1. Volume 156, Issue 1, Pages 97-108 (January 2014)
An Extracellular Bacterial Pathogen Modulates Host Metabolism to Regulate Its Own Sensing and Proliferation 
Moshe Baruch, Ilia Belotserkovsky, Baruch B. Hertzog, Miriam Ravins, Eran Dov, Kevin S. McIver, Yoann S. Le Breton, Yiting Zhou, Catherine Youting Cheng, Emanuel Hanski 
Cell 
Volume 156, Issue 1, Pages (January 2014)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 sil Is Activated In Vivo
(A) Schematic representation of the sil genes. sil core contains three polycistronic units: silA/B, TCS SilA/B; silE/D/CR, ABC transporter system (SilD/E) (plus the autoinducer peptide SilCR); and blp bacteriocin-like peptides, including BlpM. Their transcription is initiated from P1, P3, and P4 promoters, respectively. The transcript of silC is initiated from the P2 promoter. Promoters induced and noninduced by SilCR are illustrated by filled and empty flags, respectively.
(B and C) sil is activated in vivo. Biopsies were taken 6 hr after subcutaneous inoculation of mice with JS95ATApP4-gfp or JS95ATGpP4-gfp. Tissue sections were stained and analyzed by confocal microscopy (×63) for (A) and ×10 for (B). Brackets, hair follicles; empty arrows, bacteria; filled arrows, bacteria expressing GFP; triangles, muscle layer; scale bars, 100 μm.
(D) Quantification of sil activation. Mice were inoculated with JS95ATGpP4-luc or JS95ATApP4-luc. Punch biopsies were homogenized, and relative luminescence units (RLU) were normalized to the CFUs. Each value represents the mean of two determinations conducted for each punch biopsy. The highest mean value designated as 100% was obtained for a mouse challenged with JS95ATGpP4-luc. Horizontal lines, medians. The probabilities were calculated using Mann-Whitney U test. Two independent experiments were performed and yielded similar results.
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4. Figure 2
Interaction of GAS with Eukaryotic Cells Is Required for sil Activation
(A and B) FACS analyses of sil activation. HeLa cells were infected (MOI ∼1.0) with JS95ATGpP4-gfp (A) or JS95ATApP4-gfp (B). FACS analyses of GFP-labeled bacteria were performed at the indicated times as explained in the Experimental Procedures and Figure S1.
(C) Quantification of sil activation. Infection of HeLa cells was conducted with the indicated strains (Table S1). The mean fluorescence intensities (MFIs) were computed from the FACS analyses for each time point. All values represent the mean of three determinations ± SD. Two independent experiments were performed and yielded similar results (Table S4).
(D) Contact between GAS and HeLa cells facilitates sil activation. JS95ATGpP4-gfp was added to Transwell chambers as indicated. sil activation was determined by FACS analyses. The values represent the mean of three determinations ± SD. Two independent experiments were performed and yielded similar results (Table S4).
(E) Both SLS and SLO mediate sil activation. JS95ATG and the specified mutants (Table S1) were incubated with MEF cells for the indicated time periods. sil activation was determined by quantifying the level of SilCR as explained in the Experimental Procedures and Figure S1B. The fluorescence of the GFP-labeled reporter strain was normalized to the number of the bacteria (relative fluorescence). The values represent the mean of tree determinations ± SD. Two independent experiments were performed and yielded similar results (Table S4).
(F) Both SLS and SLO mediate sil activation in vivo. Mice were inoculated with JS95ATGpP4-luc, JS95ATApP4-luc, or JS95ATGΔslo,sagI−pP4-luc. Punch biopsies were taken 6 hr after injection and homogenized, and relative luminescence units (RLU) were normalized to the CFUs. Each value represents the mean of two determinations conducted for each punch biopsy. The highest mean value designated as 100% was obtained for a mouse challenged with JS95ATG. Horizontal lines, medians. The probabilities were calculated using Mann-Whitney U test. Two independent experiments were performed and yielded similar results.
See also Figures S1 and S2.
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5. Figure 3
Triggering of ER Stress in Mammalian Cells Produces a Conditioned Medium Capable of Activating sil
(A) Treatment of MEF cells with STS. MEF cells were incubated with or without 0.1 μM STS. Supernatants were collected and added to JS95ATGpP4-gfp, and the mixture was further incubated for 7 hr. sil activation was determined by FACS analyses, computing the mean fluorescence intensities of GFP-labeled bacteria. The values represent the mean of three determinations ± SD. Two independent experiments were performed and yielded similar results (Table S4).
(B) Treatment of MEF cells with DTT or TG. MEF cells were incubated with 0.5 mM DTT or 1.0 μM TG. Then, supernatants were collected and mixed with JS95ATGpP4-gfp, and sil activation was determined as in (A). The values shown represent the mean of three determinations ± SD. Two independent experiments were performed and yielded similar results (Table S4).
(C) STS and TG-mediated activation of sil bypasses the requirement for SLO and SLS. MEF cells were infected with JS95ATG or with the indicated mutants in the presence and absence of STS (0.1 μM) (left) or TG (1.0 μM) (right). At the indicated time points, sil activation was determined by quantifying the production of SilCR as conducted in Figure 2E. The values shown represent the mean of three determinations ± SD. Four independent experiments (left) and two independent experiments (right) were performed and yielded similar results (Table S4).
See also Figure S3.
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6. Figure 4 GAS Upregulates asns Transcription in Host Cells
(A) Infection of MEF cells with GAS upregulates asns transcription. MEF cells in DMEM supplemented with 5% FCS were infected with the indicated GAS strains, or TG (1.0 μM) was added. At the indicated time points, asns transcript level was determined by real-time RT-PCR and was normalized to the transcript level of β-actin using the primers described in Table S3. The real-time RT-PCR for each sample was performed in duplicates, and the values shown represent the means ± SD. Four independent experiments were performed and yielded similar results (Table S4). Inset, a zoomed image of the results obtained for MEF, MEF + JS95ATGΔslo,sagI−, and MEF + JS95ATG.
(B) Visualization of sil activation. MEF cells infected with JS95ATGpP4-gfp for the indicated time periods and stained with rhodamine-phalloidin (red), NucBlue (blue), anti-group A carbohydrate antibody (orange), and GFP-labeled bacteria (green). Overlays are presented in the lower right. Scale bar, 50 μm.
(C) TG enhances sil activation. MEF cells were incubated with TG (1.0 μM) and, after 0.5 hr, were infected with JS95ATGpP4-gfp. At the indicated time points, staining and visualization were performed as in (B).
See also Figure S4.
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7. Figure 5
ASN Is Essential for sil Activation and Promotes Bacterial Proliferation
(A) F-12 (HAM) medium supports sil activation. JS95ATGpP4-gfp was incubated for 6 hr in DMEM medium, DMEM medium supplemented with SilCR (5 ng/ml), or in F-12 (HAM) medium. sil activation was determined by FACS analyses. The values shown are the mean of three determinations ± SD. Results are representative of two independent experiments.
(B) 5AA are responsible for sil activation. The indicated reagents were added to DMEM medium, creating final concentrations equal to those present in the F-12 (HAM) medium. JS95ATGpP4-gfp was added, and the mixtures were incubated for 6 hr. sil activation was determined by FACS analyses. The values shown are the mean of three determinations ± SD. Results are representative of two independent experiments.
(C) ASN is essential for sil activation. sil activation in DMEM medium; DMEM medium supplemented with 4 amino acids (4AA) [proline (35 mg/l), aspartic acid (13 mg/l), glutamic acid (15 mg/l), and alanine (9 mg/l)]; with ASN (15 mg/l); or with 4AA plus ASN was determined by FACS analyses. All values represent the mean of three determinations ± SD. Two independent experiments were performed and yielded similar results (Table S4). (See Figure S5A for the growth curves under these conditions.)
(D) Inactivation of TrxR leads to constitutive sil activation. JS95ATGtrxR− and the parental JS95ATG strain were incubated in DMEM medium or in DMEM medium supplemented with 15 mg/l of ASN. SilCR production was quantified at the indicated time points as in Figure 3C. All values represent the mean of three determinations ± SD. Two independent experiments were performed and yielded similar results (Table S4).
(E) Plasmid curing of trxR− mutants restores the dependence of sil activation on ASN. The trxR− mutant was cured of the insertion inactivation plasmid (Table S2). JS95ATG, the trxR− mutant, and three cured clones were incubated for 6 hr in DMEM medium or in DMEM medium supplemented with 15 mg/l of ASN. SilCR production was quantified as in Figure 2E. The values shown are the mean of three determinations ± SD. Results are representative of two independent experiments.
(F) Contribution of ASN to JS95ATG growth in a medium totally depleted of ASN. DMEM medium depleted of ASN (see Experimental Procedures) was supplemented with 4AA (see C) and with the indicated concentrations of ASN. OD600 was measured at the indicated time points. The values shown are the mean of three determinations ± SD. Results are representative of three independent experiments.
See also Figure S5.
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8. Figure 6
Depletion of ASN Induces Upregulation in the Transcription of SLS and SLO Encoding Genes
(A–D) Total RNA was isolated from cultures of JS95ATG and MGAS5005 strains grown in the absence or presence of Kidrolase as explained in Experimental Procedures. For JS95ATG and its isogenic mutant deficient of trxR (TrxR−), the transcription level of sagA (A), sagB (B), sagD (C), and slo (D) were determined by real-time RT-PCR. For each gene, the amount of complementary DNA (cDNA) was normalized to that of gyrA in each RNA sample. The values shown are the mean ± SD of at least two independently isolated RNA preparations analyzed in duplicates. For MGAS5005 and its isogenic mutant deficient of trxR (TrxR−), the data are derived from RNA-seq (Table S5).
(E) Hypothetical model showing how GAS exploits host ASN metabolism for its own benefit (see Results for details).
See also Figure S6.
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9. Figure 7 The Therapeutic Effects of ASNase
(A) ASNase arrests growth in human blood. Ability of JS95ATG to grow in nonimmune human blood was quantified in the absence and presence of ASNase (Kidrolase 4.0 I.U ml). Bacterial growth (multiplication factor, MF) represents the increase in titer during 3 hr of incubation. The values shown are the mean ± SD of two independent experiments performed on blood withdrawn from two donors; each experiment was performed in duplicates.
(B) ASNase protects mice against GAS bacteremia. Survival rate of mice (n = 7 per group) after intravenous inoculation with GAS strain JS95ATG only or with additional 1 or 2 intravenous injections of ASNase (Kidrolase 200 I.U per mouse). The Kaplan-Meier analysis showed a significant difference in the rate of death of the group receiving GAS only compared to that receiving GAS and two consecutive injections of ASNase. p = ; log rank (Mantel-Cox) test. p = 0.015; log rank (Mantel-Cox) test was obtained for an additional separate experiment.
See also Figure S7.
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10. Figure S1 Assays of Functional sil, Related to Figure 2
(A) A change of ATA to ATG in the start codon of SilCR is sufficient for its production. The “Jump Start” assays were performed as previously described (Belotserkovsky et al., 2009). The values shown are the mean ± the SD results of two independent experiments each performed in triplicates.
(B) Schematic representation of the two assays performed to assess sil activation during mammalian cells infection with GAS and GGS. For the first one (left), GAS and GGS strains and mutants are transformed with pP4-gfp and GFP-labeled bacteria are quantified by FACS analysis (see Experimental Procedures). The second assay (right) is based on quantification of SilCR production using the reporter strain JS95ATApP4-gfp or its Erm-resistant derivative JS95ATAsilE−pP4-gfp. It includes culturing of the reporter strains with the tested supernatants for 2 hr in THY, which strongly amplifies the amount of GFP-labeled bacteria. In this case, the fluorescence measurements are conducted using a fluorometer and readings are normalized to the number of bacteria. This assay was mainly used when mammalian cells were infected with mutants of JS95ATG containing 2 antibiotic resistance genes or the Erm-resistant gene. This either prevented the transformation with pP4-gfp plasmid or affected the rate of bacterial growth. (The Erm resistant reporter was used in cases where the tested strain had to be grown with Erm).
(C) Two-dimensional flow cytometric FSC–SSC density-plot performed on supernatant of HeLa cells infected with JS95ATGpP4-gfp. The density plot is displayed according to the relative abundance of events, ranging from low (red), to medium (yellow), to high (green/blue). The black circle represents the gate set to detect non-aggregated GAS. Encircled are the 50,000 gated events which are above 90% of the overall number of events of the reading.
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11. Figure S2
sil Activation Requires Intact Mammalian Cells and Is Mediated by GAS SLO and SLS, Related to Figure 2
(A) Intact but not lysed HeLa cells promote sil activation. Intact HeLa cells, lysed HeLa cells (see EXTENDED EXPERIMENTAL PROCEDURES) or DMEM medium were infected with JS95ATGpP4-gfp. The level of sil activation at the indicated time points was determined by FACS analyses, computing the mean fluorescence of GFP-labeled bacteria. The values shown represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4).
(B) The activation of sil in vitro is blocked by anti-SilCR serum. HeLa cells were infected with JS95ATGpP4-gfp. Anti-SilCR serum (Belotserkovsky et al., 2009) or control serum were added at the indicated dilutions and the level of sil activation after 8 hr of incubation was determined by FACS analyses. All values represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4).
(C) Infection of MEF cells with streptococcal strains possessing naturally intact sil leads to its activation. GAS and GGS possessing naturally intact sil (Belotserkovsky et al., 2009) were transformed with pP4-gfp. MEF cells were infected with the resulting mutants and incubated for 8 hr in the presence of either anti-SilCR serum or control serum (1:100). sil activation was determined by FACS analyses, computing the mean fluorescence of GFP-labeled bacteria. The values shown are the mean of three determinations ± SD. Results are representative of two independent experiments.
(D) sil activation occurs in vivo in a strain containing naturally active sil. Punch biopsies of mice challenged with WT NS144 and its isogenic silE mutant (as a control) were taken 6 hr after injection, homogenized, and relative luminescence units (RLU) were normalized to the CFUs. Each value represents the mean of two determinations conducted for each punch biopsy. The highest mean value designated as 100% was obtained for a mouse challenged with NS144pP4-luc. Horizontal lines-medians. The probabilities were calculated using Mann–Whitney U test. Two independent experiments were performed yielding similar results.
(E) Internalization of GAS into mammalian cells is not required for sil activation. (Left) HeLa cells were untreated or treated with cytochalasin D (Ozeri et al., 2001) for an hour prior to infection with JS95ATGpP4-gfp. sil activation was determined by FACS analyses, computing the mean fluorescence intensities of GFP labeled bacteria. All values represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4). (Right) Entry of bacteria into cells after 6 hr of incubation was quantified by the standard gentamicin protection assay as described previously (Ozeri et al., 2001). The values shown are the mean ± SD of 2 independent experiments each performed in triplicate.
(F) Either expression of SLS or SLO is sufficient to trigger sil activation. For complementation of either SLO or SLS activity, the double mutant lacking both SLO and SLS (JS95ATGΔslo,sagI−) was transformed with a shuttle vector expressing either SLS (JS95ATGΔslo,sagI−pLZsagI) or SLO (JS95ATGΔslo,sagI−pLZslo) (Table S1). MEF cells were infected with the JS95ATG, and the indicated mutants and production of SilCR was quantified using the reporter assay (Figure S1B). The fluorescence of GFP-labeled reporter was normalized to the number of the bacteria. The values shown represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4).
(G) Complementation of hemolysis in the SLS and SLO double mutant by expressing SLS from a plasmid. The hemolysis of JS95ATG, JS95ATGΔslo,sagI−, or JS95ATGΔslo,sagI−pLZsagI was determined as explained in the EXTENDED EXPERIMENTAL PROCEDURES. Under non-reducing conditions SLO is not active and trypan blue specifically inhibits SLS-mediated hemolysis. The values shown are the mean ± SD of 2 independent experiments each performed in triplicate.
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12. Figure S3
Triggering of ER Stress Leads to sil Activation, Related to Figure 3
(A) Autophagy is not involved in sil activation. Atg5−/− MEF cells support sil activation. Atg5+/+ and Atg5−/− MEF cells were infected with JS95ATGpP4-gfp. As a control JS95ATGpP4-gfp was added into DMEM medium without MEF cells. At the indicated time points, samples from the culture medium were subjected to FACS analyses. The values shown represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4).
(B) Inhibitors of autophagy do not inhibit sil activation. Prior to infection MEF cells were treated for 3 hr with chloroquine (5 μM), or wortmannin (1 μM) then infected with JS95ATGpP4-gfp. As a control, JS95ATGpP4-gfp was added to DMEM medium. At the indicated time points samples from the culture medium were subjected to FACS analyses. The values shown represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4).
(C) Apoptosis is not involved in sil activation. MEF cells were pretreated with the pan-caspase inhibitor Z-VAD (100 μM) for 3 hr before infection with JS95ATGpP4-gfp. As a control JS95ATGpP4-gfp was added to DMEM medium. At the indicated time points samples from the culture medium were subjected to FACS analyses. The values shown represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4).
(D) Inhibition of TNF-α-induced necroptosis in L929 cells supports sil activation.L929 cells were treated with TNF-α (10 ng/ml) and Z-VAD (100 μM) or with TNF-α plus Z-VAD (100 μM) and necrostatin-1 (Nec-1, 100 μM). Representative phase-contrast images of untreated L929 cells and cells treated with the indicated reagents (taken after 2 hr of incubation) are shown in the upper panel (40x lens; scale bar, 50 μM). In the lower panel the culture media of L929 cells, (treated as indicated in the upper panel), were collected in the indicated times and added to JS95ATGpP4-gfp. The mixtures were further incubated for 7 hr and sil activation was determined by FACS analyses. The values shown represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4).
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13. Figure S4
Reducing the FCS Concentration Delays GAS-Mediated Cell Necrosis, Related to Figure 4
Prior to infection the DMEM medium was exchanged to DMEM supplemented either with 5 or 10% FCS. MEF cells incubated in the indicated media were infected with JS95ATGpP4-gfp. (Upper panel). At the indicated time points samples from the culture media were subjected to FACS analyses (solid line) computing the mean fluorescence intensity of GFP-labeled bacteria. The values shown represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results. Cell viability assay was performed by quantifying LDH release; maximal release represents 100% cytotoxicity (dotted line) (EXTENDED EXPERIMENTAL PROCEDURES). The values shown represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results. (Lower panel). The growth of JS95ATGpP4-gfp was determined by measuring OD600 at the indicated time points. The values shown are the mean of three determinations ± SD. Results are representative of two independent experiments.
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14. Figure S5
ASN Is Essential for sil Activation and Promotes GAS Proliferation, Related to Figure 5
(A) Growth curves of JS95ATG in DMEM medium, DMEM medium supplemented with: 4 amino acids (4AA) [proline (35 mg/l), aspartic acid (13 mg/l), glutamic acid (15 mg/l) and alanine (9 mg/l)]; with ASN (15 mg/l) or with 4AA plus ASN were determined by measuring OD600 at the indicated time points. The values are the mean triplicate determinations ± SD. Results are representative of three independent experiments.
(B) ASN is required for sil activation. (Left). DMEM medium containing 4AA (see above) was supplemented with the indicated concentrations of ASN and seeded with JS95ATGpP4-gfp. At the indicated time points samples from the culture medium were subjected to FACS analyses. The values represent the mean of 3 determinations ± SD. Two independent experiments were performed yielding similar results (Table S4). (Right). Growth of JS95ATGpP4-gfp in DMEM medium supplemented with 0.6 and 0.45 mg/l of ASN was determined by enumerating bacteria at the indicated time points. The values shown are the mean of three determinations ± SD. Results are representative of two independent experiments.
(C) The predicted structure of the surface exposed domain of TrxS resembles that of McpB. The upper PAS domain of McpB of B. subtilis (Glekas et al., 2011) stretching from AA 35 to AA 279 was subjected to structure modeling using LOMETS, I-TASSER and Phyre2 servers. The predicted surface exposed domain of TrxS stretching from AA 50 to AA 289 was modeled by the same servers. The predicted structures were overlaid using the Cealign algorithm (Shindyalov and Bourne, 1998).
(D) In vivo sil activation requires ASN. Mice were inoculated with either JS95ATGpP4-luc + PBS, JS95ATApP4-luc + PBS or JS95ATGpP4-luc + 200 I.U Kidrolase. Punch biopsies were taken 6 hr after injection, homogenized, and relative luminescence units (RLU) were normalized to the CFUs. Each value represents the mean of two determinations conducted for each punch biopsy. The highest mean value designated as 100% was obtained for a mouse challenged with JS95ATGpP4-luc. Horizontal lines-medians. The probabilities were calculated using Mann–Whitney U test. Two independent experiments were performed yielding similar results.
(E) Contribution of ASN to M1T1 growth in a medium depleted of ASN. M1T1 growth in a medium depleted of ASN by ASNase treatment (see Experimental Procedure) was supplemented with 4AA and the indicated concentrations of ASN. OD600 readings were recorded. The values shown are the mean of three determinations ± SD. Results are representative of two independent experiments.
(F) ASNase arrests GAS growth. JS95ATG and M1T1 strains were cultured in DMEM medium depleted of ASN and supplemented with the 4AA. The ASN-depleted media was seeded with either JS95ATG or M1T1 strains and GAS was grown in the absence or the presence of the indicated concentrations of ASNase (units/ml) for 24 hr. Control represents the growth of GAS in the presence of 15 mg/l of ASN. The amount of bacteria was determined by recording OD600. The values shown are the mean of three determinations ± SD. Results are representative of two independent experiments.
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15. Figure S6
Transcription of silE and blpM in JS95ATG and Heatmap of RNA-Seq, Related to Figure 6
(A) Heatmap of RNA-Seq results presented in Table S5. Heatmap of RNA-seq differential expression (p < 0.05) of WT MGAS5005 and its isogenic TrxR mutant. These strains were grown without (-) or with (+) Kidrolase and total RNA was prepared as explained in Extended Experimental Procedures. Transcripts overexpressed (yellow) and under expressed (blue) in the absence of Kidrolase compared to its presence, are shown. Map was generated using Genesis v1.7.1 software (Sturn et al., 2002) and is ordered based on WT MGAS5005.
(B and C) Transcription of sil genes. Total RNA from JS95ATG was prepared as indicated in A. The amounts of silE (B) and blpM (C) transcripts were determined by real-time RT-PCR. For each gene, the amount of cDNA was normalized to that of gyrA in each RNA sample. The values shown are the mean ± SD of at least two independently isolated RNA preparations analyzed in duplicates.
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16. Figure S7
Kidrolase Arrests Growth of M1T1 Strain in Human Blood but Does Not Affect the Survival of S. epidermidis, Related to Figure 7
(A) The ability of M1T1 MGAS5005 strain to grow in non-immune human blood was quantified in the absence and presence of ASNase (Kidrolase 4.0 I.U ml). Kidrolase was added to the blood together with the bacteria or at 0.5 and 1.0 hr after bacterial addition. Bacterial growth (multiplication factor, MF) represents the increase in titer during a 3 hr period. The values shown are the mean ± SD results of two independent experiments, performed on blood withdrawn from two donors; each experiment was performed in duplicate.
(B) (Left panel). S. epidermidis ATCC (Zhang et al., 2003) and GAS JS95ATG were grown in DMEM medium supplemented with 4 AA with or without Kidrolase (40 I.U/ml). The OD600 was measured at the indicated time points. The values shown are the mean of two determinations ± SD. Results are representative of three independent experiments. (Right panel). The Ability of S. epidrmidis to grow in human blood was quantified in the absence and presence of ASNase (Kidrolase 40 I.U/ml). MF represents the decrease in titer during 2 hr of incubation. The values shown are the mean ± SD of two independent experiments, performed on blood withdrawn from two donors; each experiment was performed in duplicates.
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