Mechanistic Insights into Impaired cGAS Activation in Staphylococcus aureus Biofilm Environments Reveal That STING Activation via 2′3′-cGAMP Restores Macrophage Immune Responses

Abstract Biofilm formation is a major cause of chronic implant-related bone infections and is associated with impaired immune responses. In a previous study, we identified the cGAS–STING pathway as a potential therapeutic target, as its activation—observed in response to planktonic Staphylococcus aureus (SA)—was absent in the corresponding biofilm setting. The present study aimed to identify potential mechanisms underlying the lack of cGAS activation in the biofilm environment. As biofilm-derived nucleases might degrade cGAS ligands, we assessed presence and activity of micrococcal nuclease in conditioned media from planktonic and biofilm-grown SA and evaluated the impact of extracellular DNases on cGAS pathway activation in macrophages. In addition, we examined altered cGAS expression, the requirement for continuous biofilm exposure and potential downstream inhibition resulting from degradation of the cGAS product. Biofilm formation was associated with dynamic nuclease expression, and exposure to the biofilm environment led to reduced cGAS levels in macrophages, accompanied by a lack of interferon response. Exogenous cGAS activation by G3-YSD failed to restore signaling, independent of nuclease activity or continuous biofilm exposure. In contrast, supplementation with the cGAS product and STING ligand 2′3′-cGAMP fully restored interferon responses and enhanced macrophage activation, indicating that increased degradation of the second messenger in the biofilm environment is not responsible for impaired pathway activation. Similar effects observed with Staphylococcus epidermidis and primary macrophages suggest a broader mechanism that is not SA- or cell line–specific. In conclusion, our data provide novel mechanistic insight into biofilm-mediated impairment of cGAS–STING signaling, revealing a previously unrecognized mechanism of immune evasion in staphylococcal biofilms. These findings extend our previous work and support the therapeutic potential of targeting STING as promising strategy to restore immune responses in chronic implant-related bone infections. Highlights Biofilm-derived factors impair cGAS–STING pathway activation and suppress interferon responses in macrophages. Impaired signaling is not primarily explained by extracellular micrococcal nuclease–mediated degradation of potential cGAS ligands. Biofilm exposure reduces cGAS expression levels and inhibits exogenous cGAS activation independently of continuous presence. Exogenous 2′3′-cGAMP fully restores interferon responses, indicating that impaired signaling is not due to degradation of the cGAS product. Direct activation of STING broadly enhances macrophage activation and by this could amplify overall immune responses. Bypassing cGAS via direct STING targeting represents a potential therapeutic strategy to overcome immune evasion in chronic implant-related bone infections.