agr-Mediated Dispersal of Staphylococcus aureus Biofilms

TLDR

The agr quorum‑sensing system regulates virulence factor expression and modulates biofilm dynamics, with agr mutants forming robust biofilms while dispersing cells display an active agr system. Detachment is driven by agr‑induced extracellular proteases, as evidenced by increased serine protease levels in detaching effluents and inhibition of detachment by the serine protease inhibitor PMSF and by loss of Aur and Spl proteases. Repressing agr is necessary for biofilm formation, whereas reactivating agr—by AIP addition or glucose depletion—triggers detachment across diverse strains, restores rifampicin sensitivity, and operates independently of ica, confirming agr‑mediated protease activity as the dispersal mechanism.

Abstract

The agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). Recent studies have suggested a role for the agr system in S. aureus biofilm development, as agr mutants exhibit a high propensity to form biofilms, and cells dispersing from a biofilm have been observed displaying an active agr system. Here, we report that repression of agr is necessary to form a biofilm and that reactivation of agr in established biofilms through AIP addition or glucose depletion triggers detachment. Inhibitory AIP molecules did not induce detachment and an agr mutant was non-responsive, indicating a dependence on a functional, active agr system for dispersal. Biofilm detachment occurred in multiple S. aureus strains possessing divergent agr systems, suggesting it is a general S. aureus phenomenon. Importantly, detachment also restored sensitivity of the dispersed cells to the antibiotic rifampicin. Proteinase K inhibited biofilm formation and dispersed established biofilms, suggesting agr-mediated detachment occurred in an ica-independent manner. Consistent with a protease-mediated mechanism, increased levels of serine proteases were detected in detaching biofilm effluents, and the serine protease inhibitor PMSF reduced the degree of agr-mediated detachment. Through genetic analysis, a double mutant in the agr-regulated Aur metalloprotease and the SplABCDEF serine proteases displayed minimal extracellular protease activity, improved biofilm formation, and a strongly attenuated detachment phenotype. These findings indicate that induction of the agr system in established S. aureus biofilms detaches cells and demonstrate that the dispersal mechanism requires extracellular protease activity.