium in complex media (S6 Fig).

It has been shown that epithelial cells contain either cytosolic bacteria, vacuolar bacteria or a mix of both populations at later times p.i. [32]. We used single-cell analysis to monitor the effect of metals on the distribution of these intracellular populations. Using mCherry-S. Typhimurium harboring pFN101 as a fluorescent reporter for cytosolic access, we analyzed the intracellular populations at 8 h p.i., in addition to the number of cytosolic (GFP+, mCherry+) and vacuolar (GFP-, mCherry+) bacteria within each infected cell. There was no significant difference in the frequency of epithelial cells that only contained cytosolic bacteria (0–1.2% of infected cells), only vacuolar bacteria (84.0–86.4% of infected cells) or a mixed population (12.4–16.0% of infected cells) between any of the treatments (Fig 4E, left panel), indicating that metal chelation does not alter the intracellular distribution of bacteria. However, the ability of Salmonella to replicate in the cytosol was noticeably affected (Fig 4E, right panel), in agreement with the CHQ resistance assay results. In untreated (control) cells, 6.4±0.89% of infected cells contained ≥100 cytosolic bacteria/cell at 8 h p.i. (n = 4 experiments). DPI treatment reduced proliferation of S. Typhimurium in the cytosol such that only 0.40±0.89% of infected cells contained ≥100 cytosolic bacteria/cell. Add-back of iron (DPI+FAC) partially restored the hyper-proliferative capacity of S. Typhimurium in the cytosol (3.3±1.9%) whereas FAC treatment alone (7.8±1.7%) was no different than control. DPI limited bacterial replication in the SCV (Fig 4E, right panel) as evidenced by a reduction in the average number of vacuolar bacteria/cell at 8 h p.i. (13.8 for untreated and 10.8 for DPI-treated, n = 4 experiments), albeit without statistical significance (p = 0.15, Kruskal-Wallis test). Add-back of iron restored vacuolar replication (11.7 bacteria/cell) whereas FAC alone (11.6 bacteria/cell) was not significantly different to untreated cells. In summary, iron restriction affects S. Typhimurium proliferation in both the cytosol and vacuole in epithelial cells but has a much more profound effect in the cytosol.

Zinc and magnesium are more limiting in the vacuole than the cytosol

We investigated whether metals other than iron affected bacterial gene expression in epithelial cells. S. Typhimurium encodes multiple transporters for divalent cations [58]. The up-regulation of sitABCD and mntH in cytosolic S. Typhimurium (Figs 3 and S3), which are induced by iron and manganese limitation (S5 Fig) [53,56] and encode high affinity Mn²⁺ transporters [59,60], suggests that Mn²⁺ availability is lower in the cytosol than SCV. Zn²⁺ import is dependent on a high affinity transport system in S. Typhimurium, ZnuABC [61], and its accessory protein, ZinT (formerly known as YodA) [62,63], and a low affinity ZIP family metal permease, ZupT [64,65]. zupT is constitutively expressed [64], in agreement with our RNA-seq analysis (S1 Dataset). By contrast, znuA and zinT are induced when zinc is limiting [66] and both genes are in the “up vacuole” shortlist (Fig 2A and S1 Dataset), consistent with increased ZnuA production upon S. Typhimurium internalization into mammalian cells [61]. We found that a PzinT-gfpmut3 reporter was exquisitely sensitive to zinc concentrations, with a 54% and 99% reduction in GFP fluorescence detected upon the addition of 0.1 μM and 1 μM Zn²⁺ to minimal media, respectively (S5 Fig). The fluorescence intensity of both vacuolar and cytosolic mCherry-S. Typhimurium harboring PzinT-gfpmut3 increased at 8 h p.i. compared to t₀, indicating the intracellular induction of zinT. However, GFP signal was considerably greater for vacuolar bacteria (Fig 5), suggesting that zinc is more limiting in the SCV (<0.1 μM). Quantification of the average MFI of GFP fluorescence confirmed the qualitative findings (5.8-fold increase for vacuolar bacteria/cytosolic bacteria; Fig 5, lower panel). A recent study utilizing a znuA transcriptional reporter to measure intracellular Zn²⁺ levels reached a similar conclusion [67].

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Fig 5. Zn²⁺ and Mg²⁺ are limiting in the vacuole, not the cytosol.

Upper panels: Epithelial cells seeded on coverslips were infected with mCherry-S. Typhimurium harboring PzinT-gfpmut3 or PmgtC-gfpmut3 transcriptional reporters. At 8 h p.i., cells were fixed and stained with Hoechst 33342 to detect DNA. Representative confocal microscopy images show induction of zinT (upper panel) and mgtC (lower panel) promoters in vacuolar bacteria. Green = transcriptional reporter, red = S. Typhimurium, blue = DNA. Scale bars are 10 μm. Lower panels: Quantification of the MFI of GFP signal by fluorescence microscopy and ImageJ. Bacteria were designated as being cytosolic (Cyto) or vacuolar (Vac) if residing within cells with ≥100 bacteria or 2–40 bacteria, respectively. t₀ represents the infection inoculum i.e. bacteria grown to late log phase in LB-Miller broth. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of two experiments. Acquisition parameters (exposure time and gain) were set up using PzinT-gfpmut3 (the highest GFP intensity) and these same parameters were applied throughout. Dashed lines indicate the range of background fluorescence in the GFP channel measured for mCherry-S. Typhimurium (no reporter plasmid).

https://doi.org/10.1371/journal.ppat.1009280.g005

S. Typhimurium also has multiple transporters for Mg²⁺ uptake. CorA is a constitutively expressed Mg²⁺ transporter, and MgtA and MtgB are inducible Mg²⁺ transporters [68]. Consistent with its constitutive expression, expression of corA was similar in both vacuolar and cytosolic bacteria (S1 Dataset). Low Mg²⁺ induces mgtA and mgtB transcription [68,69]. Expression of mgtA was not induced in the vacuole, however (S1 Dataset). Acidic pH abolishes the mgtA (but not mgtB) transcriptional response to Mg²⁺ concentration [69], which could explain why mgtA is not induced in the SCV of epithelial cells. In contrast, all three genes within the mgtCBR operon were up-regulated in vacuolar bacteria according to the RNA-seq dataset (S1 Dataset). Using mCherry-S. Typhimurium PmgtC-gfpmut3, we confirmed mgtCBR is responsive to Mg²⁺ levels (S5 Fig) and the operon is up-regulated after bacterial internalization (Fig 5). Furthermore, the average MFI of GFP signal was increased by 6.2-fold for vacuolar bacteria/cytosolic bacteria (Fig 5, lower panel) indicating that Mg²⁺ is more limiting in the SCV than cytosol, in agreement with a recent study [67]. Altogether, our studies of metal-regulated gene expression reveal that S. Typhimurium encounters distinct metal ion availabilities in the SCV and cytosol; specifically, iron and manganese are more limiting in the cytosol whereas magnesium and zinc are limiting in the vacuole.

Identification of growth-phase related, cytosol-induced genes

Our transcriptomic analysis highlighted another set of cytosol-induced genes that are highly expressed during late exponential/early stationary phase of in vitro growth in broth, and in some cases responsive to oxygen shock (Fig 2 and S1 Dataset). Many of the genes encode for T3SS1 structural proteins (orgA, spaQ, sipB, sipC, sipD) and effectors known to be translocated by T3SS1 (sipA, sopB, sopD, sopE, sopE2, sopF [SL1344_1177]). We have previously shown that prgH (prgHIJK-orgABC operon), encoding a structural T3SS1 component, is up-regulated in cytosolic bacteria [12], in agreement with our RNA-seq results (S1 Dataset). Using GFP transcriptional reporters, we confirmed the increased expression of sicA (sicA-sipBCDA operon), sopE2 and sopF (Fig 6) in cytosolic bacteria. Of note, the average MFI of cytosolic bacteria was comparable to t₀ cultures (Fig 6, lower panel), which are highly T3SS1-induced [70]. PipB, a T3SS2 effector encoded on SPI5 [71], was used as a control for the vacuole-specific induction of a type III effector. In accordance, the average MFI of GFP fluorescence was 9.8-fold higher for vacuolar bacteria harboring a PpipB-gfpmut3 reporter compared to those in the cytosol (Fig 6). We confirmed that siiA (siiABCDEF operon) and lpxR were more highly expressed in bacteria residing within the cytosol compared to the SCV (Fig 6). The siiABCDEF operon in SPI4 (S2 Fig) encodes for a type I secretion system for the secretion of SiiE, a giant non-fimbrial adhesin [72,73]. Immunostaining with anti-SiiE antibodies detected the adhesin on the surface of cytosolic bacteria (S7 Fig, upper panel), and diffusely spread throughout the cytosol (S7 Fig, lower panel). Vacuolar bacteria were negative for SiiE staining (S7 Fig). We have previously shown by electron microscopy that cytosolic bacteria have extensive filamentous material on their surface [12]. Taken together, our findings suggest the filaments could be SiiE. Like the sii operon, lpxR (SL1344_1263) is also co-regulated with SPI1 [74] and encodes an outer membrane 3’-O-deacylase of lipid A [75,76]. Transcriptional reporters did not verify cytosolic up-regulation of ygbA (SL1344_2840), which encodes a conserved hypothetical protein required for S. Typhimurium growth during nitrosative stress [77], or asnA, which is involved in asparagine biosynthesis [78] (S4 Fig).

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Fig 6. SPI1-associated genes are up-regulated in the epithelial cytosol.

(A) Epithelial cells seeded on coverslips were infected with mCherry-S. Typhimurium harboring gfpmut3 transcriptional reporters. At 8 h p.i., cells were fixed and stained with Hoechst 33342 to detect DNA. Representative confocal microscopy images show induction of sicA, sopE2, sopF, siiA and lpxR promoters in cytosolic bacteria. PipB is a type III effector translocated by T3SS2 and the PpipB-gfpmut3 reporter served as a control for vacuole-specific gene induction. Green = transcriptional reporter, red = S. Typhimurium, blue = DNA. Scale bars are 10 μm. (B) Quantification of the MFI of GFP signal by fluorescence microscopy and ImageJ. Bacteria were designated as being cytosolic (Cyto) or vacuolar (Vac) if residing within cells with ≥100 bacteria or 2–40 bacteria, respectively. t₀ represents the infection inoculum i.e. bacteria grown to late log phase in LB-Miller broth. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of 2–3 experiments. Acquisition parameters (exposure time and gain) were set up using PsicA-gfpmut3 (the highest GFP intensity) and these same parameters were applied throughout. Dashed lines indicate the range of background fluorescence in the GFP channel measured for mCherry-S. Typhimurium (no reporter plasmid).

https://doi.org/10.1371/journal.ppat.1009280.g006

We conclude that there is a generalized up-regulation of “SPI1 associated” genes in the mammalian cytosol. However, we do not observe uniform up-regulation of these genes within the cytosolic population (Fig 6), in agreement with previous reports for prgH [12,14,36]. Based on average MFIs, all genes were more highly expressed in cytosolic bacteria than vacuolar bacteria (Fig 6, lower panel)–sicA (2.4-fold), sopE2 (6.1-fold), sopF (3.8-fold), siiA (8.5-fold) and lpxR (2.3-fold)–yet with a broader range of MFI/bacterium than the iron-regulated genes (Fig 3, lower panel), and with many cytosolic bacteria having background levels of GFP fluorescence. Such heterogeneity in T3SS1 gene expression has been reported previously in broth cultures [79,80], a result we confirmed here (t₀; Fig 6, lower panel).

Extraneous genes that are induced in the epithelial cytosol

Several osmotically-sensitive genes that are up-regulated when S. Typhimurium is exposed to NaCl shock [34] were identified as candidate cytosol-induced genes, namely cysP (cysPUWA operon), soxS and proV (proVWX operon) (S1 Dataset). We qualitatively and quantitatively confirmed the cytosol-specific expression of cysP and soxS using transcriptional reporters (S8 Fig). However, the proV-gfpmut3 transcriptional fusion was not up-regulated in the cytosol (S4 Fig). The cysPUWA operon encodes a sulfate/thiosulfate permease and periplasmic binding protein [81]. SoxS is a member of the AraC/XylS family of transcriptional regulators that is required for bacterial resistance to oxidative stress [82,83].

Six additional S. Typhimurium genes were identified as being up-regulated in the cytosol according to the transcriptomics data, namely uhpT, sfbA (sfbABC operon), fruB (fruBKA operon), grxA, mtr and trpE (trpEDCBA operon) (S1 Dataset). All six genes are constitutively expressed in infection-relevant broth conditions [34] and sfbA, mtr, trpE and fruB are also up-regulated upon S. Typhimurium infection of macrophages ([43]; S1 Dataset). Infection of epithelial cells with mCherry-S. Typhimurium harboring PuhpT-gfpmut3, PsfbA-gfpmut3, PgrxA-gfpmt3 and PfruB-gfpmut3 confirmed the RNA-seq-based prediction of their induction in the cytosol (S8 Fig). However, mtr and trpE were not induced in the cytosol (S4 Fig). UhpT is a hexose phosphate transporter whose induction has been reported previously for cytosolic S. Typhimurium [36]. fruBKA, known as the fructose operon, encodes for three enzymes involved in fructose uptake [84]. GrxA, or glutaredoxin 1 (Grx1), is a redox enzyme that detoxifies oxidizing agents such as reactive oxygen species (ROS), thereby defending against oxidative stress [85]. The sfbABC operon is predicted to encode a periplasmic iron-binding lipoprotein SfbA, a nucleotide-binding ATPase SfbB and a cytoplasmic permease SfbC [86].

Identification of S. Typhimurium genes required for optimal cytosolic proliferation

Having identified S. Typhimurium genes/sRNAs that are up-regulated within the epithelial cytosol, we next tested whether any of these genes/sRNAs are required for colonization of this niche. We did not focus on the SPI1-associated effectors as their individual contributions to the cytosolic stage of the intracellular cycle have been reported previously [24,32,87,88]. Twenty-nine single or multiple gene deletion mutants were constructed and their ability to access and replicate within the cytosol was first assessed in a population-based assay by CHQ resistance (Fig 7A). Four single gene deletion mutants had a significantly reduced proportion of cytosolic bacteria at 7 h p.i. compared to wild-type bacteria (50.2±7.2%), namely ΔentC (41.9±7.0%), ΔfepB (40.6±5.9%), ΔsoxS (42.4±6.4%) and ΔznuA (26.3±6.7%). A ΔsoxSΔgrxA mutant (40.0±4.2%) was also defective, but not more than the ΔsoxS deletion mutant. Deletion of both Mn²⁺ transporters (ΔmntHΔsitA) reduced the proportion of cytosolic bacteria (40.1±6.2%), whereas single ΔmntH and ΔsitA deletion mutants were without effect. Lastly, a triple ΔmntHΔsitAΔentC mutant was more compromised (29.8±4.3%) for the percentage of cytosolic bacteria at 7 h p.i. than the ΔmntHΔsitA mutant or ΔentC mutant. None of the identified mutants had a growth defect in complex media (S6 Fig). The remaining deletion mutants had similar proportions of cytosolic bacteria as wild-type S. Typhimurium.

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Fig 7. A subset of “up cytosol” genes is required for optimal proliferation in the epithelial cell cytosol.

(A) Epithelial cells were infected with wild-type (WT) bacteria or the indicated gene deletion mutants and the proportion of cytosolic bacteria at 7 h p.i. was quantified using the CHQ resistance assay. n≥4 independent experiments for each strain. Asterisks indicate data significantly different from WT (ANOVA with Dunnett’s post-hoc test). (B) Genetic complementation. Epithelial cells were infected with WT bacteria or deletion mutants harboring an empty vector (-), pWSK29 or pWKS30, or the respective vector encoding the indicated gene(s), except for the ΔfepB mutant, which was complemented with a chromosomal copy of fepB at the glmS site. The proportion of cytosolic bacteria at 7 h p.i. was quantified using the CHQ resistance assay. Large dots indicate the mean of each experiment (n≥3). Asterisks indicate data significantly different from WT (ANOVA with Dunnett’s post-hoc test). (C) Epithelial cells were infected with WT bacteria or the indicated gene deletion mutants and the proportion of cytosolic bacteria at 90 min p.i. was quantified using the CHQ resistance assay. Large dots indicate the mean of each experiment (n≥3). (D) Epithelial cells seeded on coverslips were infected with WT bacteria or gene deletion mutants harboring pCHAR-Duo(ASV), a plasmid-borne dual reporter–constitutive mCherry expression is driven by the synthetic ProB promoter and gfpmut3.1(ASV) (encoding for destabilized GFP) is under the control of the glucose-6-phosphate responsive uhpT promoter from S. Typhimurium. The number of cytosolic (GFP+, mCherry+) and vacuolar (GFP-, mCherry+) bacteria in each infected cell was blindly scored by fluorescence microscopy. Small dots represent individual bacteria; large dots indicate the mean of each experiment; horizontal bars indicate the average of 3 experiments. Asterisk indicates data significantly different from WT (Kruskal-Wallis test). (E) Data from (D) was reanalyzed to determine the percentage of infected cells containing >50 cytosolic bacteria. Large dots indicate the mean of each experiment. Asterisks indicate data significantly different from WT (ANOVA with Dunnett’s post-hoc test).

https://doi.org/10.1371/journal.ppat.1009280.g007

Plasmid-borne complementation of ΔentC (with entCEBA), ΔgrxAΔsoxS (with soxS), ΔmntHΔsitA (with sitABCD) and ΔznuA (with znuA) mutants restored their respective phenotypes to wild-type levels in the CHQ resistance assay (Fig 7B). Chromosomal complementation by Tn7-based integration at the glmS site with fepB also restored the ΔfepB defect to wild-type levels (Fig 7B). Importantly, ΔentC, ΔfepB, ΔgrxAΔsoxS, ΔmntHΔsitA, ΔmntHΔsitAΔentC, ΔsoxS and ΔznuA mutants were comparable to wild-type bacteria for their proportion of cytosolic bacteria at 90 min p.i. (Fig 7C), indicating that the observed defects at 7 h p.i. are not simply due to delayed vacuole lysis.

We extended this population-based assay with single-cell analysis using S. Typhimurium harboring the dual fluorescence reporter plasmid, pCHAR-Duo(ASV), in which constitutive expression of mCherry is driven by the synthetic promoter ProB and destabilized GFP (gfpmut3.1(ASV)) is under the control of the S. Typhimurium uhpT promoter. All bacteria harboring this plasmid are mCherry-positive and only those bacteria in the cytosol will be GFP-positive. Epithelial cells were infected with wild-type, ΔentC, ΔfepB, ΔmntHΔsitA, ΔmntHΔsitAΔentC, ΔsoxS and ΔznuA bacteria harboring pCHAR-Duo(ASV) and the number of cytosolic (GFP+, mCherry+) and vacuolar (GFP-, mCherry+) bacteria in each infected cell at 8 h p.i. was scored by fluorescence microscopy. There was no statistical difference in the proportion of infected cells that contained cytosolic bacteria between wild-type bacteria and the tested deletion mutants–wild-type (14.7±2.5%, n = 3 experiments), ΔentC (10.7±2.5%), ΔfepB (11.3±4.6%), ΔmntHΔsitA (12.3±3.1%), ΔmntHΔsitAΔentC (9.3±4.5%), ΔsoxS (9.0±2.0%) and ΔznuA (16.7±2.1%) (Fig 7D). However, ΔentC, ΔfepB, ΔmntHΔsitA, ΔmntHΔsitAΔentC and ΔsoxS mutants were all defective for proliferation in the cytosol compared to wild-type bacteria as demonstrated by a decreased number of infected cells containing >50 cytosolic bacteria at 8 h p.i. (Fig 7E). The decreased proportion of cytosolic bacteria for the ΔznuA mutant (Fig 7A) was explained by an increased number of vacuolar bacteria/cell at 8 h p.i. (Fig 7E) rather than a defect in cytosolic proliferation per se (Fig 7D).

Overall, our data show that of the 27 bacterial genes we identified as being up-regulated in the epithelial cytosol (Figs 3, 6, S3, and S8), five are specifically required for the optimal proliferation of S. Typhimurium in this intracellular niche—entC, fepB, mntH, sitA and soxS.

Bacterial strains and plasmids

S. Typhimurium SL1344 was the wild-type strain used in this study [139]. Gene deletion mutants were constructed using sacB allelic exchange, λ red recombinase technology or P22 transduction. SL1344 ΔsitA (deleted for amino acids 4–298), ΔmntH (deleted for amino acids 1–411), ΔyjjZ (ΔSL1344_4483, deleted for amino acids 4–76) and ΔSL1344_1802 (deleted for amino acids 4–53) deletion mutants were made via sacB negative selection. Two fragments of ~1 kb upstream and downstream of the gene of interest were amplified from S. Typhimurium SL1344 genomic DNA using Phusion High-Fidelity DNA polymerase (Thermo Scientific) (primer sequences are listed in S1 Table). The two fragments were combined for a second round of amplification by overlap extension PCR. The amplicon was then digested with appropriate restriction enzymes, ligated into the suicide vector pRE112 (Cm^R) [140], and electroporated into E. coli SY327λpir. After sequence confirmation, the pRE112 plasmids were transferred to E. coli SM10λpir (Kan^R) for conjugation into SL1344 wild-type or ΔmntH bacteria (for the ΔsitAΔmntH double mutant). For the second recombination event, sacB-based counterselection on LB agar containing 5% (w/v) sucrose was used, and streptomycin-resistant, chloramphenicol-sensitive colonies were screened by PCR with primers outside of the recombination region to confirm the deletion of each gene. S. Typhimurium SL1344 ΔsufABCDSE::kan, ΔnrdHIEF::kan, ΔfhuB::kan and ΔfepB::kan strains were generated using λ red recombineering technology [141]. λ red cassettes were amplified using pKD4 as a template with the oligonucleotide pairs listed in S1 Table and electroporated into S. Typhimurium SL1344 wild-type containing the temperature-sensitive helper plasmid, pKD46. Transformants were selected on LB agar plates containing streptomycin (100 μg/ml) and kanamycin (50 μg/ml). The correct integration of the kanamycin resistance cassette was confirmed by colony PCR with a target-flanked primer and a kanamycin resistance gene-specific primer. Each mutation was transferred into a clean SL1344 wild-type background using P22 phage transduction. The following insertional mutants were constructed by P22 transduction into a SL1344 wild-type background from phage lysates prepared from the following S. Typhimurium mutants: ΔcirA::kan, ΔcysP::kan, ΔentC::kan, ΔfepA::kan, ΔfhuE::kan, ΔfoxA::kan, ΔgrxA::Cm, ΔiroBC::kan, ΔiroN::kan, ΔlpxR::kan, ΔmgtB::kan, ΔsfbA::kan, ΔsiiABCD::kan, ΔSL3990::kan, ΔsoxS::kan, ΔSTnc3080::kan, ΔuhpT::kan, ΔznuA::kan ([96,142–144]; or provided by Manuela Raffatellu or Carsten Kroeger). Selection was on LB agar plates containing kanamycin (50 μg/ml) or chloramphenicol (30 μg/ml).

For genetic complementation, the deleted gene was either restored on a low copy number plasmid or in the chromosome. Specifically, ΔentC bacteria were transformed with pWSK29-entCEBA (provided by Steve Libby and Joyce Karlinsey), ΔgrxAΔsoxS bacteria with plasmid-borne soxS under the control of its native promoter (pWKS30-soxS), ΔmntHΔsitA bacteria with pWKS30-sitABCD [145] and ΔznuA bacteria with pWKS30-znuA, which contains a DNA fragment encompassing the znuA promoter region and coding sequence. The ΔfepB mutant was complemented with a chromosomal copy of fepB by transposition at the attTn7 site [146]. Oligonucleotides used to construct pWKS30-soxS, pWKS30-znuA and pGP-Tn7-Cm-fepB are listed in S2 Table.

SL1344 wild-type bacteria constitutively expressing mCherry from the chromosome (glmS::Ptrc-mCherryST::FRT, denoted as mCherry-S. Typhimurium) or a plasmid (pFPV-mCherry) have been described previously [7,147]. mCherry expression is driven by the constitutive trc promoter or S. Typhimurium rpsM promoter, respectively. The PuhpT-gfpova plasmid, pNF101 [24], was used as a biosensor for bacterial exposure to the mammalian cytosol. Expression of the unstable GFP variant, gfp_ova (protein half-life <60 min, [148]), is under the control of the glucose-6-phosphate responsive uhpT promoter from S. flexneri. Alternatively, bacterial access to the cytosol was assessed using the bidirectional reporter plasmid, pCHAR-Duo(ASV), whereby constitutive mCherry expression is driven by the synthetic ProB promoter and gfpmut3.1(ASV) expression is under the control of the S. Typhimurium uhpT promoter. To create pCHAR-Duo(ASV), the coding sequence for the stable GFP variant, gfpmut3.1 (protein half-life >24 h), was excised from pCHAR-Duo [32] by XmaI/ApaI digestion and replaced with the coding sequence for a destabilized GFP variant, gfpmut3.1(ASV) (protein half-life ~110 min), which had been released from pGFP(ASV) (Clontech) by XmaI/ApaI digestion.

To construct fluorescent transcriptional reporters, we extracted the precise transcriptional start sites from the online SalComMac database (http://tinyurl.com/SalComMac; [34,43]). Approximately 500 bp of sequence upstream of the ATG start codon of each gene, or transcript start site of each sRNA, was amplified from SL1344 genomic DNA with the oligonucleotide pairs listed in S3 Table. Amplicons were digested with XbaI/SmaI and ligated into the corresponding restriction sites of pGFPmut3.1 (Clontech). XbaI/ApaI digestion released the gene/sRNA promoter-gfpmut3 fragment, which was then ligated into the corresponding sites of the low copy number plasmid, pMPMA3ΔPlac [70]. The pipB and sopB promoters were excised from pMPMA3ΔPlac-PpipB-GFP(LVA) and pMPMA3ΔPlac-PsopB-GFP(LVA), respectively [70], by digestion with XbaI/KpnI and ligated into the corresponding sites of pGFPmut3.1 and then transferred to pMPMA3ΔPlac as described above. The pMPMA3ΔPlac-PuhpT-gfpmut3 construct has been described previously [149]. GFP reporter plasmids were electroporated into wild-type mCherry-S. Typhimurium.

Reporter assays for metal sensitivity

For metal repression studies, wild-type mCherry-S. Typhimurium bacteria harboring gfpmut3 fusions were grown shaking overnight (220 rpm) at 37°C in LB-Miller broth containing 100 μg/ml streptomycin and 50 μg/ml carbenicillin, then washed twice in M9 salts pH 7.0 (6 g Na₂HPO₄, 3 g KH₂PO₄, 0.5 g NaCl, 1 g NH₄Cl per liter) supplemented with 1 mM MgSO₄, 0.4% (w/v) glucose and 0.01% (w/v) histidine–hereafter referred to as M9-supplemented media–and resuspended in an equal volume of M9-supplemented media. Washed bacteria were diluted 1:200 in M9-supplemented media containing 100 μg/ml streptomycin and 50 μg/ml carbenicillin and the desired concentration of cobalt (II) chloride (Alfa Aesar, Puratronic, 99.998%), iron (III) chloride (Acros Organics, 99+%), manganese (II) chloride (Alfa Aesar, Puratronic, 99.999%), nickel (II) chloride (Alfa Aesar, Puratronic, 99.9995%) or zinc chloride (Alfa Aesar, Puratronic, 99.999%). All solutions were prepared fresh in MilliQ water. Bacterial cultures (2 ml) were grown in 14 ml polystyrene round-bottom tubes (Falcon) at 37°C, shaking at 220 rpm for ~16 h. GFP fluorescence was measured in black 96-well plates (Costar) using a TECAN SPARK plate reader (excitation wavelength of 485 nm, bandwidth 20 nm; emission wavelength of 535 nm, bandwidth of 20 nm). The background fluorescence of mCherry-S. Typhimurium bacteria (no reporter plasmid) was subtracted from all readings, which were then normalized for bacterial growth (OD₆₀₀).

Measurement of bacterial growth in broth

Bacterial cultures were grown overnight in LB-Miller broth, shaking (220 rpm) at 37°C, then subcultured 1:100 into 10 ml LB-Miller broth in 125 ml Erlenmeyer flasks with shaking (220 rpm) at 37°C. Samples were collected at hourly intervals for OD₆₀₀ readings.

Mammalian cell culture

HeLa human cervical adenocarcinoma epithelial cells were purchased from American Type Culture Collection (ATCC, CCL-2) and used within 15 passages of receipt. Cells were maintained at 37°C and 5% CO₂ in the growth medium (GM) recommended by ATCC i.e. Eagle’s minimum essential medium (EMEM, Corning) containing sodium pyruvate, L-glutamine and 10% (v/v) heat-inactivated fetal calf serum (FCS; Gemini Bio Products). Tissue culture plasticware was purchased from Thermo Scientific Nunc.

Bacterial infection of mammalian cells

HeLa cells were seeded 24 h prior to infection at the following densities: (i) 5x10⁴ cells/well in 24-well tissue culture plates, (ii) 6x10⁴ cells/well on acid-washed glass coverslips in 24-well plates, or (ii) 2.6x10⁶ cells/15 cm tissue culture dish. T3SS1-induced bacterial subcultures were prepared in LB-Miller broth (Difco) [150] and cells were infected for 10 min with bacterial subcultures (MOI ~50) as described [150]. To induce autophagy, HeLa cells were shifted to Earle’s Balanced Salt Solution (EBSS, Sigma) 3 h prior to infection and maintained in EBSS until 90 min p.i. Thereafter, infected cells were switched back to regular growth medium. To inhibit autophagy, cells were incubated in growth media containing 100 nM wortmannin (WTM, Calbiochem) for 45 min prior to infection, continuing to 90 min post-infection, whereupon cells were transferred back to regular growth media. For manipulation of cellular cation levels, epithelial cells were treated 16–18 h prior to infection in growth media containing 200 μM 2,2’-dipyridyl (DPI, Sigma-Aldrich), 200 μM DPI and 200 μM ammonium iron (III) citrate (FAC, Alfa Aesar) or 200 μM FAC alone. DPI +/- FAC treatment continued throughout the infection. DPI was prepared as a 100 mM stock in 95% ethanol and stored at -80°C for a maximum of 2 weeks. FAC was prepared extemporaneously in MilliQ water. Gentamicin protection and CHQ resistance assays (chloroquine diphosphate salt, Sigma) were as described previously [150]. Monolayers were solubilized with 0.2% (w/v) sodium deoxycholate (Sigma) and serial dilutions were plated on LB-Miller agar (Remel) for enumeration of colony forming units (CFUs).

Bacterial RNA enrichment from infected cells, cDNA synthesis and Illumina sequencing

S. Typhimurium was enriched from infected HeLa cells (12 x 15 cm dishes for EBSS treatment, 6 x 15 cm dishes for WTM treatment) as described previously by Hautefort et al. (2008) [33] with minor modifications. Briefly, infected HeLa cells were washed once with 30 ml chilled phosphate-buffered saline (PBS), then lysed on ice for 10–20 min in 0.1% SDS, 1% acidic phenol, 19% ethanol in water (15 ml per 15 cm dish). Pooled lysates were collected into 50 ml conical tubes and centrifuged at 3,220 xg for 30 min, 4°C. After three washes in ice-cold wash buffer (0.1% phenol, 19% ethanol in water) (30 min, 3,220 xg, 4°C each wash), the remaining bacterial pellet was resuspended in <1 ml of wash buffer, transferred to an RNase-free microcentrifuge tube and centrifuged at 16,000 xg for 2 min. The supernatant was discarded, and the pellet resuspended in 1 ml TRIzol reagent (Life Technologies) by gently pipetting up-and-down (~60 times), then transferred to -80°C for storage. Total RNA was extracted as described [34] and the RNA concentration quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific). RNA quality was analyzed using an Agilent Bioanalyzer 2100.

RNA samples isolated from two independent experiments were sent to Vertis Biotechnologie AG (https://www.vertis-biotech.com) for cDNA synthesis and RNA sequencing. The RNA samples were fragmented with ultrasound (4 pulses of 30 sec at 4°C) followed by a treatment with antarctic phosphatase and re-phosphorylated with polynucleotide kinase. Afterwards, the RNA fragments were poly(A)-tailed using poly(A) polymerase and an RNA adapter was ligated to the 5’-phosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 20–30 ng/μl using a high-fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The four cDNA samples were pooled in equimolar amounts and size fractionated in the size range of 150–500 bp using a differential clean-up with the Agencourt AMPure kit, then the cDNA samples were sequenced on an Illumina HiSeq 2000 machine with 50 bp read length.

RNA-seq data analysis

A total of 42 to 57 million sequence reads were generated from each sample. Alignment and processing of bacterial RNA-seq reads was done using bacpipe pipeline v0.8a (https://github.com/apredeus/multi-bacpipe). Briefly, reads were aligned to a reference genome sequence of S. Typhimurium strain 4/74 that included the complete chromosome, as well as 93 kb pSLT virulence plasmid, 87 kb pCol1B9 plasmid, and 9 kb pRSF1010 plasmid (NCBI assembly GCF_000188735.1, replicon GenBank IDs CP002487.1—CP002490.1). Reads were aligned using STAR v2.7.6a with “—alignIntronMin 20—alignIntronMax 19—outFilterMultimapNmax 20” options. Ribosomal operons were predicted using Prokka v1.14.6, after which reads overlapping rRNA operons were removed from the bam file using bedtools v2.29.2 with “bedtools intersect -nonamecheck -v” options. Remaining reads were subsequently quantified using the featureCounts program from Subread v2.0.1, with “-O -M—fraction -t gene -g ID -s 1” options. Custom GTF annotation of the 4/74 strain with 282 known Salmonella non-coding RNAs was used and is available at https://github.com/apredeus/salmonella_pathways/tree/main/474_ref. From 51 to 59% of reads aligned to rRNA operons; of the remaining reads, 11.7–21.6 M aligned to the reference genome, and 10.1–18.5 M (22.7–33.9%) were assigned to an annotated gene. Raw sequencing reads have been deposited at Gene Expression Omnibus (GEO) database under series ID GSE179103. Differential expression analysis was done in R v4.0.4 using DESeq2 v1.30.1. Genes with adjusted p-value of 0.05 or below were deemed differentially expressed. TPM values were calculated in bacpipe v0.8a (see above). Plotting was done using ggplot2 v3.3.3.

Pathway enrichment analysis

A collection of 80 custom, Salmonella-specific pathways was generated from previous RNA-seq, ChIP-seq, and microarray studies of S. Typhimurium regulons [112,124,128,151,152], as well as transcriptional profiling of S. Typhimurium 4/74 under infection-relevant in vitro growth conditions [34] and inside murine macrophages [43]. A detailed description of the pathway curation process and all relevant scripts can be found at https://github.com/apredeus/salmonella_pathways. Briefly, custom curated gene sets of SPI1 plus SPI1-translocated effectors, SPI2 plus SPI2-translocated effectors, and iron transporters and siderophores were compiled using many literature sources that are listed in the “Salmonella custom pathways.xlsx” table in the repository above (supp_tables subdirectory). For data from [112,151,152] [124,128], supplementary tables from the publications with differentially expressed genes were used; up- and down-regulated genes were used to create separate pathways. Whenever the authors used a different reference strain, gene IDs were converted to 4/74 IDs using the provided ortholog table. For single-replicate RNA-seq experiments profiling 4/74 and D23580 under the seventeen in vitro conditions, the following strategy was used. Tables of TPM-normalized expression values (see supp_tables/474.tsv, D23.tsv) were used to calculate fold change (FC) of a particular condition with regard to all 16 others. Genes with log2FC of 2 or more were selected as "marker" genes for a particular in vitro condition; only up-regulated genes were considered for this specific dataset. The lists of genes up-regulated in each single-replicate experiment were generated using the calculate_fc_tpm.pl script available from the same repository. Additionally, KEGG pathways for S. Typhimurium SL1344 were obtained using the KEGGREST R package v1.28.0, and gene ontology (GO) mapping of SL1344 genes was extracted from SL1344 protein annotation by InterProScan v5.41–78. See S2 Dataset for the entire dataset. The combined pathways were used to annotate gene sets up-regulated in the vacuole and cytosol using the clusterProfiler R package v3.16.1. The resulting overlaps were visualized using ggplot2 v3.3.2.

Fluorescence microscopy

HeLa cells were seeded on acid-washed, 12 mm glass coverslips (#1.5 thickness, Fisher Scientific) in 24-well plates. Infected HeLa cells were fixed with 2.5% (w/v) paraformaldehyde in PBS for 10 min at 37°C. The immunostaining procedure has been described previously [24]. Rabbit anti-SiiE serum (kindly provided by Michael Hensel) was used at a dilution of 1:200. Digitonin permeabilization to deliver anti-Salmonella LPS antibodies (Salmonella O-Antiserum Group B Factors 1, 4, 5, 12; Difco; 1:300 dilution) directly to the cytosol was as described previously [14]. Cells were stained with Hoechst 33342 (1:10,000 in DDH₂O, Life Technologies) to label DNA and coverslips were mounted onto glass slides using Mowiol.

Samples were visualized on a Leica DM4000 upright fluorescence microscope for scoring the number of bacteria per cell, the proportion of cytosolic bacteria and quantification of transcriptional reporter activity. ImageJ software was used to quantify the activity of transcriptional reporters at the individual bacterium level. Images in the red (mCherry) and green (GFP) channels were acquired sequentially under 63x magnification on a Leica DM4000 upright fluorescent microscope using the same acquisition settings (exposure and gain) for each group of transcriptional reporters (determined by the reporter with the highest intensity in the green fluorescence channel). A minimum of 100 cytosolic and 100 vacuolar bacteria, from cells with ≥100 bacteria and 2–40 bacteria, respectively, acquired from ≥5 random fields of view were quantified for each transcriptional reporter from each experiment. From grayscale images, well-defined, individual bacteria were arbitrarily chosen on the mCherry channel (represents all bacteria) and manually outlined, converted to a binary image and the pixel intensity associated with each identified particle (i.e. one bacterium) quantified from the corresponding GFP channel image. GFP fluorescence/bacterium was subsequently plotted as the mean fluorescence intensity (MFI).

Images were acquired using confocal microscopy (Leica SP8) in sequential acquisition mode through an optical section of 0.3 μm in the z-axis. Images are maximum intensity projections of z-stacks.

Statistical analysis

All experiments were conducted on at least three separate occasions, unless otherwise indicated, and results are presented as mean ± SD. Except for the RNA-seq data (see separate paragraph on RNA-seq data analysis), statistical analyses were performed using: (i) one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test, or (ii) one-way ANOVA on ranks (Kruskal-Wallis test) for data with a non-normal distribution (i.e. number of bacteria/cell, MFI) (GraphPad Prism). A p-value of <0.05 was considered significant.