A Lipoylated Metabolic Protein Released by Staphylococcus aureus Suppresses Macrophage Activation  James P. Grayczyk, Cameron J. Harvey, Irina Laczkovich, Francis Alonzo  Cell Host & Microbe  Volume 22, Issue 5, Pages 678-687.e9 (November 2017) DOI: 10.1016/j.chom.2017.09.004 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2017 22, 678-687. e9DOI: (10. 1016/j. chom. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 LipA Suppresses TLR2-Dependent Activation of Macrophages (A) Setup of screen used to identify macrophage immunomodulatory factors. Transposon mutants were grown to an OD600 of ∼1.2, followed by addition of cell-free supernatants to BMMs for 24 hr. BMM supernatants were collected and assessed for cytokine and chemokine secretion using cytometric bead array (CBA). (B) Relative abundance of IL-6, TNF, CCL3, and CCL4 produced by macrophages after addition of cell-free supernatants from JE2 (WT), NE1757 (lspA::erm), and NE264 (lipA::erm). (C–E) Growth curves of WT, ΔlipA, or ΔlipA+lipA in TSB (C) or RPMI (D) medium with and without 25 nM lipoic acid. Coomassie-stained SDS-PAGE gels of TCA precipitated exoproteins after growth in TSB (C) or RPMI+BCFA (E). (F) IL-6, TNF, CCL3, and CCL4 production (pg/mL) by BMMs after addition of supernatant from WT, ΔlipA, or ΔlipA+lipA grown in RPMI+BCFA. Data shown are from one of at least three experiments conducted in triplicate. Means ± SD are shown (n = 3). ∗p < 0.05; ∗∗p < 0.01, by one-way ANOVA with Bonferroni-Sidak post-test. (G) BMM production of IL-6 and TNF (pg/mL) after addition of cell-free supernatant from WT, ΔlipA, or ΔlipA+lipA to WT, TLR2−/−, TLR4−/−, or MyD88−/− BMMs. Data shown are from one of at least three experiments conducted in triplicate. (−), media alone. Means ± SD are shown (n = 3). ∗∗p < 0.01; ∗∗∗p < 0.001, by one-way ANOVA with Bonferroni-Sidak post-test. (H) NFκB activation after treatment of RAW 264.7 cells, containing an NFκB-inducible secreted embryonic alkaline phosphatase reporter, with cell-free supernatant from WT, ΔlipA, or ΔlipA+lipA. Relative reporter activity (absorbance 450 nm) from one of two experiments conducted in triplicate is shown. Means ± SD are shown. ∗∗p < 0.01; ∗∗∗∗p < 0.0001, by one-way ANOVA with Bonferroni-Sidak post-test. (I) Whole-cell lysates or TCA-precipitated exoproteins from the indicated S. aureus strains collected after growth in RPMI+BCFA followed by immunoblotting for lipoic acid-containing proteins. See also Figure S1 and Table S1. Cell Host & Microbe 2017 22, 678-687.e9DOI: (10.1016/j.chom.2017.09.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 The Release of Lipoyl-E2-PDH by S. aureus Requires Lipoic Acid Synthesis and Likely Involves Atl (A) Model of the S. aureus lipoic acid synthesis and salvage pathways. ACP, acyl carrier protein; GcvH, H protein of the glycine cleavage system; E2s, E2 subunits of PDH, OGDH, and BCODH; LipM, octanoyl transferase; LipA, lipoyl synthetase; LipL, lipoyl transferase; LplA1/A2, lipoate protein ligases. (B and E) Whole-cell lysates or TCA-precipitated exoproteins from the indicated S. aureus lipoic acid biosynthesis and salvage mutants (B) or atl and secA2 mutants (E) collected after growth in RPMI+BCFA followed by immunoblotting for lipoic acid-containing proteins. (C) IL-6, TNF, CCL3, and CCL4 production (pg/mL) by BMM after addition of supernatant from the indicated strains. Data shown are from one of at least three experiments conducted in triplicate. Means ± SD are shown (n = 3). ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, by one-way ANOVA with Bonferroni-Sidak post-test. (D) IL-6 and TNF production (pg/mL) by BMM after addition of T3, T5, or T9 supernatant from the indicated strains. Data shown are from one of at least three experiments conducted in triplicate. (−), media alone. Means ± SD are shown (n = 3). ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, by one-way ANOVA with Bonferroni-Sidak post-test. NS, not significant. See also Figure S2. Cell Host & Microbe 2017 22, 678-687.e9DOI: (10.1016/j.chom.2017.09.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Lipoyl-E2-PDH Blunts TLR1/2-Dependent Activation of Macrophages (A and B) IL-6 and TNF production (pg/mL) after addition of ΔlipA supernatant (A) or 3 ng/mL Pam2CKS4 (Pam2) and 30 ng/mL Pam3CSK4 (Pam3) (B) to BMMs in the presence of free lipoic acid (3 or 0.3 mM). (−), media alone. Data shown are from one of at least three experiments conducted in triplicate. Means ± SD are shown (n = 3). ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, by one-way ANOVA with Bonferroni-Sidak post-test. (C and D) IL-6 and TNF production (pg/mL) after addition of 10 nM lipoyl-E2-PDH (LA-PDH) (C) or synthetic tripeptides DKLA and DKA (D) to BMMs in the presence of 1 ng/mL Pam2 or 3 ng/mL Pam3. Data shown are from one of at least three experiments conducted in triplicate. Means ± SD are shown (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, by one-way ANOVA with Bonferroni-Sidak post-test. (E) Coomassie-stained SDS-PAGE gel of 500 ng purified SitC. IL-6, TNF, CCL3, and CCL4 production (pg/mL) after addition of SitC (0.1 ng/mL for CCL3/CCL4 and 1.0 ng/mL for IL-6/TNF) to BMMs in the presence of 10 nM synthetic tripeptides DKLA and DKA. Data shown are from one of at least three experiments conducted in triplicate. Means ± SD are shown (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, by one-way ANOVA with Bonferroni-Sidak post-test. See also Figure S3. Cell Host & Microbe 2017 22, 678-687.e9DOI: (10.1016/j.chom.2017.09.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 LipA-Dependent Suppression of Macrophage Activation Correlates with S. aureus Virulence (A and B) Serum CCL3 and CCL4 levels (pg/mL) 16 (A) or 72 hr (B) post-infection. Means ± SD are shown (WT and ΔlipA, n = 10; ΔlipA+lipA, n = 7). ∗p < 0.05, by one-way ANOVA with Bonferroni-Sidak post-test. NS, not significant. (C and D) Pro-inflammatory macrophages in the peritoneal cavity at 16 (C) and 72 hr (D) post-infection. Macrophages were gated on CD11b+ F4/80+ Ly6G− cells followed by assessment of CCR5+ I-A/I-Ehi cells. Flow cytometry plots are representative of 4–8 animals per group. Scatterplots display percent CCR5+/I-A/I-Ehi cells within the CD11b+ F4/80+ Ly6G− gate. Bars display the median. ∗∗p < 0.01; ∗∗∗p < 0.001, by one-way ANOVA with Bonferroni-Sidak post-test. NS, not significant. (E and F) Bacterial burden (E) and medians of bacterial burden (F) in the peritoneal cavity and kidneys of mice 16 (WT, n = 17; ΔlipA, n = 19; ΔlipA+lipA, n = 12) and 72 hr (WT, n = 11; ΔlipA, n = 12; ΔlipA+lipA, n = 14) post-intraperitoneal infection. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001, by non-parametric one-way ANOVA (Kruskal-Wallis test) with Dunn’s post-test. Dashed lines, limit of detection. n = 7 and n = 6 in the 72 hr dataset are the number of animals with undetectable CFU (E). NS, not significant. (G) Survival/outgrowth of WT S. aureus after infecting WT (n = 8), ΔlipA (n = 8), or PBS (n = 8) elicited F4/80+ cells. ∗p < 0.05, ∗∗∗∗p < 0.0001, by two-way ANOVA with Tukey’s post-test. See also Figure S4. Cell Host & Microbe 2017 22, 678-687.e9DOI: (10.1016/j.chom.2017.09.004) Copyright © 2017 Elsevier Inc. Terms and Conditions