Single-cell approach dissecting agr quorum sensing dynamics in Staphylococcus aureus

Abstract

Abstract Quorum sensing (QS) enables bacteria to coordinate collective behaviors by secreting and sensing diffusible signals. Understanding QS at single-cell resolution is essential because population-level measurements often obscure regulatory heterogeneity. In Staphylococcus aureus , the accessory gene regulator ( agr ) system is a major QS-controlled virulence regulator activated by autoinducing peptides (AIPs). Four agr- types exist, each defined by distinct AIPs and capable of cross-inhibition, yet their activation dynamics and interaction hierarchies remain poorly understood. Using microfluidics, time-lapse microscopy, and deep-learning-based image analysis, we quantified agr -activation in congenic and native agr -type strains. Agr -types differed in sensitivity to their homologous AIPs: agr -III was largely unresponsive, whereas agr -IV was highly sensitive with elevated basal activation. Agr -activation distribution was frequently bimodal (simultaneous existence of agr -ON and agr- OFF cells), driven by subpopulations that never activated or switched to agr -OFF despite constant stimulation. Combining homologous and heterologous AIPs, AIP‑IV suppressed pre‑activated agr -I, while AIP-I had no inhibitory effect on agr ‑IV, indicating asymmetric cross‑inhibition. In spatially segregated cocultures, diffusional crosstalk resulted in four reproducible agr -interaction regimes, stable-dominance, stable-, delayed-, or unstable-dual-activation, determined by agr -type pairing and influenced by genetic background. Our approach links single-cell signaling to population outcomes and uncovers agr- type-specific asymmetries with potential consequences for strain competition and virulence.