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

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.