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Extracellular ATP (eATP) acts as a Danger Associated Molecular Pattern. In mammalian cells eATP binds to purinergic receptors located on the cell surface, starting signalling cascades and modulating inflammation. We have previously shown that intestinal epithelial cells secrete ATP through connexin hemichannels during infection with bacterial pathogens, triggering inflammation. Additionally, gut bacteria release ATP during growth. Whether intestinal bacteria can regulate the eATP in the gut to dampen inflammation, and how, is poorly studied. Here, we show that Escherichia coli is able to enzymatically degrade eATP to hypoxanthine. To identify the factors involved, we screened a genome-wide mutant library of non-pathogenic E. coli BW25113. This revealed the key enzymes in the eATP degradation pathway, and identified potential transporters and regulators. The roles of the factors were confirmed using biochemical and genetic techniques, with special emphasis on the eATP degrading factor, which was purified and biochemically characterized. Degradation of eATP occurs in the bacterial envelope, and the responsible enzyme is not secreted. However, outer membrane vesicles carry the eATP-degrading enzyme, which could thus act remotely. We further bioinformatically identified homologues in various strains of pathogenic Gammaproteobacteria and tested their ability to degrade eATP. Finally, we confirmed the role of the eATP-degrading factor in modulating eATP concentrations in bacterial cultures, enterocyte infection models and in vivo mouse models during homeostasis and perturbation. This work expands our mechanistic knowledge of how bacteria regulate the eATP concentration in the gut, and the ensuing impact on the development of inflammation. 

In animals and plants, extracellular ATP (eATP) functions as a signal and regulates the immune response. During inflammation, intestinal bacteria are exposed to elevated eATP originating from the mucosa. However, whether bacteria respond to eATP is unclear. Here, we show that non-pathogenic Escherichia coli responds to eATP by modifying its transcriptional and metabolic landscapes. A genome-scale promoter library showed that the response is dependent on time, concentration, and medium and ATP specific. Second messengers and genes related to metabolism, biofilm formation, and envelope stress were regulated downstream of eATP. Metabolomics confirmed that eATP triggers enrichment of compounds with bioactive properties in the host or bacteria. Combined genome-scale modeling revealed modifications to global metabolic and biomass building blocks. Consequently, eATP altered the sensitivity to antibiotics and antimicrobial peptides. Finally, in pathogens, eATP controlled virulence factor expression. Our results indicate that eATP is an environmental cue in prokaryotes, which broadly regulates physiology, antimicrobial resistance, and virulence.

The endogenous danger signal extracellular ATP (eATP) acts as proinflammatory mediator in immune and inflammatory diseases and promotes coagulation. However, its role during viral infection is poorly understood and clinical data on eATP are scarce. Here, we employed a recently established approach to perform same-day measurements of eATP in blood plasma in a cohort of 400 SARS-CoV-2 positive patients (the CoVUm cohort), established at the Norrlands University Hospital in Umeå, Sweden. Periodic eATP quantifications spanned the acute disease phase to one year after infection. This showed that eATP levels in SARS-CoV-2- positive patients were elevated during acute disease with variations between severity groups. Strikingly, average eATP levels remained high in all severity groups for up to a year. Heightened eATP positively correlated with initiation of humoral responses, markers of thrombotic, heart, and kidney dysfunction and of chronic but not acute inflammation. Furthermore, eATP levels peaked after anti-SARS-CoV-2 vaccination. Together, these findings show the multifaceted and dynamic role of eATP in COVID-19 and provide new angles to understand the dysregulated coagulation and immune responses.