Single-cell approach dissecting agr quorum sensing dynamics in Staphylococcus aureus
- Julian Bär
- Mariane Pivard
- Samuel G. V. Charlton
- Andrew G. Kempchinsky
- Alejandro Gómez-Mejia
- Giovanni Stefano Ugolini
- Srikanth Mairpady Shambat
- Eleonora Secchi
- Annelies S. Zinkernagel
2026-05-20
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.