Staphylococcus aureus Multiplexes Death-Effector Deoxyribonucleosides to Neutralize Phagocytes

Abstract

Adenosine synthase A (AdsA) is a key virulence factor of Staphylococcus aureus, a dangerous microbe that causes fatal diseases in humans. Together with staphylococcal nuclease, AdsA generates deoxyadenosine (dAdo) from neutrophil extracellular DNA traps thereby igniting caspase-3-dependent cell death in host immune cells that aim at penetrating infectious foci. Powered by a multi-technological approach, we here illustrate that the enzymatic activity of AdsA in abscess-mimicking microenvironments is not restricted to the biogenesis of dAdo but rather comprises excessive biosynthesis of deoxyguanosine (dGuo), a cytotoxic deoxyribonucleoside generated by S. aureus to eradicate macrophages of human and animal origin. Based on a genome-wide CRISPR-Cas9 knock-out screen, we further demonstrate that dGuo-induced cytotoxicity in phagocytes involves targeting of the mammalian purine salvage pathway-apoptosis axis, a signaling cascade that is concomitantly stimulated by staphylococcal dAdo. Strikingly, synchronous targeting of this route by AdsA-derived dGuo and dAdo boosts macrophage cell death, indicating that S. aureus multiplexes death-effector deoxyribonucleosides to maximize intra-host survival. Overall, these data provide unique insights into the cunning lifestyle of a deadly pathogen and may help to design therapeutic intervention strategies to combat multidrug-resistant staphylococci.

Targeting host deoxycytidine kinase mitigatesStaphylococcus aureusabscess formation

“…More precisely, we incubated wild-type S. aureus Newman or its adsA variant in the presence or absence purine deoxyribonucleoside monophosphates (dAMP or dGMP) to obtain conditioned culture media, which were filter-sterilized and added to vehicle-or (R)-DI-87-exposed human U937 macrophages. In line with earlier studies (Thammavongsa et al, 2013;Winstel et al, 2018;Winstel et al, 2019;Tantawy et al, 2022), macrophage killing required purine deoxyribonucleoside monophosphate-conditioned media and adsAproficient staphylococci as only wild-type S. aureus Newman and dAMP-or dGMP-supplemented media triggered phagocyte cell death in this approach (Figure 2K-L). However, (R)-DI-87-exposed cells could not be killed in these experiments supporting the idea that pharmacological inhibition of host dCK can shield macrophages from S. aureus-and AdsA-driven cell death (Figure 2K-L).…”

“…Of note, (R)-DI-87-mediated inhibition of mammalian dCK in U937 or U937 MФ efficiently prevented dAdo-or dGuo-induced cell death in a dose-dependent manner (Figure 2A-D; supplementary Figure 1A-D). Moreover, these effects resembled the phenotype of dCK-deficient U937 or U937 MФ, which were found to be refractory to dAdo-or dGuo-mediated cytotoxicity (Figure 2A-D) (Winstel et al, 2018;Tantawy et al, 2022). In addition, we observed that pre-treatment of primary human CD14 + monocytes or human monocyte-derived macrophages (HMDMs) with (R)-DI-87 blocked dAdo-or dGuotriggered cytotoxicity suggesting that dCK inhibition may suppress the toxigenic properties of staphylococcal death-effector deoxyribonucleosides (Figure 2E-H).…”

“…Isolation of murine bone marrow-derived macrophages. Bone marrow-derived macrophages (BMDMs) were isolated from C57BL/6 mice as previously described (Tantawy et al, 2022). In brief, mice were euthanized to remove the femur and tibia which were sterilized by using 70% ethanol.…”

“…Protein purification. Purification of recombinant S. aureus AdsA was performed as described elsewhere (Thammavongsa et al, 2009;Tantawy et al, 2022). Briefly, a glutathione S-transferase (GST)-tagged and recombinant version of S. aureus AdsA (rAdsA) was expressed in E. coli BL21 using the pGEX-2T plasmid system (GE Healthcare) and purified via glutathione S-transferase affinity chromatography (Thammavongsa et al, 2009;Tantawy et al, 2022).…”

Anti-Inflammatory Metabolites in the Pathogenesis of Bacterial Infection

“…The Gram positive bacterium Staphylococcus aureus exploits adenosine accumulation through several mechanisms. S. aureus expresses a surface protein adenosine synthase A (AdsA) that generates adenosine from ATP, ADP and deoxyadenosine, as well as the cytotoxic deoxyguanosine ( Winstel et al., 2018 ; Tantawy et al., 2022 ). In a model of renal abscess and septicemia, the resulting NETosis provides S. aureus with a source of DNA and nucleosides for adenosine synthesis.…”