# A Salmonella subset exploits erythrophagocytosis to subvert SLC11A1-imposed iron deprivation

Béatrice Roche, Beatrice Claudi, Olivier Cunrath, Christopher K E Bleck,

Minia Antelo-Varela, Jiagui Li, Dirk Bumann

► To cite this version:

Béatrice Roche, Beatrice Claudi, Olivier Cunrath, Christopher K E Bleck, Minia Antelo-Varela, et al. A Salmonella subset exploits erythrophagocytosis to subvert SLC11A1-imposed iron deprivation. Cell Host & Microbe, 2025, 33, pp.632 - 642.e4. 10.1016/j.chom.2025.04.013. hal-05069071

HAL Id: hal-05069071

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Submitted on 15 May 2025

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# Cell Host & Microbe

## A *Salmonella* subset exploits erythrophagocytosis to subvert SLC11A1-imposed iron deprivation

**Graphical abstract**

<figure><img src="image_2.png" /></figure>

**Authors**

Béatrice Roche, Beatrice Claudi,
Olivier Cunrath, Christopher K.E. Bleck,
Minia Antelo-Varela, Jiagui Li,
Dirk Bumann

**Correspondence**

dirk.bumann@unibas.ch

**In brief**

Iron sequestration is a key host mechanism to control pathogen growth in infected tissues. However, Roche et al. show that a subpopulation of *Salmonella* evades iron restriction by targeting iron-rich vacuoles in macrophages that ingest and degrade red blood cells. Facile iron access enables faster replication of this subset.

**Highlights**

* The metal transporter SLC11A1 sequesters iron from *Salmonella*-containing vacuoles

* Some *Salmonella* evade iron starvation in vacuoles harboring red blood cell remnants

* Iron-starved *Salmonella* replicate poorly

* The iron-replete subset primarily drives overall *Salmonella* growth

<figure><img src="image_3.png" /></figure>
Short article

# A *Salmonella* subset exploits erythrophagocytosis to subvert SLC11A1-imposed iron deprivation

Béatrice Roche,<fnref n="1" />,<fnref n="2" /> Beatrice Claudi,<fnref n="1" /> Olivier Cunrath,<fnref n="1" />,<fnref n="3" /> Christopher K.E. Bleck,<fnref n="1" />,<fnref n="4" /> Minia Antelo-Varela,<fnref n="1" /> Jiagui Li,<fnref n="1" /> and Dirk Bumann<fnref n="1" />,<fnref n="5" />,*

## SUMMARY

Solute carrier family 11 member 1 (SLC11A1) is critical for host resistance to diverse intracellular pathogens. During infection, SLC11A1 limits *Salmonella's* access to iron, zinc, and magnesium, but only magnesium deprivation significantly impairs *Salmonella* replication. To understand the unexpected minor impact of iron, we determined *Salmonella's* iron access in infected SLC11A1-deficient and normal mice. Using reporter strains and mass spectrometry of *Salmonella* purified from the spleen, we found that SLC11A1 caused growth-restricting iron deprivation in a subset of *Salmonella*. Volume electron microscopy revealed that another *Salmonella* subset circumvented iron restriction by targeting iron-rich endosomes in macrophages degrading red blood cells (erythrophagocytosis). These iron-replete bacteria dominated overall *Salmonella* growth, masking the effects of the other *Salmonella* subset's iron deprivation. Thus, SLC11A1 effectively sequesters iron, but heterogeneous *Salmonella* populations partially bypass this nutritional immunity by targeting iron-rich tissue microenvironments.

## INTRODUCTION

Iron is an essential nutrient for most organisms. During infections, hosts often limit pathogen growth by sequestering iron, a process called nutritional immunity.<fnref n="1" /> One example is the solute carrier family 11 member 1 (SLC11A1, also known as NRAMP1), which removes iron from endosomes<fnref n="2" />⁻<fnref n="4" /> and is critical for host defense against intracellular pathogens such as *Mycobacterium tuberculosis*, *Leishmania* spp., and *Salmonella*.<fnref n="2" /> In *Salmonella*-infected mice, SLC11A1 reduces iron, zinc, and magnesium availability in *Salmonella*-containing vacuoles in macrophage. However, only magnesium deprivation—likely occurring indirectly,<fnref n="5" /> possibly through altered vesicle trafficking<fnref n="3" />,⁶—restricts *Salmonella* replication.<fnref n="3" />

The minor impact of SLC11A1-mediated iron sequestration is unexpected because iron is (1) an essential nutrient for both *Salmonella*⁷ and the host; (2) a cofactor of NADPH oxidase, which is essential for host control of *Salmonella*⁸,⁹; (3) an amplifier of oxidative stress through the Fenton reaction¹⁰,¹¹; and (4) a regulator of antibacterial immunity through key transcription factors and cytokine signaling.¹²⁻¹⁷ Iron overload worsens *Salmonella* infection, and *Salmonella* mutants with defective iron uptake are less virulent, suggesting iron limitation.¹¹,¹³⁻¹⁵,¹⁷⁻²⁴ However, this contradicts data showing that host iron-deficiency and anemia accelerate *Salmonella* replication⁹,²⁵⁻²⁷ and are risk factors for disease

and complications of invasive salmonellosis in humans and mice.²⁰,²¹,²⁷⁻³³ Anemia itself may be promoted by *Salmonella*-induced hematophagocytosis, the ingestion and degradation of blood cells (including erythrocytes) by macrophages,³²,³⁴,³⁵ which can increase iron access for intracellular *Salmonella*³⁶ and suppress antibacterial immunity.³⁷

Many studies have investigated how iron modulates systemic salmonellosis,¹¹,¹³⁻¹⁵,¹⁷⁻²⁴,³⁸ but the results remain difficult to interpret because of methodological limitations. Commonly used *Salmonella* mutants with defective iron uptake require micromolar iron,<fnref n="3" /> whereas wild-type *Salmonella* thrive on nanomolar iron.<fnref n="3" /> Commonly used mouse strains such as C57BL/6 (B6) and BALB/c are deficient for SLC11A1, leading to disrupted iron homeostasis and macrophage dysfunction.<fnref n="2" />,³⁹⁻⁴⁵ Moreover, iron availability varies within infected tissues,⁴⁶,⁴⁷ but most studies measured bulk properties such as tissue iron levels, bacterial loads, or overall inflammation, potentially masking critical bacterial subsets.⁴⁸,⁴⁹

## RESULTS

### SLC11A1 diminishes iron access for a subset of *Salmonella*

To determine *Salmonella* iron access at the single-cell level, we constructed *Salmonella* reporters carrying transcriptional

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹Biozentrum, University of Basel, 4056 Basel, Switzerland</footnote>

<footnote n="2">²Institut de Biologie Moléculaire et Cellulaire, UPR 9002 CNRS, 67084 Strasbourg, France</footnote>

<footnote n="3">³Biotechnologie et signalisation cellulaire, Université de Strasbourg, 67412 Illkirch, France</footnote>

<footnote n="4">⁴HHMI Janelia Research Campus, Ashburn, VA 20147, USA</footnote>

<footnote n="5">⁵Lead contact</footnote>

<footnote>*Correspondence: dirk.bumann@unibas.ch
https://doi.org/10.1016/j.chom.2025.04.013</footnote>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 1.** SLC11A1 partially restricts *Salmonella* access to iron</figcaption></figure>

(A) Blue fluorescence (BF) of *Salmonella* carrying *P*<sub>ryhB-2-bfp-ova</sub> in spleen of SLC11A1-deficient mice (B6, C57BL/6) or B6 *Slc11a1*<sup>r</sup> mice with restored SLC11A1 (pooled data for three to four mice per group). For comparison, a *Salmonella* *in vitro* culture at low iron level is shown (dashed line). The arrows indicate sorting gates for *Salmonella* subsets with different BF. The right graphs show for individual mice the fraction of cells with BF >3,000 and median fluorescence intensities (MFIs) (the lines represent means; unpaired two-tailed t test comparing values for B6 and B6 *Slc11a1*<sup>r</sup> mice).

(B) Proteome comparison of *Salmonella* subsets with low or high *P*<sub>ryhB-2-bfp-ova</sub> activity sorted from spleen of six B6 *SLC11A1*<sup>r</sup> mice using gates shown in (A). Each circle represents one protein. Data points above the dashed line have a false discovery rate q < 0.05 (orange, proteins associated with iron starvation; blue, magnesium starvation; pink, zinc starvation). See also Table S1.

(C) Fluorescence properties of *Salmonella* carrying a dual reporter construct with *P*<sub>ryhB-2-bfp-ova</sub> and *P*<sub>mgtCQBRUCIGR<sup>r</sup>gfp-ova</sub> in spleen of C57BL/6 *SLC11A1*<sup>r</sup> mice. Pooled data from three independently infected mice are shown, with each dot representing a single *Salmonella* cell. Median data for individual mice are shown in Figure S1F. Histograms of *in vitro* fluorescence intensities are shown for comparison.
See also Figure S1.

fusions of known iron-responsive promoters to *gfp-ova* encoding unstable GFP⁵⁰ (to reflect current promoter activities). The promoter *P*<sub>ryhB-2</sub> showed the strongest induction by iron starvation (~100-fold above autofluorescence) but was unresponsive to zinc, manganese, or magnesium starvation (Figures S1A and S1B). In the spleens of SLC11A1-deficient BALB/c and B6 mice, *Salmonella* exhibited low *P*<sub>ryhB-2</sub> activity (Figure 1A), suggesting ample iron availability.³,⁵¹ In contrast, in B6 *SLC11A1*<sup>r</sup> mice with functional SLC11A1,³ ~40% *Salmonella* showed high *P*<sub>ryhB-2</sub> activity (Figure 1A).

To determine the stimuli inducing *P*<sub>ryhB-2</sub> *in vivo*, we purified *Salmonella* subsets with low or high *P*<sub>ryhB-2</sub> activity from spleen using flow cytometry.³,⁵²,⁵³ Mass spectrometry revealed that

six of 1,372 quantified proteins were more abundant in *P*<sub>ryhB-2</sub>-high *Salmonella* (Figure 1B). All six proteins were associated with iron starvation,⁷,⁵⁴,⁵⁵ confirming reduced iron access in the *P*<sub>ryhB-2</sub>-high subset. All other proteins were similarly abundant between subsets, including those responsive to alternative stimuli activating *P*<sub>ryhB-2</sub> (oxidative/nitrosative stress and carbon starvation⁵⁶,⁵⁷; Figure S1C; Table S1). Thus, the heterogeneous *P*<sub>ryhB-2</sub> activity indicated that SLC11A1 deprived some, but not all, *Salmonella* of iron within otherwise comparable microenvironments.

Heterogeneous iron deprivation might reflect uneven SLC11A1 activity. To test this idea, we examined SLC11A1-dependent magnesium and zinc deprivation.³ Iron-starved and
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<figure><img src="image_20.png" /><figcaption>**Figure 2.** Macrophage erythrophagocytosis provides iron to a subset of *Salmonella*</figcaption></figure>

(A) Confocal fluorescence micrograph of iron-replete *Salmonella* (low $P_{r,yhB-2-gfp}$ activity; constitutive mCherry) in a F4/80-positive red pulp macrophage that contains a large LAMP1/2-positive endosome (LE).

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iron-replete subsets displayed a similar abundance of magnesium transporters, suggesting similar magnesium starvation³ (MgtA, 190 ± 20 copies per cell vs. 210 ± 70; MgtB, 1,990 ± 300 vs. 2,000 ± 300; Figure 1B). MgtC encoded within the *mgtCQBRUcigR* operon along with MgtB was slightly, but non-significantly, more abundant in iron-starved *Salmonella* (166 ± 50 copies per cell vs. 117 ± 50 copies), consistent with ~30% higher $P_{mgtCQBRUcigR}$ activity (Figures 1C and S1D-S1F), reflecting the operon's complex regulation.⁵⁸ However, these differences were small compared with the ~6.6-fold difference in $P_{ryhB-2}$ activity between iron-starved and iron-replete subsets (Figure S1F), suggesting minimal variation in magnesium availability. Zinc transporters ZinT, ZnuA, and ZnuC and the ribosomal protein RpmE2, which replaces the zinc-containing RpmE during zinc starvation, showed ~2-fold higher abundance in iron-starved *Salmonella* (Figures 1B and S1C; Table S1), suggesting slightly reduced zinc availability in this subset. These data suggest similar SLC11A1-driven metal sequestration in vacuoles harboring iron-starved or -replete *Salmonella*. Thus, differential SLC11A1 activity did not explain the heterogeneous iron availability.

## Iron-replete *Salmonella* colocalize with LEs containing red blood cells

To identify the cause of *Salmonella*'s heterogeneous iron access, we analyzed spleen microenvironments by confocal microscopy. Most *Salmonella* resided in LAMP1/2 (lysosomal-associated membrane proteins 1 and 2)-positive vacuoles within F4/80-positive red pulp macrophages.⁵²,⁵⁹ Iron-replete *Salmonella* (low $P_{ryhB-2}$ activity) were almost exclusively in macrophages containing large endosomes (LEs) (Ø > 2.5 µm), whereas iron-starved *Salmonella* (high $P_{ryhB-2}$ activity) resided in macrophages with either LE or small endosome (SE) (Figures 2A–2D). Thus, LEs marked host cells providing facile iron access.

Volume electron microscopy (serial block-face scanning electron microscopy [SBEM])⁶⁰ revealed that large macrophage endosomes contained homogeneous electron-dense material resembling red blood cells (Figures 2E–2G), indicating erythrophagocytosis (ingestion and degradation of aged or damaged red blood cells), a common function of red pulp macrophages⁶¹ that is stimulated by *Salmonella* infection.³⁴,⁶²,⁶³ SBEM identified *Salmonella* based on their rod shape and electron density (Figures S2A and S2B). Some *Salmonella* resided directly in endosomes containing red-blood-cell-derived contents (Figures 2G–2I, 2K–2N, S2C, and S2D), highlighting erythrophagocytosis as a potential iron source for *Salmonella* (as in cell-culture infec-

tions³⁶). One mouse red blood cell contains ~500 Mio iron atoms⁶⁴ or ~2,500-fold the iron requirements of a *Salmonella* cell (~200,000 iron atoms).⁶⁵ SLC11A1 transports iron from the red-blood-cell-degrading endosomes to the cytosol for subsequent redistribution to the bone marrow.⁴³ This process can extend over 20 h,⁶⁶,⁶⁷ and, during this time, *Salmonella* would be able to access this iron surplus, explaining the presence of iron-replete *Salmonella* in erythrophagocytosing macrophages. Red blood cells also contain ~3 Mio zinc atoms⁶⁸ (~15-fold the *Salmonella* requirements, 190,000 atoms⁶⁵) and ~67 Mio magnesium atoms⁶⁹,⁷⁰ (~2.5-fold the *Salmonella* cell requirements, 26 Mio atoms⁶⁵). These data are consistent with a strong impact of erythrocyte remnants on *Salmonella*'s iron access but small effects on zinc and magnesium availability (Figures 1B and S1C; Table S1). Other *Salmonella* resided in electron-light endosomes with no connection to red-blood-cell-derived endosomes (Figures S2E and S2F), explaining the presence of iron-starved *Salmonella*. Thus, *Salmonella* partially subverted iron-dependent nutritional immunity by targeted iron-rich endosomes in erythrophagocytosing macrophages.

## Iron-starved *Salmonella* make limited contributions to overall *Salmonella* growth

To test the relevance of iron access for *Salmonella* fitness, we selectively eliminated iron-starved *Salmonella* using a conditional suicide construct⁷¹ expressing the toxin gene *doc*⁷²,⁷³ from $P_{ryhB-2}$ using a stringent ON/OFF mechanism⁷⁴ (to prevent baseline *doc* expression in iron-replete *Salmonella*; Figures 3A and S3A). *gfp* under $P_{ryhB-2}$ control reported iron availability and all *Salmonella* cells constitutively expressed mCherry. A control strain carried inactive *doc*<sup>H66Y D70N A76E72</sup> and constitutive BFP (blue fluorescent protein; Figures 3A and S3B).

The *doc*-strain and the inactive control grew similarly under iron-rich conditions, whereas only the inactive control strain grew under iron starvation, confirming selective targeting of iron-starved *doc-Salmonella* (Figure S3C). In B6 SLC11A1<sup>r</sup> mice, the control strain exhibited normal broad $P_{ryhB-2}$ activities, whereas the *doc*-strain predominantly showed low $P_{ryhB-2}$ activity, lacking the ~40% iron-starved cells with high activity (Figures 3B and S3D). Thus, the *doc*-construct selectively eliminated iron-starved *Salmonella* in vivo.

We infected mice with a ~1:1 mixture of both strains and observed similar spleen loads at day 6 (Figures 3C and S3E). This was unexpected, as the loss of 40% of the *doc*-population should have reduced their net growth. The control strain divided 10.3 ± 2.3 times over 6 days of infection (~1,200-fold increase).

(B) Iron-starved *Salmonella* (*high* $P_{ryhB-2-gfp}$ activity) in a macrophage that contains LEs.

(C) Iron-starved *Salmonella* (*high* $P_{ryhB-2-gfp}$ activity) in a macrophage that contains only small endosomes (SEs).

(D) Quantification of iron-replete (GFP<sup>low</sup>) and iron-starved (GFP<sup>high</sup>) *Salmonella* in red pulp macrophages with large endosomes (Mφ-LE), or only small endosomes (Mφ-SE), or F4/80-negative cells. Each dot represent data from one of three independently infected mice, the bars represent mean values (N: 137, 263, and 300 *Salmonella* cells; two-way ANOVA for testing the interaction between GFP level and cell type).

(E) SBEM image of a macrophage phagocytosing a ingested red blood cell (RBCi) in spleen ("erythrophagocytosis"). Free RBCs reside in the interstitial space.

(F) Macrophage with two RBCi.

(G) Macrophage with an endosome that contains several *Salmonella* (yellow asterisks) and material derived from an RBCi. Similar images were obtained for dozens of *Salmonella* in three independently infected mice.

(H) 3D reconstruction of the *Salmonella*-containing endosome shown in (G) based on layers of the 3D tomogram (I–N). The *Salmonella* cells are labeled by numbers. The scale shows the vertical z axis.

(I–N) Layers of 3D tomogram of the *Salmonella*-containing endosome shown in (G) and (H).

See also Figure S2.
A

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<figure><img src="image_23.png" /><figcaption>**Figure 3.** Iron-starved *Salmonella* have limited impact on overall *Salmonella* growth</figcaption></figure>

(A) Construct for eliminating iron-starved *Salmonella*. *P*<sub>ryhB-2</sub> drives expression of *gfp-ova* encoding an unstable variant of the green fluorescent protein. The same operon contains the toxin gene *doc* embedded in a stringent ON/OFF module consisting of two inhibitory feedback loops (*lac* encodes the *lac* repressor inhibiting the *P*<sub>A1/4os</sub> promoter, the small RNA *MicC* represses *lacI* translation). *mCherry* is expressed from the constitutive *P*<sub>ybaJ</sub> promoter to detect all *Salmonella*. A control construct carries inactive *doc*<sup>H66Y D70N A76E</sup> and *bfp* instead of *mCherry*.

(B) Green fluorescence of the strain with active *doc/mCherry* (red) and the control strain with *doc inactive/BFP* (blue) in spleen of B6 *SLC11A1*<sup>r</sup> mice. Pooled data for four independently infected mice are shown. The inset shows the lacking fraction of bright cells in the strain with active *doc*. Each circle represents an individual mouse, the line represents the mean.

(C) Spleen loads at day 6 after infection with a ~1:1 mixture of the two strains (paired t test on log-transformed data).

See also Figure S3.

Assuming a similar division rate but a 40% loss per division (resulting in only 1.2 instead of 2 surviving daughter cells), the *doc*-strain should have grown only ~6.5-fold (1.2<sup>10.3</sup>), predicting 200-fold lower loads. Instead, both strains reached similar loads, indicating that overall *Salmonella* growth was driven primarily by iron-replete cells, with limited contributions from iron-starved cells.

## Iron starvation restricts Salmonella replication

To determine the single-cell impact of iron starvation, we combined a *P*<sub>ryhB-2-bfp</sub> fusion with the TIMER<sup>bac</sup> reporter, which indicates bacterial replication rates by shifting fluorescence spectra<sup>3,59,75-83</sup> (Figure S4A). During these experiments, we unexpectedly observed more variable iron-starvation responses (Figures 4A and 4B). We traced this issue to inconsistent iron levels in the mouse diet (nominally 209 mg iron/kg; actual values as high as 239 mg/kg; earlier actual levels unknown). We therefore obtained mouse diets with defined iron contents (30 mg/kg, close to recommended levels,<sup>84</sup> or 5 mg/kg) from another vendor and compared mice raised on these different diets.

In SLC11A1-deficient B6 mice on a high-iron diet, *Salmonella* showed low baseline *P*<sub>ryhB-2</sub> activity, which slightly increased at moderate or low dietary iron (Figures 4A and 4B). Differences were more pronounced in the liver, another major systemic target of *Salmonella*, yet >85% of *Salmonella* retained low *P*<sub>ryhB-2</sub> activity even under low dietary iron, suggesting that most bacteria had ample iron access regardless of diet.

In B6 *Slc11A1*<sup>r</sup> mice, *Salmonella* exhibited higher *P*<sub>ryhB-2</sub> activity (extending our data; Figure 1A), especially with reduced dietary iron and in the liver (Figures 4A and 4B). We analyzed these mice at day 6 after infection with ~1,000 colony-forming units (CFUs) when tissue loads approximated those in B6 mice at day 4.<sup>3</sup> Similar results were obtained at day 4 after infection with ~40,000 CFUs (Figures S4B and S4C), confirming that mouse genotype rather than infection duration determined these phenotypes. Together, these data show that (1) dietary iron influ-

ences *Salmonella*'s iron access and (2) *Salmonella* faces greater iron restriction in liver than spleen, consistent with predominant erythrocyte degradation in the spleen (although the liver can also remove damaged erythrocytes<sup>56</sup>).

Data from the dual *P*<sub>ryhB-2/TIMER<sup>bac</sup></sub> reporter (Figure S4A) revealed similarly rapid *Salmonella* replication in SLC11A1-deficient B6 mice in spleen across dietary iron levels (Figures 4C-4F; TIMER<sup>bac</sup> cannot be used in liver due to uneven oxygenation<sup>76</sup>). This was consistent with the dominance of iron-replete *Salmonella* in B6 mice regardless of diet (Figures 4A-4C). Small iron-starved subsets at moderate and low dietary iron showed slightly slower replication (Figures 4C and 4F), with limited impact on overall replication (Figure 4E). Despite constant *Salmonella* replication rates, high dietary iron increased *Salmonella* loads in spleen and liver<sup>27</sup> (Figure 4G), suggesting impaired host control under iron-overload conditions and perturbed iron homeostasis in the absence of SLC11A1.<sup>12-17,42</sup>

In B6 *Slc11A1*<sup>r</sup> mice, *Salmonella* replicated more slowly,<sup>3</sup> especially the iron-starved subset (Figures 4D-4F, S4D, and S4F), partly explaining this subset's limited contribution to *Salmonella*'s overall growth (Figure 3C). Dietary iron levels affected only slightly the overall and subset replication rates (Figures 4D, 4E, S4C, and S4D). Similarly, bacterial loads were comparable across dietary iron levels (Figures 4G, S4G, and S4H), indicating that SLC11A1-mediated iron homeostasis<sup>42</sup> limited the influence of dietary iron levels on anti-*Salmonella* host immunity (i.e., oxidative bursts, cytokine signaling, Fenton reaction, etc.).<sup>2,39-45</sup> At high dietary iron levels, nearly all *Salmonella* cells achieved iron sufficiency (Figures 4A, 4B, and 4D), yet their replication remained slow (Figure 4E), supporting the importance of iron-independent magnesium deprivation in restricting *Salmonella* growth in B6 *Slc11A1*<sup>r</sup> mice.<sup>3</sup>

In contrast to wild-type *Salmonella*, *Salmonella feoABC entC sitABC mntH*,<sup>3</sup> lacking all high-affinity iron transporters, exhibited strong diet-dependent fitness defects<sup>3</sup> (Figures 4H and
<figure><img src="image_24.png" /><figcaption>Figure 4. Iron starvation limits replication of a *Salmonella* subset</figcaption></figure>

(A) BF of *Salmonella* carrying a dual *P*<sub>ryhB-2</sub>-bfp-ova/TIMER<sup>bac</sup> reporter. Data are shown for mice raised on diets with different iron contents (Hi, 209–239 mg iron/kg; 30 mg/kg; 5 mg/kg). Data for individual mice are shown in (B).

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S4E). This mutant requires at least 1 µM iron for growth (~50 µM optimal³), concentrations available only in mice on a high-iron diet. The discrepancy with wild-type *Salmonella* (which thrive on nM iron⁵; Figures 4E and S4F) demonstrates the limited informative value of transport-defective mutants.

DISCUSSION

SLC11A1-mediated iron sequestration has limited impact on *Salmonella* infection,³ but our single-cell analysis revealed that SLC11A1 actually did deprive some *Salmonella* cells of iron, particularly at moderate to low dietary iron levels. These iron-starved *Salmonella* upregulated high-affinity iron transporters but still struggled to obtain sufficient iron for normal replication, limiting their contribution to overall *Salmonella* growth. However, another *Salmonella* subset overcame iron limitation by exploiting erythrophagocytosis—the removal of aging or damaged red blood cells that could clog small capillaries.⁸⁷ *Salmonella* resided directly in endosomes containing remnants of degraded red blood cells in red pulp macrophages. These endosomes provided ample iron, undermining SLC11A1's iron sequestration and all other mechanisms for iron-dependent nutritional immunity. Red blood cells also provided some zinc, slightly relieving *Salmonella*'s zinc starvation, and minimal magnesium (compared with *Salmonella*'s requirements). Some *Salmonella*-infected macrophages ingest lymphocyte fragments,⁶² but this has probably limited impact on iron availability compared with erythrophagocytosis because iron concentration in lymphocytes is ~1,000-fold lower (~1 mg/L; ~1 fg iron⁸⁸ in a cell volume of ~1 pL⁸⁹) than in erythrocytes (1,000 mg/L; ~45 fg iron⁶⁴ in a cell volume of ~45 fL⁸⁹).

In summary, SLC11A1 is a potent contributor to iron-dependent nutritional immunity in intracellular compartments, especially at low dietary iron levels. However, *Salmonella* bypass iron restriction by targeting iron-loaded macrophages undergoing erythrophagocytosis. Other SLC11A1-restricted pathogens such as mycobacteria and *Leishmania* predominantly target host cells not involved in erythrophagocytosis, suggesting a potentially more important role of SLC11A1-mediated iron deprivation in addition to possible zinc and magnesium limitations. The strategy and methods developed in this study could help to test this idea.

Limitations of the study

We studied intravenously infected mice. *Salmonella* replication dynamics and overall growth in spleen are comparable between the natural oral and intravenous infections,⁷⁶ but infection levels are less variable across intravenously infected mice, allowing us to use fewer animals. We focused on *Salmonella*'s iron access and its consequences, but it remains unclear how *Salmonella* targets the endosomes of erythrophagocytosing macrophages.

RESOURCE AVAILABILITY

Lead contact

Requests for further information and resources should be directed to, and will be fulfilled by, the lead contact, Dirk Bumann, [dirk.bumann@unibas.ch](mailto:dirk.bumann@unibas.ch).

Materials availability

All materials are available upon request, without restriction, from the [lead contact](mailto:dirk.bumann@unibas.ch).

Data and code availability

Proteomics data have been deposited at MassIVE (mass spectrometry interactive virtual environment; https://massive.ucsd.edu/) as MassIVE: MSV000094024 and are publicly available as of the date of publication. All other data are available upon request from the [lead contact](mailto:dirk.bumann@unibas.ch). This paper does not report original code.

ACKNOWLEDGMENTS

This research was supported by Swiss National Science Foundation grants 310030_156818 (D.B.), 310030_182315 (D.B.), and NCCR_180541 AntiResist (D.B.). We thank Jasmin Künneke for analyzing imaging data, Janine Bögli (Biozentrum, FACS Core Facility) for sorting, Carola Alampi and Cinzia Tiberi (Biozentrum, BioEM lab) for scanning electron micrographs, Christel Genoud (Friedrich-Miescher-Institute, Basel) for help with SBEM, Alexander Harms (ETH Zurich) for plasmid pAH154_doc_v2, and Belén Calles (Centro Nacional de Biotecnología-CSIC, Madrid) for plasmid pS238D.M.

AUTHOR CONTRIBUTIONS

D.B. and B.R. conceived and designed the study. B.R. performed most of the experiments and analyzed the data, assisted by B.C., O.C., and J.L. C.K.E.B. performed SBEM. M.A.V. performed proteome analysis. D.B. supervised the research and analyzed data. B.R. and D.B. wrote the manuscript, with comments from all authors.

(B) Fraction of *Salmonella* cells with high BF in mice raised on diets with different iron contents. Each symbol represents an individual mouse, lines represent means. Statistical differences between mouse lines, as well as diets and organs (separately for each mouse line), were analyzed using two-way ANOVA.

(C) *Salmonella* division rate and BF in the spleen of B6 mice raised on diets with different iron contents. Pooled data for 3-4 individual mice are shown. Data for individual mice are shown in (B), (E), and (F).

(D) *Salmonella* division rate and BF in the spleen of B6 *Slc11A1*⁻/⁻ mice raised on diets with different iron contents. Pooled data for 5-10 individual mice are shown. Data for individual mice are shown in (B), (E), and (F). The two lower panels show difference maps of mice raised on diets containing 30 mg iron/kg or 5 mg/kg compared with mice raised on high dietary iron.

(E) Median division rates of *Salmonella* in the spleens of mice raised on diets with different iron contents. Each symbol represents an individual mouse, lines represent means. Statistical differences between mouse lines as well as diets (separately for each mouse line) were analyzed using two-way ANOVA.

(F) Division rates of *Salmonella* subsets with dim (D) or bright (B) BF in the spleens of mice raised on diets with different iron contents. Each symbol represents an individual mouse, line represent medians. Statistical differences were analyzed separately for each mouse line using two-way ANOVA.

(G) Net growth of *Salmonella* in spleen and liver of mice raised on diets with different iron contents. Each symbol represents an individual mouse, lines represent geometric means. Fold increase values were calculated by dividing the final organ loads by the estimated initial organ loads (~20% of the inoculum⁸⁵). Statistical differences between mouse lines, as well as diets and organs (separately for each mouse line), were analyzed using two-way ANOVA.

(H) Relative fitness of a *Salmonella* mutant lacking high-affinity iron uptake (compared with wild-type *Salmonella*) in the spleens of mice raised on diets with different iron contents. Each symbol represents an individual mouse, lines represent geometric means. Statistical differences were analyzed using two-way ANOVA.

See also Figure S4.
DECLARATION OF INTERESTS

The authors declare no competing interests.

STAR★METHODS

Detailed methods are provided in the online version of this paper and include
the following:

● KEY RESOURCES TABLE

● EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

○ Mice

○ Bacterial strains, plasmids and growth media

● METHOD DETAILS

○ Mouse infections

○ Flow cytometry

○ Proteomics

○ Immunohistochemistry and confocal microscopy

○ Serial blockface scanning electron microscopy

● QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.chom.2025.04.013.

Received: February 20, 2024

Revised: March 19, 2025

Accepted: April 15, 2025

Published: May 14, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td><strong>Antibodies</strong></td><td></td><td></td></tr><tr><td>Rat anti-mouse F4/80:biotin</td><td>Bio-Rad</td><td>Cat# MCA497B; RRID:AB_322046</td></tr><tr><td>Rat anti-mouse CD107a(LAMP-1):BV421</td><td>BioLegend</td><td>Cat# 121617; RRID:AB_10896064</td></tr><tr><td>Rat anti-mouse CD107b(LAMP-2):BV421</td><td>BD Biosciences</td><td>Cat# 564249; RRID:AB_2738702</td></tr><tr><td><strong>Bacterial and virus strains</strong></td><td></td><td></td></tr><tr><td>Salmonella enterica serovar Typhimurium SL1344 hisG<sup>Leu69</sup></td><td>Claudi et al.<sup>76</sup></td><td>N/A</td></tr><tr><td>Salmonella feoABC entC sitABC mntH</td><td>Cunrath and Bumann<sup>3</sup></td><td>N/A</td></tr><tr><td><strong>Chemicals, peptides, and recombinant proteins</strong></td><td></td><td></td></tr><tr><td>Chelex 100 sodium form</td><td>Sigma</td><td>Cat.-Nr. 95621-100G-F</td></tr><tr><td>Iron maltoside (MonoFer)</td><td>Pharmacosmos</td><td>N/A</td></tr><tr><td>Tryptone</td><td>LLG</td><td>Cat.-Nr. 6271005</td></tr><tr><td>Yeast Extract</td><td>Oxoid</td><td>Cat.-Nr. LP0021B</td></tr><tr><td>Agar</td><td>AppliChem</td><td>Cat.-Nr. A0949</td></tr><tr><td>Phosphate-buffered saline, pH 7.2</td><td>ThermoFisher</td><td>Cat.-Nr. 20012019</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>Cat.-Nr. T9284</td></tr><tr><td>Streptavidin-Alexa 647</td><td>ThermoFischer</td><td>Cat.-Nr. S21374</td></tr><tr><td><strong>Critical commercial assays</strong></td><td></td><td></td></tr><tr><td>KOD Hot Start Master Mix</td><td>Novagen</td><td>Cat.-Nr. 71842-4</td></tr><tr><td>GoTaq® G2 Master Mix</td><td>Promega</td><td>Cat.-Nr. M7822</td></tr><tr><td>Gibson Assembly Master Mix</td><td>NEB</td><td>Cat.-Nr. E2611L</td></tr><tr><td><strong>Deposited data</strong></td><td></td><td></td></tr><tr><td>Mass spectrometry data</td><td>This study</td><td>MassIVE: MSV000094024</td></tr><tr><td><strong>Experimental models: Organisms/strains</strong></td><td></td><td></td></tr><tr><td>Mouse: BALB/cAnNCrl SPF</td><td>Charles River; bred at mouse facility of University of Basel</td><td>N/A</td></tr><tr><td>Mouse: C57BL/6J SPF</td><td>Jackson Laboratories; bred at mouse facility of University of Basel</td><td>N/A</td></tr><tr><td>Mouse: C57BL/6J Slc11A1<sup>f/f</sup> SPF</td><td>Cunrath and Bumann<sup>3</sup></td><td>N/A</td></tr><tr><td><strong>Oligonucleotides</strong></td><td></td><td></td></tr><tr><td>See Table S2</td><td></td><td></td></tr><tr><td><strong>Recombinant DNA</strong></td><td></td><td></td></tr><tr><td>See Table S3</td><td></td><td></td></tr><tr><td><strong>Software and algorithms</strong></td><td></td><td></td></tr><tr><td>SnapGene 4.0.3</td><td>GSL Biotech LLC, Boston, USA</td><td>snapgene.com</td></tr><tr><td>ImageJ 1.53q</td><td>Schneider et al.<sup>90</sup></td><td>https://imagej.net/ij/</td></tr><tr><td>Huygens Professional 21.10</td><td>Scientific Volume Imaging B.V., Hilversum, Netherlands</td><td>https://svi.nl/Huygens-Software</td></tr><tr><td>FACSDIVA V8.0.1</td><td>Beckton Dickinson</td><td>N/A</td></tr><tr><td>FlowJo 10.6.1</td><td>Beckton Dickinson</td><td>N/A</td></tr><tr><td>GraphPad Prism 9.3.1</td><td>GraphPad Software, La Jolla, California, USA</td><td>https://www.graphpad.com</td></tr><tr><td>OriginPro 2019 (64-bit) 9.6.0.172</td><td>OriginLab Corporation, Northampton, USA</td><td>www.originlab.com</td></tr><tr><td>3dmod 4.12.32</td><td>Wasserstein and Lazar<sup>91</sup></td><td>http://bio3d.colorado.edu/ftp/ Windows3dmod/</td></tr><tr><td>SpectroNaut 16.1.220730.53000</td><td>Biognosys AG. Schlieren, Switzerland</td><td>https://biognosys.com</td></tr><tr><td>Perseus 2.0.7.0</td><td>Wiśniewski et al.<sup>92</sup></td><td>https://maxquant.net/perseus/</td></tr></tbody></table>
## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Mice

All animal experiments were approved (license 2239, Kantonales Veterinäramt Basel) and performed according to local guidelines (Tierschutz-Verordnung, Basel) and the Swiss animal protection law (Tierschutz-Gesetz). Mice (BALB/cAnNCrl, C57BL/6J, or C57BL/6J *Slc11A1*<sup>r/r</sup><sup>3</sup>) were housed at 22°C (-2°C/+3°C), relative humidity of 55 +/- 10%, and a 12h/12h dark/light cycle. We infected mice at 10 to 15 weeks of age and used both female and male mice which show comparable results for *Salmonella* infections.<sup>3</sup> Most mice were raised on standard diet (M/R Haltung Extrudat 3439 Ex, Granovit) that should contain 209 mg iron/kg. However, we later realized that iron content was inconsistent and some batches contained 239 mg iron/kg. We then raised additional mice after weaning on another diet (C1038 iron-deficient diet with 5 mg iron/kg or 30 mg iron/kg, Altromin).

We estimated sample size by a sequential statistical design. We first infected two to three mice based on effect sizes and variation observed in our previous studies.<sup>3,93</sup> and used the results to estimate group sizes for obtaining statistical significance with sufficient power. The experiments were not randomized. For animal welfare reasons, researchers were not blinded to mouse genotype during study and data collection. Specifically, *Slc11A1<sup>s</sup>* mice need to be sacrificed at day 4 post-infection to prevent high severity grades, while *Slc11A1<sup>r</sup>* mice reach similar bacterial loads only at day 6 post-infection (or at day 4 after high-dose infection). All other data were collected and analyzed without blinding but objectively, using automated instruments without bias and analysis definitions that were uniformly applied to all data sets.

### Bacterial strains, plasmids and growth media

*Salmonella* strains used in this study were based on SME51, a prototrophic *his*G<sup>Leu69</sup> derivative of *Salmonella enterica* serovar Typhimurium SL1344.<sup>76,94,95</sup> The *Salmonella feoABC entC sitABC mntH* mutant has been described previously.<sup>3</sup>

Reporter strains carried low-copy episomal pSC101 derivatives (Table S3) with constitutive expression of *mCherry*, *mtagbfp2*,<sup>96</sup> or *timer*<sup>pac76</sup> from the *P*<sub>ybaJ</sub> promoter<sup>97</sup> (chromosomal coordinates in SL1344 GenBank FQ312003.1: 528,632-528,081), and fusions of promoters *P*<sub>mgtCQBRUCigR</sub> (3,987,050-3,986,453) or *P*<sub>ryhB-2</sub> (1,309,793-1,310,035) driving expression of *gfp-ova* coding for a degradable variant of the green fluorescent protein GFP.mut2<sup>50,98</sup> or expression of *mtagbfp2-ova*. The conditional elimination construct combined a stringent ON/OFF module from plasmid pS238D.M<sup>74</sup> with *P*<sub>ryhB-2</sub> and the *doc* toxin gene of phage P1 from plasmid pAH154_doc_v2.<sup>99</sup> All plasmids were constructed using Gibson assembly<sup>100</sup> using primers listed in Table S2.

Bacteria were grown in Lennox lysogeny broth (LB) or in MES-MM (100 mM MES-KOH pH 5.5, 0.4% glycerol, 15 mM NH<sub>4</sub>Cl, 1.5 mM K<sub>2</sub>SO<sub>4</sub>, 3 mM KH<sub>2</sub>PO<sub>4</sub>) containing 90 mg/L streptomycin and 50 mg/L kanamycin. For metal depletion, MES-MM was treated with 5g/100 mL Chelex 100 for 4h. Chelex 100 was removed by filtering and the metal-depleted medium was stored in plastic bottles. Immediately before the experiments, metals were added back as needed (2 µM FeCl<sub>3</sub>, 1 µM ZnCl<sub>2</sub>, 0.5 µM MnCl<sub>2</sub>, 0.2 mM MgSO<sub>4</sub>, 50 µM CaCl<sub>2</sub>, and 0.25 µM CuCl).

In vitro growth in 96-well plates (Greiner, PS flat-bottomed microplate) at 37°C with shaking was monitored by OD<sub>600</sub> measurements at 15 min intervals in an Epoch2 microplate reader (BioTek).

## METHOD DETAILS

### Mouse infections

BALB/cAnNCrl, C57BL/6J, or C57BL/6J *Slc11A1*<sup>r/r</sup><sup>3</sup> mice were infected by tail-vein injection of 100 µL phosphate-buffered saline containing 500-5,000 CFU *Salmonella* grown to late-log phase in Lennox LB containing 10 mM MgCl<sub>2</sub> (which increased consistency across experiments). For some experiments in C57BL/6J *Slc11A1*<sup>r</sup> mice, we used a high infection dose of ~40,000 CFU. The inoculum size was determined by plating for each infection. We used both female and male mice. Mice were scored daily for disease signs (unprovoked behavior: normal – 0, minor changes – 1, less mobile and isolated – 2, restless and very still – 3; provoked behavior: responsive & alert – 0, unresponsive & not alert – 3; physical appearance: normal – 0, lack of grooming – 1, piloerection – 2, hunched up – 3, eyes half closed – 4; clinical signs: normal respiratory rate – 0, slight changes – 1, decreased rate and abdominal breathing – 2, marked abdominal breathing & cyanosis – 3; hydration status: normal – 0, dehydrated – 5; strength (grip test): grab and pull – 0, grab but no pull – 2, neither grab nor pull – 4).

Four days (BALB/c, C57BL/6, high-dose infection of C57BL/6 *Slc11A1*<sup>r</sup>) or six days (low-dose infection of C57BL/6 *Slc11A1*<sup>r</sup>) post-infection, the mice were euthanized with CO<sub>2</sub> and the spleen and the liver were prepared. *Salmonella* load was determined by plating on lysogeny broth agar. Fitness was calculated as log<sub>2</sub>(FI) with FI corresponding to the fold increase starting from the estimated initial spleen or liver colonization (around 20% of the total inoculum<sup>85</sup>) to the final organ load. The fitness value of co-administered wild-type *Salmonella* was set to 1 for determining the relative fitness of the mutant. We also determined the more commonly used read-out "competitive index" (CI) by dividing the output ratio (mutant/wild-type) by the inoculum ratio (mutant/wild-type). In contrast to relative fitness, CI values increasingly diverge from 1 with ongoing division. Because *Salmonella* divided more slowly in C57BL/6 *Slc11A1*<sup>r</sup> mice compared to C57BL/6 mice<sup>3</sup> (Figure 4E), CI values underestimated the mutant phenotypes in C57BL/6 *Slc11A1*<sup>r</sup> mice.

### Flow cytometry

The spleen and liver were each homogenized in 10 mL ice-cold phosphate-buffered saline containing 0.2% Triton X-100. All samples were kept on ice until and during analysis. Large host cell fragments were removed by repeated centrifugations at 100xg for 10 min at
4°C. Relevant spectral parameters were recorded in a FACS Fortessa II (Becton, Dickinson) with 'low' speed, using a threshold on sideward scatter (SSC) to exclude electronic noise. We used the following channels: mTagBFP2, excitation 405 nm, emission 460-480 nm ("blue"); GFP and green TIMERbac component, excitation 488 nm, emission 502-525 nm ("green"); mCherry and red TIMERbac component, excitation 561 nm, emission 595-664 nm ("red") (with 633 nm laser switched off); yellow and orange autofluorescence, excitation 488 nm, emission 533-551 nm, 573-613 nm; orange autofluorescence, excitation 445 nm, emission 573-613 nm. All parameters were measured as 'height'. Data were processed with FlowJo 10.6.1 and MATLAB R2020b. TIMERbac fluorescence log color-ratios were converted into *Salmonella* division rates based on previously established calibration data.⁷⁶

Fluorescent *Salmonella* cells were purified from spleen homogenates using a FACSAria Fusion cell sorter (BD Biosciences) using the following channels: mTagBFP2, excitation 405 nm, emission 425-475 nm; green TIMERbac component, excitation 488 nm, emission 502-525 nm; red TIMERbac component, excitation 561 nm, emission 595-664 nm (with 633 nm laser switched off). Sorted *Salmonella* cells were collected by centrifugation for 10 min at 21,300xg at 4°C. The supernatant was removed and an aliquot was plated for enumerating cell loss in the supernatant. These data were subtracted from plating results for an aliquot of sorted cells taken prior to centrifugation, in order to calculate the cell numbers in the pellet. The pellets were stored at -80°C.

## Proteomics

Sorted *Salmonella* cells (1 to 6.5 Mio per sample) were resuspended in lysis buffer (5% sodium dodecyl sulfate, 10 mM tris(2-carboxyethyl) phosphine, 100 mM triethyloammonium bicarbonate) and incubated at 95°C for 10 min. Samples were then sonicated with 20 cycles On/Off cycles (30 s, 30 s) using a Bioruptor (Diagenode). The protein content was determined by tryptophan-based fluorescence assay (excitation at 278 nm, emission 354 nm; Infinite M Plex, Tecan). Samples were alkylated by addition of 20 mM iodoacetamide and incubation at 25°C for 30 min with gentle shaking. Material containing 20 µg protein was processed using S-Trap (ProtiFi). Peptide concentration was determined using a UV-based assay (Infinite M Nano, Tecan).

Peptides (0.2 ng per sample) were separated on a Dionex UltiMate 3000 system (ThermoFisher Scientific) coupled online to an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific) using ID capillary columns (20 cm long, diameter 75 µm) packed in-house with 1.9 µm Reprosil-Pur C18 beads (Dr. Maisch, Ammerbuch, Germany). The column temperature was maintained at 50 °C using an integrated column oven interfaced online with the mass spectrometer. Formic acid (FA) 0.1% was used to buffer the pH in the two running buffers used. The total gradient time was 60 min and went from 2% to 12% acetonitrile (ACN) in 5 min, followed by 45 min to 35%, and 10 min at 50%. This was followed by a washout by 95% ACN, which was kept for 20 min, followed by re-equilibration with 0.1% FA buffer. Flow rate was kept at 300 nL/min. Spray voltage was set to 2,500 V, funnel RF level at 40, and heated capillary at 275 °C. For data-independent acquisition full MS resolution was set to 120,000 and full MS normalized AGC target was 300% with an IT of 45 ms. Mass range was set to 350-1400. Normalized AGC target value for fragment spectra was set at 1000%. A total of 63 windows of 9 Da were used with an overlap of 1 Da. Resolution was set to 15,000 and IT to 22 ms. Normalized CE was set at 28%. All data were acquired in centroid mode using positive polarity and advanced peak determination was set to on.

For data processing and protein identification, raw data were imported into SpectroNaut (16.1.220730.53000, Biognosys) and analyzed with directDIA. Searches were carried out against a fasta file including the proteomes of *Salmonella enterica* serovar Typhimurium SL1344 and *Mus musculus* as well as common human contaminants. Cysteine carbamidomethylation was set as fixed modification. Methionine oxidation, methionine excision at the N terminus were selected as variable modifications. Results were filtered for a 1% false discovery rate (FDR) on spectrum, peptide, and protein levels. Mass spectrometry data have been deposited to the ProteomeXchange Consortium via the MassIVE (https://massive.ucsd.edu/) partner repository with the data set identifier MSV000094024. Proteomics data analysis was performed with Perseus (1.6.14.0). Protein abundance was calculated using protein group quantities (PG quantity) based on the "total protein approach" concept.⁹² The calculated false discovery rate q was based on data permutations and significance-analysis-of-microarrays statistics¹⁰¹ using a s0 value of 0.1.

## Immunohistochemistry and confocal microscopy

Infected mice were anesthetized with 100 mg/kg ketamine and 16 mg/kg xylazine. Mice were transcardially perfused with 15 ml ice-cold PB buffer (0.1 M phosphate buffer pH 7.2 without NaCl) containing 10 U/ml heparin (Sigma), followed by 35 ml 4% paraformaldehyde (PFA) in PB with heparin.¹⁰² Perfusion-fixed spleen was prepared, cut into four pieces, and post-fixed in 4% PFA in PB overnight.

Samples were washed thrice in PB for 15 min at 4°C and embedded in 4% ROTI®Garose with low melting and gelling temperature (Roth) in PB buffer.¹⁰² Spleen sections with 50 µm thickness were cut using a Compresstome (Precisionary). To reduce autofluorescence⁵⁹ and for long-term storage, the sections were put in ice-cold cryoprotectant (300 g sucrose, 10 g polyvinyl-pyrrolidone 40, 400 ml ethylene glycol, 400 ml PB, after dissolving add PB to 1 l).¹⁰³ One hour after sections had sunken to the bottom of the tube, the tube was placed for at least seven days in a freezer at -20°C.

Sections were warmed to room temperature and washed for 5 min with TBS (20 mM Tris base, 150 mM NaCl, adjusted to pH 7.4 with HCl). For antigen retrieval, sections were incubated in pre-warmed 10 mM sodium citrate pH 8.5 at 37°C for 45 min. Sections were washed with TBST (0.1% Triton X-100 in TBS), blocked for 1 h with blocking buffer (10 mg/mL BSA, 2% mouse serum in TBST), and washed again in TBST. Sections were incubated with 10 µg/mL biotinylated F4/80 (clone: A3-1, AbD Serotec MCA497B), 4 µg/mL anti-CD107a (LAMP-1)-BV421 (clone: 1D4B, Biolegend 121617), and 4 µg/mL anti-CD107b (LAMP-2)-BV421 (clone: ABL-93, BD 564249) in blocking buffer at 4°C overnight, washed in TBST, and stained with 10 µg/mL Streptavidin-Alexa 647 (ThermoFischer S21374) in TBST for 1 h at room temperature. Sections were washed in TBS and mounted in mounting medium (DAKO).
Imaging was done with a SpinSR spinning-disk confocal super-resolution microscope (Olympus) equipped with a GFP narrow band emission filter (500-520 nm) using the 60x objective with additional 3.2x magnification in Super Resolution mode (back-projected pinhole radius 130 nm, distance 2.53 µm). Images were deconvolved with Huygens Professional (https://svi.nl/Huygens-Professional) with the “standard” profile and default settings from the MOSAIC file. Images were analyzed with ImageJ 1.53q.<sup>90</sup>

## Serial blockface scanning electron microscopy

Samples were prepared according to the National Center for Microscopy and Imaging Research protocol, University of California, San Diego (https://www.protocols.io/view/preparation-of-biological-tissues-for-serial-block-36wgq7je5vk5/v2). Infected mice were anesthetized with 100 mg/kg ketamine and 16 mg/kg xylazine, and were perfusion-fixed with 150 mM cacodylate buffer pH 7.4 containing 2.5% glutaraldehyde, 2% paraformaldehyde with 2 mM calcium chloride. Spleen was prepared and post-fixed for 3h in the perfusion solution. Spleen pieces were cut with a vibratome in 50 µm thick sections. The sections were washed 5 times in cold cacodylate buffer containing 2 mM calcium chloride and stained for 1 h on ice in cacodylate buffer containing 2 mM calcium chloride, 1.5% potassium ferrocyanide, and 2% osmium tetroxide. The sections were washed 5 times in distilled water, treated with 1% thiocarbohydrazide for 20 min at room temperature, and washed 5 times in distilled water. Sections were then incubated with 2% osmium tetroxide for 30 min at room temperature, washed 5 times in distilled water, and stained overnight with 1% uranyl acetate at 4°C. After washing 5 times in distilled water, sections were treated with Walton's lead aspartate for 30 min at 60°C. Sections were washed 5 times in distilled water and dehydrated in raising ethanol concentrations followed by anhydrous ice-cold acetone. Sections were then embedded in Durcupan ACM resin (Sigma Aldrich), Resin blocks were trimmed to an area of ~1 mm² and were imaged using a serial block-face scanning electron microscope (Merlin 3View; Zeiss) with an imaging area of 830 x 830 µm², a pixel size of 10 x 10 nm² and 300 to 700 slices of ~70 nm thickness. Image stacks were aligned with ImageJ 1.53q⁹⁰ using the plugin MultiStack-Reg.¹⁰⁴ Image analysis and 3D reconstruction was done with 3dmod 4.12.32.¹⁰⁵

## QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical tests were performed with GraphPad Prism 9.3.1 as indicated in the figure legends. The respective comparisons are denoted in the Figure panels. We log-transformed CFU data to approximate normal distributions.⁵³ Comparison of two groups were done by t-test. Matched measurements from the same infected mouse (such as subsets with differential promoter activity, or mutant and wild-type Salmonella loads) were analyzed by paired tests. Comparisons of three or more groups were done by one-way ANOVA. The impact of two factors was analyzed by two-way ANOVA. Proteome data were analyzed with Perseus (1.6.14.0).⁹² For these data, the false-discovery rate q was calculated based on data permutations and significance-analysis-of-microarrays statistics¹⁰¹ using a s0 value of 0.1. A q-value of 0.05 indicates that on average 5 out of 100 data points with such a q-value are false discoveries. All tests were two-tailed. We deliberately avoided specifying a particular significance threshold for *P*-values (e.g., $\alpha$ = 0.05, 0.01, or 0.001) to avoid excessive emphasis on arbitrary cut-offs.⁹¹