Ethics

All animal experiments were approved by the Institutional Animal Care and Use Committee (protocols #16-1119 and #22-0307). Procedures were performed according to all institutional policies, the Animal Welfare Act, NIH guidelines, and American Veterinary Medical Association guidelines on euthanasia. Adult (6–8-week-old) female C57BL/6 J and derivative mice, were maintained at Washington University School of Medicine animal facilities. 10BiT IL-10 reporter mice were maintained in a specific pathogen-free facility²¹.

Bacterial strains

Where indicated, S. pyogenes HSC5⁵⁵ and isogenic mutant ΔPdh⁵ were cultured in Todd Hewitt + 1% Yeast Extract (THY) broth⁵⁶ or C medium supplemented with 0.2% glucose⁵⁶. Quantitation of S. pyogenes CFUs utilized THY medium solidified by the addition of 1.4% agar⁵⁶, which was incubated at 37 °C anaerobically using a commercial atmospheric container (GasPak™ EZ, catalog #BD 260001). All cultures were seeded from overnight cultures in C medium to an initial OD₆₀₀ = 0.05, which were then incubated for the various times indicated in the text.

Mouse subcutaneous ulcer model of infection

Infection of 6–8-week-old C57BL/6 and 10BiT⁺ mice followed a well-established protocol⁵. As sex does not influence infection outcomes⁵⁷, only female mice were utilized in order to minimize the number of mice used⁵⁷. Briefly, mice received a subcutaneous injection of ~ 10⁷ CFU of the indicated bacterial strains into the thigh, and the areas of the resulting ulcers determined following 72 h of infection from analysis of digital images using ImageJ as described⁵⁷. Mice were euthanized by CO₂ with a flow rate of 30–70% of the chamber’s volume per minute, followed by cervical dislocation. Bacterial burdens in lesions were assessed by excision and homogenization of infected tissues and spot-plating aliquots of the tissue homogenate⁵⁸. CFU counts were normalized based on the entire infected tissue volume. Procedures of H8 treatment were shown in Fig. 6E.

Macrophage infection

RAW 264.7 macrophages were grown in Dulbecco’s Modified Eagles Medium (DMEM, Sigma) with 10% fetal bovine serum supplemented with antibiotics (1% Penicillin-Streptomycin) at 37 °C in an atmosphere of 5% CO₂. For infection, the bacterial inoculum was prepared as described above for murine infection, and after replacing media with antibiotic-free media, bacteria were added to macrophages cultured in 12-well dishes at an MOI of 10:1 which were then immediately subjected to centrifugation at 500 × g for 1 min. and placed in an incubator for 1 h. Wells were then washed twice with Dulbecco’s PBS, then DMEM containing antibiotics (100 μg/ml Gentamicin) was added for 5 minutes and washed 3 x with PBS before the cells were incubated for an additional 2–4 h in antibiotic-free DMEM. For supplementation, acetate/formate (Sigma-Aldrich, #32319, 71539) or acetylation inhibitors (TSA: Sigma-Aldrich, T1952; TMP195: Selleck, #S8502; Tub A: Selleck, #S8049; Ent: Selleck, #S1053; H8 is custom-synthesized using the formula from Fig. 6A^14,16,17) were added at the beginning of infection. Cells were harvested using a cell scraper and collected by centrifugation at 1000 × g for 5 min, were resuspended in 1 ml dH₂O at pH 11, and lysed using a cup sonicator for 20 sec. Bacteria in the resulting suspension were collected by centrifugation (6000 × g, 5 min) and resuspended in 100 μl of C-media for determination of CFUs. Net fold-change in CFU was determined by comparison to cells harvested after the addition of an antibiotic-containing medium.

Luminol assay

Bioluminescence images were captured using the In Vivo Imaging System (IVIS) Spectrum (Perkin Elmer, Santa Clara, CA, USA) and analyzed using IVIS imaging software (Perkin Elmer, version 2.6.1). Ten minutes before imaging, animals received an intraperitoneal (IP) injection of 200 mg/kg luminol sodium salt (Sigma-Aldrich, A4685).

Flow cytometry

To prepare single-cell suspensions, infected ulcer with minimal surrounding tissue were removed aseptically and placed in ice-cold PBS. Samples were mechanically dissociated using a GentleMACS^TM dissociator in C Tubes (Miltenyi Biotec, 130-096-334) according to the manufacturer’s protocol⁵⁹. The resulting cell suspension was filtered through a 40 µm cell strainer (Corning Falcon, 352340). For staining, all antibodies were used at a dilution of 1:200. Single-cell suspensions were preincubated with anti-CD16/CD32 Fc Block antibody (BioXCell, BE0307) in PBS for 10 min at RT before staining with the following antibodies. Antibodies were obtained from BioLegend and included: FITC anti-CD45.2 (109806), BV605 anti-MHC II (107639), APC anti-CD64 (139306), PB anti-Ly6C (128014), and BV510 anti-Thy1.1 (202535). The following anti-mouse antibodies were obtained from Tonbo Biosciences: PE anti-F4/80 (50-4801-U100), PercpCy5.5 anti-Ly6G (65-1276-U025), APC-Cy7 anti-CD11c (25-0114-U025), PE-Cy7 anti-CD11b (60-0112-U100). Alexa Fluor^TM 700 anti-CD3ε was from Invitrogen (56-0033-82). Validation information for these antibodies is available at vendor websites. Cells were stained for 20 min at 4 °C, washed, and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 min at 4 °C. Cell counts were determined by hemocytometer³⁶. Flow cytometry data were acquired on an LSR Fortessa cytometer (BD) and analyzed using FlowJo software (TreeStar, version 10.8.1). Gating strategies are depicted in Supplementary Fig. 1.

Transmission electron microscopy

For ultrastructural analyses, samples were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Ted Pella Inc, Redding, CA) in 100 mM sodium cacodylate buffer for 2 hr at room temperature and then overnight at 4 °C. Samples were washed in sodium cacodylate buffer and postfixed in 1% osmium tetroxide (Ted Pella Inc.) for 1 hr at room temperature. After three washes in dH₂O, samples were stained en bloc in 1% aqueous uranyl acetate (Electron Microscopy Sciences, Hatfield, PA) for 1 h. Samples were then rinsed in dH₂0, dehydrated in a graded series of ethanol, and embedded in Eponate 12 resin (Ted Pella Inc). Ultrathin sections were cut to a thickness of 95 nm with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA) equipped with an AMT 8-megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA).

Macrophage or neutrophil depletion

To deplete macrophages, mice were intraperitoneally injected with 1 mg liposomal clodronate or control liposomes (Fisher Scientific, NC1488571)⁶⁰. To deplete neutrophils, mice were intraperitoneally injected with 500 µg monoclonal anti-Ly6G (BioXCell, BE0075-1) or anti-Gr1 (BioXCell, BE0075) antibody, or rat IgG2a isotype control (BioXCell, BE0089) diluted in sterile PBS 18 h before infection⁶¹. Depletion efficiency was assessed by FACS as described in the text and Supplementary Fig. 4.

Western blot analyses

RAW 264.7 macrophages were infected for 4 hours before being collected by gentle scraping, along with uninfected controls. Total cell lysates were heated at 100 °C for 10 min, then centrifuged at 13,000 × g for 10 min at 4 °C. Equal amounts of protein (30 µg per lane) were mixed with Laemmli sample buffer containing β-mercaptoethanol, separated on 10% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) and incubated overnight at 4 °C with primary antibodies anti-H3K9 (Cell Signaling Technology, cat.#9649 T, 1:1000) or anti-H3 (Cell Signaling Technology, cat.#4499 T, 1:1000). Blots were washed in TBS-T and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000) for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescence (Thermo Fisher Scientific, cat.#32106) and imaged with a ChemiDoc MP System (Bio-Rad)⁶².

Cytokine quantification

At the 4 h time point, supernatants from infected and uninfected RAW 264.7 cells were harvested by gentle scrape and centrifuge at 1000 × g for 5 mins, and stored at − 80 °C. The following day, the concentration of IL-10 and TNFα in supernatants were determined by ELISA according to the manufacturer’s protocol (R&D Systems, cat.#DY417, #DY410). Concentrations were determined by comparison to a standard curve generated using purified proteins provided in the kit.

Measurement of SCFAs and Acetyl-CoA

Following 4 h of infection, RAW 264.7 cells were harvested by gentle scrape, subjected to centrifugation (1000 × g, 5 min), and the resulting supernatant fluids and cell pellets stored separately at − l80 °C. The concentrations of acetate (Sigma-Aldrich, MAK086), lactate (Sigma-Aldrich, MAK064), and formate (Sigma-Aldrich, MAK059) were then determined in cell supernatants. The concentration of acetyl-CoA from cell pellets was measured by a colorimetric assay using commercial kits (Sigma-Aldrich, MAK039) following the manufacturer’s protocol. SCFA and acetyl-CoA levels were compared to uninfected RAW 264.7 cells.

Fluorescent microscopy

Infected skin ulcers were fixed in 10% buffered formalin (Thermo Fisher Scientific) for 24 h at room temperature, followed by overnight storage in 70% ethanol at 4 °C. Samples were paraffin-embedded and sectioned (5 µm) by the Anatomic and Molecular Pathology Core Labs at Washington University. Sections were deparaffinized in xylene, rehydrated through graded ethanol, and subjected to antigen retrieval in citrate buffer (pH 6.0) at 95 °C for 20 min. After cooling, sections were blocked in 5% goat serum in PBS with 0.1% Tween-20 (PBS-T) for 1 h to prevent nonspecific binding. Primary antibodies diluted in PBS-T with 1% BSA were applied overnight at 4 °C. After washing, fluorescently labeled secondary antibodies (1:500 in PBS-T) were added for 1 h at room temperature. Nuclei were counterstained with DAPI, and sections were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific)⁶³. PE anti-F4/80 (Tonbo, 50-4801-U100), FITC anti-Streptococcus Group A (Invitrogen, PA1-73056), Alexa Fluor^TM 647 anti-Ly6G (BioLegend, 127609), CD163 (Invitrogen, #16646-1-AP), MDC (Invitrogen, #57226) antibodies were used. Fluorescent images were acquired using a Zeiss Axio Imager 2 microscope (Jena, Germany) equipped with a digital camera. Fluorescent intensity was measured with ImageJ software (https://imagej.nih.gov/ij/).

RNA sequencing

Total RNA from infected and treated RAW 264.7 cells (4-hpi) were isolated using a Direct-Zol RNA MiniPrep Plus kit (Zymo Research, cat.#R2071) per the manufacture’s protocol. RNA was quantified using a Qubit 2.0 Fluorometer, and integrity was checked on an Agilent TapeStation 4200. RNA sequencing libraries were prepared with the NEBNext Ultra II RNA Library Prep Kit for Illumina (cat.#E7760S). mRNAs were enriched using Oligo(dT) beads, fragmented, and converted into cDNA, which was end-repaired, adenylated, adapter-ligated, indexed, and enriched via PCR. Libraries were validated on a TapeStation, quantified with Qubit and qPCR, and sequenced on an Illumina HiSeq 4000 (2x150bp PE). Raw data were converted to fastq files and de-multiplexed using bcl2fastq (Illumina, v2.17) with one allowed index mismatch. Reads were trimmed with Trimmomatic (v0.36) and mapped to the Mus musculus genome (ENSEMBL) using a STAR aligner (v2.5.2b). Unique gene hit counts were generated with Subread (v1.5.2). Differential expression was analyzed with DESeq2 (v3.15) using Wald tests, identifying genes with adjusted P-values < 0.05 and absolute log2 fold-changes > 1. Pathway enrichment analysis was performed using Reactome, with P-values corrected for multiple testing via the Benjamini–Hochberg procedure⁵.

Single cell RNA-seq

DNA was prepared after the Gel Beads in Emulsion (GEM) generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11–16 cycles before being size-selected using SPRIselect beads (Beckman Colter, B23319). Samples were then run on a Bioanalyzer (Agilent, #2100) to determine the cDNA concentration. Gene Expression libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 5’ Reagent Kits User Guide (v2 Chemistry Dual Index), with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 5’ Kit v2, 16 rxns (PN-1000263), Chromium Next GEM Chip K Single Cell Kit, 48 rxns (PN-1000286), Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used. The concentration of each library was determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer’s protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 151 × 10 × 10 × 151 sequencing recipe according to the manufacturer’s protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression library. Transcript alignment, counting, and inter-library normalization were performed using the Cell Ranger pipeline (10x Genomics, default settings, Version 2.1.1, GRCh38 reference). Data were analyzed using the Biomage online tool (https://www.biomage.net/).

Statistical analyses

Data are derived from at least 3 independent experiments, with values presented representing the mean ± SEM for each group. The difference between experimental groups were tested for significance using a non-parametric statistical test (two-tailed Mann-Whitney U test) available in GraphPad Prism software where *, **, and *** indicates p < 0.05, < 0.01, and < 0.001, respectively. For all tests, the null hypothesis was rejected for p > 0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.