Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection

Caspase-3 controls the apoptotic pathway, a form of programmed cell death designed to be immunologically silent. Polymorphisms leading to reduced caspase-3 activity are associated with variable effects on tumorigenesis and yet arise frequently. Staphylococcus aureus is a human commensal and a frequent cause of soft tissue and bloodstream infections. Successful commensalism and virulence can be explained by the secretion of a plethora of immune evasion factors. One such factor, AdsA, destroys phagocytic cells by exploiting the apoptotic pathway. However, human CASP3 variants with loss-of-function alleles shield phagocytes from AdsA-mediated killing. This finding raises the possibility that some caspase-3 alleles may arise from exposure to S. aureus and other human pathogens that exploit the apoptotic pathway for infection.

Although neutrophils use extracellular traps (NETs) to entangle staphylococci (11,12), NETs are degraded by secreted staphylococcal nuclease (Nuc) and thereby fail to exert bactericidal activity (13). Nuclease digestion of NETs releases 5=-and 3=-monophosphate nucleotides that are converted by S. aureus adenosine synthase A (AdsA), a sortase-anchored surface protein, into deoxyadenosine (dAdo) (14). dAdo is toxic to macrophages and other immune cells (14,15). In mice, S. aureus mutants lacking adsA exhibit diminished survival in host tissues and defects in the pathogenesis of bloodstream infections (16). AdsA-mediated dAdo production has been proposed to trigger caspase-3-induced apoptosis of mouse and human macrophages. In this model, phagocyte access to the staphylococcal abscess community, the core of staphylococcal abscess lesions, is prevented thereby promoting bacterial survival within the lesion (14). The mechanism of staphylococcal dAdo cytotoxicity was investigated using CRISPR/ Cas9 mutagenesis to show that destruction of human U937 macrophages involves uptake of dAdo via the human equilibrative nucleoside transporter 1 (hENT1), dAdo conversion to dAMP by deoxycytidine kinase (DCK) and adenosine kinase (ADK), and subsequent dATP formation (17). Here, we investigate the subsequent activation of caspase-3-induced cell death upon dATP formation. We show that CASP3 Ϫ/Ϫ macrophages are resistant to AdsA-derived dAdo and that animals lacking CASP3 expression in hematopoietic cells, including macrophages and dendritic cells, are less susceptible to S. aureus infection. We also explore how single nucleotide polymorphisms (SNPs) in human CASP3 may protect macrophages from staphylococcal dAdo and may account for the varied susceptibility toward S. aureus disease in the human population.

RESULTS
Deoxyadenosine triggers caspase-3 activation in human macrophages. Earlier work revealed a correlation between staphylococcal dAdo and caspase-3 activation in macrophages surrounding abscess lesions (14). To further explore how S. aureus exploits caspase-3 activation during infection, human U937-derived macrophages were treated with 10 M dAdo. Next, caspase-3 activity was examined in macrophage cell lysates by measuring the hydrolysis of the peptide substrate Ac-DEVD-pNA. Caspase-3 activity was undetectable in cell lysates of human macrophages left untreated (Fig. 1A). Treatment with dAdo resulted in increased caspase-3 activity in cell lysates, consistent with previous studies, and in agreement with phagocyte cell death ( Fig. 1A and B) (14,17). To account for nonspecific hydrolysis of the Ac-DEVD-pNA substrate, macrophage cell lysates were cotreated with the caspase-3 inhibitor Ac-DEVD-CHO. This treatment resulted in the loss of caspase-3 activity, confirming that dAdo provokes the specific activation of caspase-3 in human phagocytes (Fig. 1A).
Caspase-3 is required for deoxyadenosine-mediated killing of macrophages. To assess whether the activation of caspase-3 is responsible for phagocyte cell death, U937-derived macrophages were incubated with dAdo alone or with increasing concentrations of Z-DEVD-FMK, a membrane-permeable caspase-3 inhibitor. Preincubation with the inhibitor prevented dAdo-mediated cell death in U937-derived macrophages in a dose-dependent manner (Fig. 1B). In light of these findings, CRISPR/Cas9 mutagenesis was used to disrupt the caspase-3-encoding gene CASP3 located on human chromosome 4 (Fig. 1C). Sanger sequencing of exon 5 targeted by the single guide RNA (sgRNA) used here confirmed the biallelic disruption of CASP3 in U937 cells (Fig. 1C). Extracts of U937-derived CASP3 Ϫ/Ϫ macrophages (referred to as CASP3 Ϫ/Ϫ macrophages) were also analyzed by immunoblotting with caspase-3-specific antibodies, which confirmed that caspase-3 production had been abolished (Fig. 1D). CASP3 Ϫ/Ϫ macrophages were found to be refractory to dAdo-mediated toxicity in a manner similar to SLC29A1 Ϫ/Ϫ macrophages (17) that can no longer transport dAdo into the cell (Fig. 1E). Next, a plasmid encoding an sgRNA/Cas9-resistant allele of CASP3 under the control of the EF1␣ promoter was transferred into CASP3 Ϫ/Ϫ macrophages (referred to as CASP3 Ϫ/Ϫ [ϩCASP3 WT ] macrophages). This process restored caspase-3 production and dAdo susceptibility ( Fig. 1D and E; see also Fig. S1 in the supplemental material). Together, these experiments demonstrate that caspase-3 contributes to dAdo-mediated killing of phagocytes and suggest that CASP3 Ϫ/Ϫ macrophages should be resistant to S. aureus-derived dAdo. To test this conjecture, cultures of S. aureus Newman (wild type [WT]) or the adsA variant were resuspended in chemically defined medium supplemented with thymus DNA to generate conditioned culture medium. This was achieved by centrifugation of cultures to remove bacteria, followed by filter sterilization of supernatants which were then added to U937-derived macrophages or their genetic variants. In agreement with earlier work, killing of U937-derived macrophages required both S. aureus expressing adsA and host DNA (Fig. 1F) (14,17). CASP3 Ϫ/Ϫ macrophages were resistant to staphylococcal dAdo in a manner comparable to SLC29A1 Ϫ/Ϫ macrophages (Fig. 1F). Genetic complementation (CASP3 Ϫ/Ϫ [ϩCASP3 WT ]) restored susceptibility to S. aureus-derived dAdo in this assay, confirming that caspase-3 is required for dAdo-mediated killing of phagocytes (Fig. 1F).
Conditional knockout mice lacking caspase-3 exhibit diminished susceptibility toward S. aureus disease. C57BL/6 mice with a floxed caspase-3 allele (CASP3 fl/fl ) have been crossed with Tie2-Cre ϩ (endothelial/hematopoietic [EϩH]) mice to obtain conditional knockout animals with endothelial/hematopoietic tissue-specific deletion of caspase-3 (CASP3 fl/fl Tie2-Cre ϩ mice) (18). These animals were used to examine the contribution of caspase-3 to S. aureus pathogenesis. Control CASP3 fl/fl and CASP3 fl/fl Tie2-Cre ϩ animals were infected by intravenous inoculation of S. aureus strain Newman (10 7 CFU). Five days postinfection, animals were euthanized. Kidneys were removed and visible abscess lesions counted before plating tissues on agar to measure bacterial loads. The analysis was conducted independently for cohorts of female and male animals. In contrast to CASP3 fl/fl female mice, bacterial loads and abscess numbers were significantly reduced in kidneys of CASP3 fl/fl Tie2-Cre ϩ female animals ( Fig. 2A and B). Similarly, conditional knockout male animals were more resistant to S. aureus infection, displaying fewer abscess lesions and a significant reduction in bacterial loads in kidneys compared to CASP3 fl/fl control males ( Fig. 2C and D). To test whether staphylococci manipulate host apoptosis during infection, groups of female and male animals were also challenged with S. aureus Newman lacking adsA. Conditional knockout animals no longer displayed increased resistance to S. aureus infection (Fig. 2). Further, infection with S. aureus adsA phenocopied CASP3 loss in agreement with the notion that AdsA is required for the persistence of abscess lesions in tissues (Fig. 2) (16). In summary, these data indicate that caspase-3 contributes to S. aureus abscess formation and disease pathogenesis in vivo in a manner requiring staphylococcal AdsA.
Caspase-3 affects macrophage infiltration into S. aureus abscess lesions. Differences in abscess development in the kidneys of infected animals may stem from caspase-3 deficiency in hematopoietic cells. In this model, the loss of caspase-3 would protect murine phagocytes from S. aureus-derived dAdo, allowing for the infiltration of macrophages to the staphylococcal abscess community, a process otherwise restricted by staphylococcal dAdo and AdsA (14). To explore this possibility, kidneys of CASP3 fl/fl or CASP3 fl/fl Tie2-Cre ϩ animals infected with S. aureus Newman were thin-sectioned and examined using immunohistochemistry. As expected, renal abscess lesions of CASP3 fl/fl mice revealed staphylococcal abscess communities surrounded by cuffs of immune cells composed mainly of Ly-6G-positive neutrophils and mostly lacking F4/80-positive macrophages ( Fig. 3A to P) (14). On the contrary, F4/80-positive macrophages were observed to be diffused throughout the neutrophil cuff of lesions from CASP3 fl/fl Tie2-Cre ϩ animals ( Fig. 3A to P). To better assess macrophage infiltration, immunohistochemistry images of multiple abscesses were used to delineate the total surface area of lesion (anti-Ly-6G-positive) and surface area free of macrophages (anti-F4/80-negative). The data were used to calculate the percent area of lesions occupied by macrophages (Fig. 3Q). Wild-type CASP3 fl/fl animals restricted macrophages from accessing abscess lesions following infection with strain Newman; as expected, this restriction was lost upon infection with the adsA mutant (Fig. 3Q). Similarly, abscess lesions in CASP3 fl/fl Tie2-Cre ϩ mice infected with Newman contained significantly more macrophages, and macrophage recruitment no longer required adsA (Fig. 3Q). Thus, the CASP3 mutation in mice phenocopies the S. aureus adsA mutation ( Fig. 3A to Q). Next, bone marrowderived macrophages (BMDM) were isolated from CASP3 fl/fl and CASP3 fl/fl Tie2-Cre ϩ mice. Immunoblotting confirmed the lack of caspase-3 in CASP3 fl/fl Tie2-Cre ϩ BMDM extracts (Fig. 3R). When exposed to dAdo, BMDM lacking caspase-3 exhibited increased viability compared to wild-type (CASP3 fl/fl ) macrophages (Fig. 3S). Together, these findings indicate that caspase-3 deficiency protects macrophages from AdsA-derived dAdo and accounts for their increased infiltration into staphylococcal abscesses.

DISCUSSION
The continued replication of staphylococci during infection is accompanied by the release of bacterial products (formyl peptides, lipoproteins, and peptidoglycan) and the  (50 M). Data are the mean (ϮSD) values from three independent determinations. Statistically significant differences were analyzed with one-way ANOVA and Tukey's multiple-comparison test (Q), or by a two-tailed Student's t test (S); ns, not significant (P Ͼ 0.05); **, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.0001. concurrent damage of host tissues (5-7). Cellular damage triggers the release of otherwise-sequestered intracellular components, such as N-formylated mitochondrial peptides, nucleosomes, S100 proteins, heat shock proteins, and purines (ATP and ADP), all of which are known to potently stimulate inflammation (20)(21)(22)(23)(24)(25)(26). Nonetheless, within deep-seated abscesses, S. aureus bacteria escape phagocytic clearance to establish persistent abscess lesions (7,27,28). Earlier work revealed that S. aureus AdsA catalyzes the dephosphorylation of ATP, ADP, and AMP, which effectively increases the concentration of adenosine (16,29). The activity of AdsA is reminiscent of host ectonucleoside triphosphate diphosphohydrolases and 5=-nucleotidases, which sequentially convert ATP to adenosine (30). Extracellular ATP and ADP stimulate purinergic receptors, leading to proinflammatory responses, whereas adenosine binding to cognate G protein-coupled receptors results in an anti-inflammatory response (31,32). This mechanism allows the host to control the amplitude of inflammatory responses. Similarly, S. aureus mitigates extensive inflammation in abscess lesions and the nonending recruitment of neutrophils by producing AdsA, which increases the concentration of the anti-inflammatory mediator adenosine and reduces the concentration of proinflammatory purines. With and various alleles of CASP3 (gray or pink bars) after treatment with dAdo (D) or after treatment with culture medium (RPMI) that had been conditioned by incubation with either wild-type S. aureus Newman or its adsA mutant in the presence of host DNA, as indicated with ϩ and -signs (E). (C to E) Gray indicates functional CASP3 alleles that support caspase-3 activity, whereas pink depicts nonfunctional alleles that do not restore caspase-3 activity in CASP3 Ϫ/Ϫ -derived macrophages. All samples received adenosine deaminase inhibitor (50 M dCF). Data are the mean (ϮSD) from three independent determinations. Statistically significant differences were analyzed with one-way ANOVA and Tukey's multiple-comparison test; ns, not significant (P Ͼ 0.05); *, P Ͻ 0.05; **, P Ͻ 0.01; ****, P Ͻ 0.0001.
Here, we demonstrate that by doing so, staphylococci selectively kill macrophages through apoptosis, a noninflammatory cell death pathway that cannot alert the immune system. Thus, S. aureus evolved AdsA to subvert two host immune surveillance pathways and establish persistent lesions. By combining CRISPR/Cas9 mutagenesis and a renal abscess mouse model, we show that caspase-3 is required for dAdo-mediated killing of phagocytes. The immunohistochemical examination of renal tissues suggests that loss of caspase-3 renders macrophages resistant to S. aureus-derived dAdo. As a result, macrophages accumulate within abscess lesions and presumably accelerate the removal of necrotic neutrophils and remnants of NETs (Fig. 5). If so, macrophagemediated engulfment of NETs together with entangled staphylococci probably elicits robust proinflammatory and pathogen-specific immune responses. Invading macrophages may also discharge their cellular content in order to form microbe-immobilizing macrophage extracellular traps (METs) (33)(34)(35) or directly combat replicating S. aureus in the deeper cavity of the abscess lesion, thereby supporting neutrophils in the phagocytic clearance of staphylococci (Fig. 5).
Conditional mutant animals with an endothelial/hematopoietic tissue-specific deletion of CASP3 were used in this study, as mice lacking CASP3 lineage dependently display neurodevelopmental abnormalities (36)(37)(38). Nonetheless, sequence analyses of human genomes reveal extensive genetic polymorphisms in CASP3. Some variants are associated with human cancers (39,40), chronic periodontitis (41), and Kawasaki disease (42), raising the question of what factor may favor the maintenance of these SNPs in the human population. Here, we report that genetic polymorphisms in human CASP3 protect macrophages from S. aureus-derived dAdo. Since humans exhibit varied susceptibility toward S. aureus infections (43,44), we propose that recurrent staphylococcal disease and excessive generation of dAdo in abscess lesions may have contributed to the selection of some SNPs in CASP3. For instance, CASP3-inactivating SNPs, such as rs371145290 (c.653C¡T, p.Ser218Leu), which predominantly occur in individuals of European ancestry, may hamper the development of abscesses as well as other staphylococcal diseases, such as endophthalmitis (45), necrotizing pneumonia (46), or mastitis (47), that have been shown to be associated with increased caspase-3 activity. Mortality rates in septic patients have also been shown to correlate with caspase-3 levels in human sera (48,49). Thus, CASP3 variants with reduced apoptotic activity may also influence the outcome of life-threatening sepsis. Other human pathogens synthesize dAdo, e.g., members of the genus Streptococcus (50-52), which also colonize large segments of the human population, or produce various stimuli which trigger host cell apoptosis (53). CASP3 polymorphisms may also arise under the selective pressure of other pathogens that exploit caspase-3 activation for disease, for example, Legionnaires' disease (54) or viral encephalitis (55,56). Overall, interference with caspase-3 activation may determine host susceptibility toward certain infectious diseases, thereby affecting the clinical outcome of acute and recurrent infections. Thus, caspase-3, staphylococcal AdsA and its homologues represent attractive targets for new immunomodulatory therapeutic strategies to combat multidrug-resistant pathogens, including methicillin-resistant S. aureus (MRSA).

MATERIALS AND METHODS
Bacterial strains and growth media. Bacterial strains were grown in Luria broth (LB; Becton, Dickinson) or tryptic soy broth (TSB; Becton, Dickinson) supplemented with the appropriate antibiotics (100 g/ml ampicillin or 50 g/ml kanamycin). All strains used in this study are listed in Table S1.
Lentivirus production. Lentiviral particles were produced by using the ViraPower kit (Thermo Fisher), according to the manufacturer's instructions. Lentiviral particles were harvested 48 to 72 h postinfection and concentrated by using a Lenti-X concentrator (TaKaRa), according to the manufacturer's instructions. Lentiviral particles were suspended in DMEM, supplemented with 10% FBS and 1% bovine serum albumin, and stored at -80°C.
Lentiviral transduction of U937 cells. Lentiviral transduction of U937 cells was performed as described before (17). Briefly, U937 cells grown in RPMI 1640 medium supplemented with 10% hi-FBS were transduced via spinfection in the presence of 8 g/ml Polybrene (Sigma, St. Louis, MO, USA) at a multiplicity of infection (MOI) of approximately 0.3. Viral titers were determined by transducing U937 cells (1.0 ϫ 10 6 cells/ml) with various volumes of lentiviral particles, along with a nonvirus-containing control via spinfection (1,000 ϫ g for 2 h at room temperature). U937 cell pellets were suspended in RPMI 1640 medium containing 10% hi-FBS and incubated for 48 h at 37°C under 5% CO 2 . Cells were centrifuged, counted, and split into duplicate wells, with one well containing 2.5 g/ml puromycin (Gibco). After 3 days, cells were counted, and the transduction efficiency was calculated as the cell count from wells containing puromycin divided by the cell count from wells without puromycin and multiplied by 100. The virus volume yielding an MOI closest to 0.3 was chosen for all experiments.
CRISPR/Cas9 mutagenesis of U937 cells. A LentiCRISPR v2 plasmid (57) containing a CASP3 targeting sgRNA (ATGTCGATGCAGCAAACCTC) was purchased from GenScript (Piscataway, NJ, USA), maintained in Escherichia coli Stbl3 cells, and used to produce lentiviral particles (Table S2). CRISPR/ Cas9-mediated mutagenesis was performed as described previously (17). Briefly, U937 cells were transduced by spinfection and selected with puromycin (2.5 g/ml) for 7 days to complete gene editing. Next, single cells were isolated and clonally expanded. Genomic DNA was isolated using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany). The genomic region targeted by the sgRNA and Cas9 was amplified by PCR with primers listed in Table S3 and cloned via the Zero Blunt TOPO PCR cloning kit (Thermo Fisher). Candidate plasmids from various E. coli clones were subjected to sequencing to confirm biallelic gene disruptions. All plasmids used in this study are listed in Table S2.
Analysis of human SNPs in CASP3. For analysis of human SNPs and complementation studies in U937 CASP3 Ϫ/Ϫ cells, a genetically engineered CASP3 gene refractory to sgRNA/Cas9 mutagenesis was synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA) without changing the amino acid sequence (Fig. S1). The sgRNA-resistant CASP3 gene was amplified via PCR and subcloned into pLVX-IRES-Neo (TaKaRa) at the XhoI and BamHI sites using primers listed in Table S3. The resulting pLVX-CASP3-IRES-Neo plasmid was further modified to replace the endogenous cytomegalovirus (CMV) promoter with the EF1␣ promoter amplified from pEF1/V5-His B (Thermo Fisher) via PCR, using the primers listed in Table S3. The new plasmid (pLVX-EF1␣-CASP3-IRES-Neo) was maintained in E. coli Stbl3 cells and used for complementation studies. The plasmid was also used as a template to introduce various human SNPs in CASP3 by site-directed mutagenesis using the primers listed in Table S3. The resulting plasmids were transferred into U937 CASP3 Ϫ/Ϫ cells by lentivirus-based transduction. Cells were selected with 500 g/ml Geneticin (Gibco). Two databases, ExAC (http://exac.broadinstitute.org/) and dbSNP (https://www.ncbi.nlm.nih.gov/snp/), were screened for candidate SNPs in human CASP3.
Isolation of murine bone marrow-derived macrophages. To isolate murine BMDM, the mice were euthanized. Subsequently, the femur and tibia were removed, sterilized with 70% ethanol, and washed with sterile phosphate-buffered saline (PBS). The ends of the bones were removed to flush out the bone marrow with RPMI 1640 containing penicillin-streptomycin. Next, the bone marrow was resuspended and passed through a nylon filter (BD; 40 m) to remove debris and unwanted tissue. Cells were centrifuged for 10 min at 200 ϫ g. The cell pellet was resuspended in 3 ml red blood cell (RBC) lysis buffer (BioLegend). RBCs were lysed for 5 min at room temperature (RT), according to the manufacturer's instructions. Cells were separated by centrifugation (10 min, RT, 200 ϫ g). RBC-free cell pellets were resuspended in RPMI 1640 without penicillin-streptomycin, and cells were enumerated by using a Caspase-3 Activation by Staphylococcus aureus ® hemocytometer. Cells were adjusted to 3.0 ϫ 10 5 cells/ml in BMDM medium (RPMI 1640 supplemented with 20% FBS, 1 mM pyruvate, 2 mM glutamine, 0.55 mM ␤-mercaptoethanol, and 10% filter-sterilized supernatant from macrophage colony-stimulating factor [CSF]-transfected 3T3-CSF cells) and seeded into 150-mm bacteriological dishes. At 3 days postextraction, cells were incubated with an additional 30 ml of BMDM medium which was entirely replaced on day 6 postextraction. BMDM were used at days 7 to 9 postextraction.
Cytotoxicity assays. dAdo-mediated cytotoxicity was analyzed as described elsewhere (14,17). Briefly, 4.0 ϫ 10 5 U937 cells per well were seeded in a 24-well plate and incubated for 48 h at 37°C under 5% CO 2 in RPMI 1640 medium supplemented with 10% hi-FBS and 160 nM phorbol 12-myristate 13-acetate (PMA). U937-derived macrophages were washed once and further incubated in growth medium (RPMI 1640 containing 10% hi-FBS) lacking PMA for 24 h. Alternatively, 3.5 ϫ 10 5 BMDM per well (obtained from CASP3 fl/fl or CASP3 fl/fl Tie2-Cre ϩ mice) were seeded in a 24-well plate and incubated for 24 h at 37°C under 5% CO 2 in BMDM medium. U937-derived macrophages or BMDM were washed again, and media were replaced by growth or BMDM medium containing 50 M pentostatin (2=-deoxycoformycin [dCF]) and 10 M dAdo, as indicated in the figure legends. Cells were further incubated (U937-derived macrophages for 24 h and BMDM for 72 h) and detached using either trypsin-EDTA solution (U937derived macrophages) or 1ϫ PBS containing 1 mM EDTA (BMDM). Where indicated, a small-molecule inhibitor of caspase-3 (Z-DEVD-FMK; R&D Systems) was added 1 h prior to dAdo treatment. Dead cells were stained with trypan blue and counted by using a microscope to calculate killing efficiency. Cytotoxicity of S. aureus-derived dAdo was analyzed as described earlier, with minor modifications (14,17). In brief, wild-type S. aureus Newman or adsA mutant cells were grown overnight in TSB, diluted 1:100 in RPMI 1640 medium, and grown at 37°C to 5.0 ϫ 10 7 CFU/ml. Next, 6.0 ϫ 10 7 CFU were incubated in RPMI 1640 containing 28 g/ml thymus DNA (Sigma) for 3 h at 37°C. Controls lacked bacteria or thymus DNA or included the S. aureus adsA mutant that cannot generate dAdo (14). Bacteria were removed by centrifugation, and the resulting filter-sterilized culture supernatants were incubated with 4.0 ϫ 10 5 U937-derived macrophages (24-well plate) in the presence of 50 M dCF. Cells were incubated for 18 h at 37°C under 5% CO 2 . Cells were detached using trypsin-EDTA solution, and killing efficiency was quantified with trypan blue staining.
Immunoblotting. U937-derived macrophages or BMDM were detached using trypsin-EDTA solution (U937) or 1ϫ PBS containing 1 mM EDTA (BMDM), washed twice in ice-cold 1ϫ PBS, and lysed for 20 min in ice-cold lysis buffer ( ). During this procedure, cells were kept on ice. Cell lysates were centrifuged for 10 min at 18,000 ϫ g and 4°C. Supernatants were mixed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) loading buffer and boiled at 95°C for 10 min. Proteins were separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes for immunoblot analysis with the following rabbit primary antibodies: anti-caspase-3 (anti-CASP3; for U937, antibody ab32351, and for BMDM, antibody ab13847, both from Abcam) and anti-GAPDH (loading control, PA1-987; Thermo Fisher; GAPDH, glyceraldehyde-3-phosphate dehydrogenase). Immunoreactive signals were revealed with a secondary antibody conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA, USA); horseradish peroxidase activity was detected with enhanced chemiluminescent (ECL) substrate.
Analysis of caspase-3 activity. Caspase-3 activity was determined using the colorimetric caspase-3 detection kit (Sigma). Briefly, U937-derived macrophages were incubated in growth medium for 24 h at 37°C under 5% CO 2 with dCF (50 M) and dAdo (10 M). Cells (1.0 ϫ 10 7 ), washed twice in ice-cold 1ϫ PBS, and lysed on ice for 20 min in lysis buffer (Sigma kit). Lysates were centrifuged (18,000 ϫ g for 10 min, 4°C) and supernatants incubated with the acetyl-DEVD-pNA substrate of caspase-3, according to the manufacturer's instructions. The caspase-3 inhibitor Ac-DEVD-CHO was used in control experiments (Sigma kit). Caspase-3 activity was measured in micromoles pNA released per minute per milliliter of cell lysate.
Animal experiments. All animal protocols were reviewed, approved, and performed under regulatory supervision of the University of Chicago's Institutional Biosafety Committee and Institutional Animal Care and Use Committee. CASP3 fl/fl or CASP3 fl/fl Tie2-Cre ϩ mice (C57BL/6 genetic background) (18) were obtained from Richard Flavell (Yale University, New Haven, CT) and Anthony Rongvaux (Fred Hutchinson Cancer Research Center, Seattle, WA). Mice were bred in a barrier facility at the University of Chicago. Prior to use, all animals were genotyped via PCR using the primers listed in Table S3, as described before (18). For disease studies, overnight cultures of wild-type S. aureus Newman or its adsA variant were diluted 1:100 in TSB and grown to an optical density at 600 nm of 0.5. Staphylococci were separated by centrifugation (10 min, RT, 8,000 ϫ g), washed twice in sterile PBS, and adjusted to 10 8 CFU/ml. Mice were anesthetized by intraperitoneal injection of 80 to 120 mg ketamine and 3 to 6 mg xylazine per kilogram of body weight. One hundred microliters of bacterial suspension (10 7 CFU) was administered intravenously via retro-orbital injection into 6-to 8-week-old and sex-matched CASP3 fl/fl or CASP3 fl/fl Tie2-Cre ϩ mice. At 5 days postinfection, the mice were euthanized. Kidneys were dissected and homogenized in sterile PBS containing 0.1% Triton X-100. Serial dilutions were prepared and plated on tryptic soy agar (TSA) for enumeration of staphylococci. For histopathology and immunohistochemistry, dissected kidneys were fixed in 10% formalin (Fisher Scientific), embedded into paraffin, and thin sectioned. Thin sections of renal tissues were stained by the Human Tissue Resource Center (University of Chicago) with hematoxylin and eosin, or with anti-Ly-6G (neutrophils, ab210204; Abcam) or anti-F4/80 (macrophages; MCA497GA; AbD Serotec) antibodies and examined by microscopy.
Histopathologic scoring. Microscopic images of renal tissue thin sections stained with hematoxylin and eosin or with anti-Ly-6G (neutrophils) or anti-F4/80 (macrophages) antibodies were analyzed using the CaseViewer software (version 2.3). To calculate the macrophage-infiltrated area per abscess lesion, the total and macrophage-free (anti-F4/80-negative) abscess areas were determined. The macrophageinfiltrated area per abscess is given in the percentage relative to the total abscess area.
Sequencing chromatograms and statistical analysis. Sequencing chromatograms were generated with DNAStar version 12.0.0 (DNAStar Software, Inc., Madison WI, USA). Statistical analysis was performed with Prism version 7.04 (GraphPad Software, Inc., La Jolla, CA, USA). Statistically significant differences were calculated by using statistical methods, as indicated. P values of Ͻ0.05 were considered significant.