Volume 19, Issue 6, Pages 814-825 (June 2016) Salmonella Mitigates Oxidative Stress and Thrives in the Inflamed Gut by Evading Calprotectin-Mediated Manganese Sequestration  Vladimir E. Diaz-Ochoa, Diana Lam, Carlin S. Lee, Suzi Klaus, Judith Behnsen, Janet Z. Liu, Nicholas Chim, Sean-Paul Nuccio, Subodh G. Rathi, Jennifer R. Mastroianni, Robert A. Edwards, Christina M. Jacobo, Mauro Cerasi, Andrea Battistoni, André J. Ouellette, Celia W. Goulding, Walter J. Chazin, Eric P. Skaar, Manuela Raffatellu  Cell Host & Microbe  Volume 19, Issue 6, Pages 814-825 (June 2016) DOI: 10.1016/j.chom.2016.05.005 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2016 19, 814-825DOI: (10.1016/j.chom.2016.05.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 STM Uses Mn Transporters to Evade CP-Mediated Growth Inhibition In Vitro (A–D) STM strains grown in LB + (A) WT CP, (B) site II CP mutant, (C) site I CP mutant, or (D) site I/site II CP double mutant; also, when indicated (+Mn) + 50 μM MnCl2. CFUs were determined by plating dilutions on selective media. Bars denote mean CFU/ml culture of three or more biological replicates ± SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01. ND, not detected. Dashed line denotes limit of detection. See also Figures S1, S2, and S3 and Tables S1 and S2. Cell Host & Microbe 2016 19, 814-825DOI: (10.1016/j.chom.2016.05.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Mn Transporters Enhance STM’s Competitive Advantage in the Inflamed Gut (A) C57BL/6 mice coinfected 1:1 with STM WT and indicated mutant (n ≥ 7/group). Competitive index (C.I.) = output ratio (CFU STM WT/mutant) divided by input ratio. Bars denote mean C.I. ± SEM. (B) CFUs in colon contents of individual mice from (A). Bars denote geometric mean. (C) ICP-MS measurement of fecal Mn levels in mock (n = 4) or STM-infected (n = 4) mice 96 hr p.i. Bars denote geometric mean ± SD. (D) Mice coinfected 1:1 with commensal E. coli JB2 and STM strains. Bars denote mean C.I. (CFU STM/E. coli) ± SEM. (E) CFUs in colon contents of individual mice from (D). ∗p ≤ 0.05, ∗∗p ≤ 0.01. NS, not significant. See also Figure S4. Cell Host & Microbe 2016 19, 814-825DOI: (10.1016/j.chom.2016.05.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Role of Mn Uptake in Mice Lacking CP or IL-22 during STM Infection (A–C, E, and G) S100a9+/+ (white bars) or S100a9−/− (black bars) mice coinfected 1:1 with STM WT and indicated mutant (n ≥ 5/group). (D, F, and H) Il22+/+ (white bars) or Il22−/− (gray bars) mice coinfected 1:1 with indicated STM strains (n ≥ 7/group). (A–D) Bars denote mean C.I. (CFU STM WT/mutant) ± SEM. ∗p ≤ 0.05. (E and F) Transcript levels of Il17a, Il22, and Cxcl1 relative to uninfected WT littermates. Bars denote mean of four or more replicates ± SEM. (G and H) Immunoblots of cecal tissue extract from STM coinfected mice. MPO, myeloperoxidase. See also Figure S5 and Table S3. Cell Host & Microbe 2016 19, 814-825DOI: (10.1016/j.chom.2016.05.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Mn Sequestration by CP Is Less Effective in Anaerobiosis (A–C) STM strains grown anaerobically in (A) LB, (B) LB + 125 μg CP/ml, or (C) LB + 250 μg CP/ml. Aerobic growth (as in Figure 1A) shown for comparison. Bars denote mean CFU/ml culture of three or more biological replicates ± SEM. ∗∗p ≤ 0.01. Dashed line denotes limit of detection. Cell Host & Microbe 2016 19, 814-825DOI: (10.1016/j.chom.2016.05.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 STM Uses SodA to Resist CP-Mediated Growth Inhibition (A–C) STM strains grown aerobically in (A) LB, (B) LB + 250 μg CP/ml and when indicated (+Mn) + 50 μM MnCl2, or (C) LB + 500 μg CP/ml. (D–F) STM strains grown aerobically in (D) LB, (E) LB + 250 μg site I CP mutant/ml, or (F) LB + 500 μg site I CP mutant/ml. Bars denote mean CFU/ml culture of three or more biological replicates ± SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01. Dashed line denotes limit of detection. See also Figure S3. Cell Host & Microbe 2016 19, 814-825DOI: (10.1016/j.chom.2016.05.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Mn-Dependent Antioxidant Enzymes SodA and KatN Promote STM Growth in the Inflamed Gut (A, C, and G) Mice coinfected 1:1 with indicated STM strains (n ≥ 4/group). Bars denote mean C.I. ± SEM. For (A) and (G), C.I. = CFU STM WT/mutant; for (C), mutant/sitA mntH zupT. (B, D, and H) CFUs in colon contents of individual mice (B) from (A), (D) from (C), or (H) from (G). Horizontal bars denote geometric mean. (E) UV/vis absorption spectra of SodA WT and E170A reveal Mn(III) and Fe(III) coordination, respectively. (F) Growth in M9 by STM strains. Data are mean of three replicates ± SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01. See also Figure S6. Cell Host & Microbe 2016 19, 814-825DOI: (10.1016/j.chom.2016.05.005) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Mn Helps STM Resist Neutrophil-Sourced ROS and CP in the Inflamed Gut (A–C) C57BL/6 mice treated (Tx) with α-Ly-6G antibody to deplete neutrophils were coinfected 1:1 with indicated STM strains. (A, D, and G) Bars denote mean C.I. (CFU STM WT/mutant) ± SEM. (B, E, and H) CFUs in colon contents of individual mice (B) from (A), (E) from (D), and (H) from (G). (D–F) Cybb+/+ mice (white bars) and Cybb−/− mice (gray bars) coinfected as in (A). (C and F) Immunoblots of cecal tissue extract from STM coinfected mice. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗∗p ≤ 0.0001. See also Figure S7. Cell Host & Microbe 2016 19, 814-825DOI: (10.1016/j.chom.2016.05.005) Copyright © 2016 Elsevier Inc. Terms and Conditions