Reagents The following reagents: cytochalasin D, staurosporine (STS), chloroquine wortmannin, necrostatin-1 (Nec-1), thapsigargin (TG), dithiothreitol (DTT) Dulbecco’s Modified Eagles Medium (DMEM), E-64, and all antibiotics were purchased from Sigma-Aldrich. Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD) was purchased from R&D systems, TNFα from Peprotech, and fetal calf serum (FCS), and nutrition medium F-12 (HAM) from Biological Industries. If not otherwise specified all other reagents were of the highest purity available.

In Vivo sil Activation Eight mm punch biopsies (Acuderm), taken from the injection site from euthanized mice, were immersed in 4% paraformaldehyde solution for 4 hr at room temperature (RT). Then, the samples were transferred to 30% sucrose solution for 16 hr at 4°C, embedded in Tissue-Tek OCT Compound (Sakura) and kept at −20°C. Transverse sections 20 μm thick were cut using a Leica CM3000 cryostat and thaw-mounted on Superfrost∗ Plus and ColorFrost∗ Plus Microscope slides (Thermo). The preparations were stained with 2 μg/ml DAPI (Invitrogen) and 5 μg/ml rhodamine-phalloidin (Invitrogen) for 0.5 hr, mounted with Permafluor solution (Thermo) and analyzed using a fluorescent confocal microscope (Zeiss LSM 710). The images were analyzed using ZEN 2009 Light Edition software, while keeping the intensity of GFP (green) at an equal level for all examined samples. Concurrently, biopsies were homogenized in 0.5 ml PBS for 30 s on ice, using Polytron PT2100 (Kinematica). Two samples from each homogenate of 100 μl were transferred to a 96-well plate and the luminescence of firefly luciferase (Luc) (Promega) was determined with Glomax multi-detection system (Promega) after addition of 50 μl of luciferin (Promega). The amount of bacteria present in the corresponding biopsies was enumerated and Luc activity was normalized according to the colony forming units (CFU) determinations.

Mammalian Cells The human HeLa epithelial cell line (HeLa ATCC Catalog No. CCL-2), the mouse embryonic fibroblasts (MEF), and Atg5−/− MEF, the mouse subcutaneous fibroblast L cell line clone 929 (ATCC Catalog No. CCL-1) and the mouse leukemic monocytes/macrophages Raw 264.7 cells (ATCC Catalog No. TIB-71), unless otherwise specified, were cultured in DMEM, containing 10% (v/v) FCS (termed here DMEM medium). The Lung alveolar adenocarcinoma A549 cells (ATCC Catalog No. CCL-185) were cultured in F-12 (HAM) supplemented with 10% FCS (v/v) [F-12 (HAM) medium]. All cell lines were grown at 37°C in an atmosphere containing 5% CO 2 .

Immunofluorescence of Mammalian Cells MEF cells were fixed with 4% paraformaldehyde in PBS at RT, and then permeabilized with 0.1% saponin (Sigma). Cells were washed with PBS and blocked for 0.5 hr at RT with Image-iT FX Signal Enhancer (Invitrogen). Anti-Streptococcus pyogenes group A carbohydrate antibody (Abcam) was incubated with the permeabilized cell preparation (1: 250) at 4°C overnight and unbound antibodies were washed away. A secondary donkey anti-goat IgG Alexa Fluor 647 (Invitrogen) antibody was added (1 μg/ml) for 1 hr at RT, washed away and cells were stained with Alexa Fluor 568 phalloidin and NucBlue (Invitrogen), according to manufacturer’s instructions. Immunofluorescently stained cells were analyzed using the laser scanner of the Nikon A1 confocal microscope. The series of z stack images were generated with a 0.8 μm step size, using a Plan Apo VC 60X/1.4 oil objective.

Lysis of HeLa Cells HeLa cells were cultured in a 24-well plate in DMEM medium. The cells were washed with cold PBS, scraped into fresh cold DMEM medium and adjusted to 2.5 × 105 cells/ml. Lysis was performed on ice by two cycles of sonication, each of 15 s (12 microns peak-to-peak), using the exponential probe of Soniprep 150 Plus (MSE). Complete lysis was verified by microscopic visualization.

LDH Activity Assay MEF cells were grown in DMEM medium that lacks phenol red indicator. Cells were infected with JS95 ATG pP4-gfp as described in the text. At desired time points, supernatants were withdrawn and the LDH activity was determined using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega), according to the manufacturer’s instructions.

Bacterial Strains and Culturing (The bacterial strains used in this study are listed in Table S1 ). For cloning, we used Escherichia coli strains JM109 and SCS110, which were cultured in Luria-Bertani broth (LB), (Becton, Dickinson) at 37°C with agitation. GAS and GGS were cultured either in Todd-Hewitt broth (Becton, Dickinson) supplemented with 0.2% yeast extract (Difco, THY) or in DMEM medium containing various combinations of the following AA (proline, 35 mg/l; aspartic acid, 13 mg/l; glutamic acid, 15 mg/l; alanine, 9 mg/l; ASN, 15 mg/l), at 37°C in sealed tubes, or in 24-well plates without agitation at 37°C in a 5% CO 2 incubator. To produce solid LB and THY, Bacto Agar (Becton, Dickinson) was added to a final concentration of 1.4%. When necessary, antibiotics were added at the following concentrations: for GAS and GGS: 250 μg/ml, kanamycin (Km); 50 μg/ml, spectinomycin (Spec); 1 μg/ml, erythromycin (Erm); for E. coli: 100 μg/ml, ampicillin (Amp); 50 μg/ml, Spec; 750 μg/ml, Erm; 50 μg/ml, Km.

Determination of SLS and SLO Hemolytic Activities For determination of SLS activity, tested bacteria were grown in THY to an early log phase (OD 600 = 0.3), washed with PBS, concentrated to OD 600 = 8 (109 CFU/ml), diluted 4,000-fold into 24-well plates containing DMEM medium supplemented with 5AA and then grown at 37°C in an atmosphere containing 5% CO 2 . At different time points, supernatants were removed centrifuged to eliminate bacteria and mixed with an equal volume of 2.5% (v/v) defibrinated sheep erythrocytes in PBS. The mixture was incubated for 1 hr at 37°C, centrifuged (3000 x g for 5 min) and readings of the supernatants at OD 540 were determined. Values of complete hemolysis representing 100% were determined by lysing the equivalent erythrocytes suspension with 1% Triton X-100. The addition of trypan blue (13 μg/ml) completely inhibited hemolysis, indicating that under the aerobic conditions the contribution of SLO (which is oxygen-sensitive) to hemolytic activity was negligible. For determination of SLO-mediated hemolytic activity, tested bacteria were grown overnight at 37°C anaerobically, in a 24-well plate containing DMEM supplemented with 5AA, 10 μM of the protease inhibitor E-64, and when necessary appropriate antibiotics. The overnight cultures were centrifuged and the supernatants of 0.5 ml were reduced by treatment with 20 mM L-cysteine for 10 min at RT. An equal volume of 4% (v/v) defibrinated sheep erythrocytes in PBS was added, and the samples were incubated at 37°C for 1 hr. After centrifugation, OD 540 readings were recorded, and 100% hemolysis was determined as described above.

Chromatography-Tandem Mass Spectrometry ASN was depleted from the DMEM medium by treatment with ASNase (described in Experimental Procedures) and treated medium was filtered through a 3,000 NMWL membrane (Amicon, MILLIPORE). The filtered solution was subjected to Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) on an Accela HPLC linked to a TSQ Quantum Access Max mass spectrometer (Thermo Scientific) via a heated electrospray ionization (H-ESI) interface. 2 O were purchased from Biolab Ltd. whereas the ion-pairing agent HFBA (Heptafluorobutyric acid) was purchased from Sigma-Aldrich. The chromatographic separation was performed using a gradient program at a flow rate of 1 ml/min over a total run time of 8 min. An outline of the mobile phase gradient program is summarized in HPLC conditions: Separations were performed on a Hypersil GOLD C8 (Thermo Scientifics), 5 μm, 150 × 4.6 mm column. For this test LC/MS-grade acetonitrile, methanol and HO were purchased from Biolab Ltd. whereas the ion-pairing agent HFBA (Heptafluorobutyric acid) was purchased from Sigma-Aldrich. The chromatographic separation was performed using a gradient program at a flow rate of 1 ml/min over a total run time of 8 min. An outline of the mobile phase gradient program is summarized in Table S6 . Solvent A is composed of an aqueous solution containing 0.1% formic acid and 0.3% HFBA. Solvent B is composed of 0.1% formic acid in methanol. The column temperature was set to 35°C, the autosampler tray temperature was maintained at 5°C and the injection volume was 5 μl. MS/MS conditions: The mass spectrometer was operated in positive ionization mode and detection and quantification were performed using multiple reactions monitoring (MRM). The pressure of the nitrogen sheath gas and auxiliary gas were set at 25 and 1 (arbitrary units). The ionization spray voltage, capillary transfer tube temperature, tube lens and skimmer offset were set at 4.5 kV 300°C, 87V and 10V, respectively. The vaporizing temperature within the H-ESI source was maintained at 300°C. The scan time was 0.05 s, with a scan width of 0.1 m/z. TSQ Tune Software (Thermo Scientific) was used for the optimization of tuning parameters. Data acquisition and processing were carried out using the Xcalibur program (Thermo Scientific). For ASN (133.08 m/z), the retention time was 3.15 min and 3 transitions were monitored: 74.25 m/z (collision energy (CE) 16V), 87.22 m/z (CE 16V) and 116.12 m/z (CE 10V). Quantitative calibration was performed before every batch of samples was analyzed; the calibration curves (0–100 μM) were obtained by linear least-squares regression of the measured peak area versus the ASN concentration. All coefficients of determinations were found to be R2 ≥ 0.9985.

RNA Preparation-GAS Bacteria were grown in THY to an early log phase (OD 600 = 0.3), washed with PBS, concentrated to OD 600 = 8 (109 CFU/ml), diluted 4,000-fold into 24-well plates containing DMEM medium supplemented with 5AA and then grown at 37°C in an atmosphere containing 5% CO 2 . At early log phase, Kidrolase (40 I.U/ml) or PBS were added. Samples were withdrawn at 30 and 60 min after Kidrolase addition, centrifuged at 5,000 × g for 10 min and pellets were mixed with RNAprotect (QIAGEN) and immediately frozen in liquid nitrogen. For RNA seq, total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research) using a modified procedure to improve GAS cell disruption. Briefly, cells from frozen pellets were resuspended in 1 ml of Trizol supplemented with approximately 300 mg of acid-washed glass beads (Sigma Life Science) and disrupted by vortexing for 5 min. Beads were collected by brief centrifugation and 700 μl of cell lysate was used for RNA purification as recommended by the manufacturer. RNA samples were treated with the Turbo DNase free kit (Ambion) to avoid genomic DNA contamination. Ribosomal RNA removal was achieved using the Ribo-Zero Magnetic (Gram-Positive Bacteria) kit (Epicentre) and rRNA-depleted RNA was then purified with the RNAClean XP kit (Agencourt). Sample quality was assessed using a 2100 Bioanalyzer (Agilent) and sample quantity was determined using a NanoDrop 8000 spectrophotometer (Thermo Scientific). Ravins et al., 2000 Ravins M.

Jaffe J.

Hanski E.

Shetzigovski I.

Natanson-Yaron S.

Moses A.E. Characterization of a mouse-passaged, highly encapsulated variant of group A streptococcus in in vitro and in vivo studies. For real-time RT-PCR, RNA was isolated by hot acidic phenol extraction as previously described (). Contaminating DNA was removed from the samples by DNase treatment according to the manufacturer’s instructions (RQ1 RNase free DNase, Promega). DNA-free total RNA was used for cDNA synthesis using MMLV reverse transcriptase (Promega), according to the manufacturer’s protocol. Standard real-time RT-PCR reactions were conducted using SYBR-green mix (Absolute SYBR GREEN ROX MIX, ABgene) and fluorescence detection was performed using Rotor-Gene 3000 A (Corbett life Science, QIAGEN) according to manufacturer’s instructions. The real-time RT-PCR primers ( Table S3 ) were designed using Primer Express software v2.0 (Applied Biosystems). The cDNA amount of gyrA was used for normalization. The data were analyzed according to the standard curve method (Rotor-gene analysis software 6.0) and are presented as abundance of transcript amount relative to that of gyrA.

RNA Preparation-MEFs MEFs were infected with GAS strains or treated with TG as described in the text. Cells were washed, scrapped into 1 ml of cold PBS and centrifuged. RNA was purified from the cell pellet by SV Total RNA isolation system (Promga) and subjected to cDNA synthesis using M-MLV reverse transcriptase (Promega,), according to the manufacturer’s protocols. Standard real-time RT-PCR reactions were conducted as described for GAS. The real-time RT-PCR asns primers ( Table S3 ) were designed using Primer Express software v2.0 (Applied Biosystems). The cDNA amount of β-actin was used for normalization. The data were analyzed according to the standard curve method (Rotor-gene analysis software 6.0) and are presented as abundance of transcript amount relative to that of β-actin.

RNA-Seq and Data Analysis For RNA-seq MGAS5005 was cultured in DMEM + 10% FCS fortified with 5 AA as described above. RNA-seq directional libraries were generated using the ScriptSeq v2 RNA-seq Library Preparation kit (Illumina) according to the manufacturer’s recommendations. Briefly, 1 μg rRNA-depleted RNA was fragmented and used for reverse-transcription with random primers containing a 5′-tagging sequence. The 5′-tagged cDNA was then modified at its 3′ end by a terminal-tagging reaction to generate di-tagged, single-stranded cDNA that was then purified using the AMPure System (Agencourt). The purified di-tagged cDNA was used as a template to generate second strand cDNA containing Illumina adaptor sequences, to incorporate Index barcodes and amplify the library by limited-cycle PCR. The resulting RNA-seq libraries were then purified using the AMPure System (Agencourt). RNA-seq library quality was verified using a 2100 Bioanalyzer (Agilent). 100 bp single-read DNA sequencing was then performed at the Institute for Bioscience and Biotechnology Research (IBBR) Sequencing Facility at the University of Maryland, College Park using the Illumina HiSeq 1500 platform. Data were generated in the standard Sanger FastQ format and raw reads have been deposited under BioProject PRJNA218856 with the Sequence Read Archive (SRA) at the National Center for Biotechnology Institute. 2) of 0.79. RNA-seq data analysis was performed by ContigExpress ( www.contigexpress.com ) as follows: quality control of raw sequences was done with FastQC v 0.10.0 and mapped to the MGAS5005 GAS reference genome (NC_007297) with Bowtie2 v 2.1.0 under standard single read and ‘very sensitive’ settings. The resulting mapping files were subjected to gene expression quantification in Fragments Per Kilobase of transcript per Million (FPKM) and differential expression analysis using Cufflinks v 2.1.0. Settings for upper quartile normalization and fragment bias correction were utilized with a Q-value cutoff of 0.05. Gene expression quantification was performed using htseq-count v 0.5.3 with read counts extracted for the first synthesized strand, the second strand, and both strands. Read counts from the first synthesized strand in duplicates were used for differential expression analysis using DESeq v1.12.0. Independent validation using real-time RT-PCR produced a coefficient of correlation (R) of 0.79.