Methicillin-resistant Staphylococcus pseudintermedius synthesizes deoxyadenosine to cause persistent infection

Abstract

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an emerging zoonotic pathogen of canine origin that causes an array of fatal diseases, including bacteremia and endocarditis. Despite large-scale genome sequencing projects have gained substantial insights into the genomic landscape of MRSP, current knowledge on virulence determinants that contribute to S. pseudintermedius pathogenesis during human or canine infection is very limited. Using a panel of genetically engineered MRSP variants and a mouse abscess model, we here identified the major secreted nuclease of S. pseudintermedius designated NucB and adenosine synthase A (AdsA) as two synergistically acting enzymes required for MRSP pathogenesis. Similar to Staphylococcus aureus, S. pseudintermedius requires nuclease secretion along with the activity of AdsA to degrade mammalian DNA for subsequent biosynthesis of cytotoxic deoxyadenosine. In this manner, S. pseudintermedius selectively kills macrophages during abscess formation thereby antagonizing crucial host immune cell responses. Ultimately, bioinformatics analyses revealed that NucB and AdsA are widespread in the global S. pseudintermedius population. Together, these data suggest that S. pseudintermedius deploys the canonical Nuc/AdsA pathway to persist during invasive disease and may aid in the development of new therapeutic strategies to combat infections caused by MRSP.

Staphylococcus aureus Multiplexes Death-Effector Deoxyribonucleosides to Neutralize Phagocytes

“…To investigate whether the AdsA-dependent hydrolysis of dGMP and coupled biosynthesis of dGuo can be recapitulated by using viable staphylococci, we generated a S. aureus Newman adsA mutant panel and made use of a previously described experimental approach ( 38 ). Specifically, we incubated wild-type S. aureus Newman and the appropriate adsA mutant with purified dGMP and assayed for the release of inorganic phosphate using a commercially available malachite green-based colorimetric assay.…”

“…Following this two-pronged strategy, staphylococci effectively block macrophage access to the bacterial abscess community located in the core of mature lesions without causing traitorous inflammatory signals, ultimately shaping the pathological architecture of abscesses in the skin or organ tissues. Since AdsA is also catalyzing the biosynthesis of immunosuppressive adenosine in blood ( 31 ), and homologues of AdsA were previously identified in other clinically relevant Gram-positive microbes including emerging methicillin-resistant Staphylococcus pseudintermedius , Bacillus anthracis , and several pathogenic streptococci ( 31 , 38 , 65 – 69 ), development of small molecule inhibitors or therapeutic monoclonal antibodies that neutralize AdsA may help to combat infections caused by MRSA and other drug-resistant bacterial pathogens.…”

“…The migratory positions of dGuo and dGMP were identified with pure dGuo and dGMP standards (Sigma) that were visualized under UV light at 254 nm. rAdsA-derived dGuo was further analyzed and quantified by LC-MS/MS analytics as described before ( 38 ). Briefly, rAdsA (1.4 µg/µl) was incubated with dGMP at 37°C in reaction buffer as described above.…”

“…Next, samples were centrifuged for 10 min at 16,000 x g. Hydrolysis of dGMP and the associated release of inorganic phosphate in resulting culture supernatants was analyzed using a colorimetric phosphate detection kit according to the manufacturer’s instructions (Abcam). AdsA-dependent formation of dGuo was further analyzed by using S. aureus -derived cell wall extracts using a method described elsewhere ( 31 , 38 ). Briefly, S. aureus overnight cultures were diluted 1:100 in TSB medium and grown to an optical density (600 nm) of 1.0.…”

Molecular Prerequisites for Neutrophil Extracellular Trap Formation and Evasion Mechanisms of Staphylococcus aureus

“…dAdo is a cytotoxic deoxyribonucleoside which exquisitely kills macrophages during abscess formation by targeting the purine salvage pathway and apoptotic signaling cascade ( 17 , 49 , 80 ). In this manner, S. aureus and related staphylococci not only prevent NET-mediated killing within abscesses, but rather exploit excretion of NETs to suppress phagocyte entry into deeper cavities of infectious foci, ultimately enhancing pathogen survival and establishment of persistent infections ( 17 , 49 , 81 ). Overall, staphylococcal Nuc represents a key determinant utilized by staphylococci to prevent NET-associated enmeshment and killing.…”