Staphylococcus aureus targets the purine salvage pathway to kill phagocytes

Significance Immune evasion by the human pathogen Staphylococcus aureus includes the synthesis of cytotoxic deoxyadenosine (dAdo) from neutrophil extracellular traps to eliminate macrophages. The signaling pathway that triggers dAdo-mediated apoptosis in macrophages is not known. By using a genome-wide CRISPR-Cas9 knockout screen, we demonstrate that S. aureus-derived dAdo targets the purine salvage pathway to provoke caspase-3–dependent cell death in human macrophages, thereby antagonizing host immune responses and promoting the establishment of invasive disease. Interference with the staphylococcal dAdo signaling pathway may boost macrophage survival and phagocytic clearance of bacteria, providing new strategies for the treatment of S. aureus infections.

Next generation sequencing (NGS), Sanger sequencing and bioinformatics. DNA samples obtained from the CRISPR-Cas9 GeCKO screen were used to prepare a sgRNA library with a two-step PCR protocol (1,2). The first PCR was used to amplify the sgRNA containing cassette. Amplified products were subjected to a second PCR with a primer pair encoding a unique 8-bp barcode required for multiplexing along with a stagger sequence to increase library complexity, as described [http://genome-engineering.org/gecko; (1,2)]. Products of the second PCR were purified, pooled, diluted and mixed with 10% PhiX, and sequenced with HiSeq2500 The raw sequencing data were processed and analyzed using customized CRI CRISPR-Cas9 library screen pipelines. Briefly, sequencing reads were first de-multiplexed by using the barcode in the reverse primer, and processed by Cutadapt (3) to remove sequences from beginning to sgRNA priming site primers. Trimmed reads were used to map sgRNA sequences to pooled GeCKO v2 libraries A and B. Read counts of sgRNA for each sample were quantified by MAGeCK v5.6.0 (4). Count data were filtered and normalized, and essential sgRNA and genes were ranked by MAGeCK (4).
DNA samples obtained from the CRISPR-Cas9 GeCKO screen were also used to analyze the distribution of individual sgRNAs via Sanger sequencing. Here, the sgRNA cassette was PCRamplified from the dAdo-resistant U937 cell population, and cloned via the Zero Blunt™ TOPO™ PCR Cloning Kit (Thermo Fisher). 80 independent E. coli-derived plasmids containing the sgRNA cassette were sequenced, and used to identify genes targeted by the individual sgRNA. All sgRNA sequences recovered were mapped to pooled GeCKO v2 libraries A and B.
Immunoblotting. U937 cells were washed twice in ice-cold 1  PBS, and lysed for 30 min in ice-cold lysis buffer (1  PBS and 0.5 % Triton X-100) containing the protease inhibitor cocktail from Pierce. During this procedure, cells were sonicated 6 times (5 seconds pulse each) by using an ultrasonicator instrument. Cell lysates were centrifuged for 10 min at 15,000 g and 4°C.
Dead cells were stained with Trypan Blue, and counted by using a microscope to calculate killing efficiency.
To analyze dAdo-mediated cytotoxicity of U937-derived macrophages, 4.0  10 5 U937 cells per well were seeded in a 24-well plate, and incubated for 48 h at 37°C under 5% CO2 in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum and 160 nM phorbol 12- aureus Newman or mutant strains were grown overnight in TSB, diluted 1:100 in RPMI 1640 medium, and grown at 37°C to 5.0  10 7 colony forming units (CFU)/ml. Next, 6.0 x 10 7 CFU were incubated in RPMI 1640 containing 28 µg/ml Thymus DNA (Sigma) for 3 h at 37°C. Controls were equally processed, and lacked bacteria or DNA, or included S. aureus mutants that cannot generate dAdo precursors (nuc) or cannot produce dAdo (adsA). Bacteria were removed by centrifugation, culture supernatants filter-sterilized and incubated with 2.0  10 5 U937 wildtype or mutant cell lines per well, or with 4.0  10 5 U937-derived macrophages (24-well plate) in the presence of 50 µM dCF. Cells were incubated for 24 h at 37°C under 5% CO2, and killing efficiency was quantified with Trypan Blue staining.
Analysis of caspase-3 activity. Caspase-3 activity was determined using the colorimetric caspase-3 detection kit (Sigma). Briefly, wild-type and mutant U937 cells were incubated for 24 h at 37°C under 5% CO2 with dCF (50 µM) and dAdo (5 and 10 µM for U937 cells and U937derived macrophages, respectively). Cells (1.0  10 7 ) were washed once in PBS and lysed on ice for 20 min in lysis buffer (Sigma kit). Lysates were centrifuged (18,000 g for 10 min at 4°C) and supernatants incubated with acetyl-DEVD-pNA, a substrate of caspase-3. The capase-3 inhibitor Ac-DEVD-CHO was used in control experiments (Sigma kit). Caspase-3 activity was measured in μmol of pNA released per min per ml of cell lysate.