agr-Mediated Dispersal of Staphylococcus aureus Biofilms

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. A biofilm is a surface-attached community of cells bound together by an extracellular matrix. In a bacterial infection, biofilm-encased cells are protected from antibiotic therapy and host immune response, and these encased cells can develop into a chronic infection. Staphylococcus aureus is a prominent bacterial pathogen known to form biofilms on many medical implants and host tissues. In this report, we demonstrate that repression of the S. aureus quorum-sensing system is required to form a biofilm, and quorum-sensing reactivation in established biofilms disperses the cells. Genetic and molecular analysis demonstrates that quorum-sensing is activated before and required for the detachment mechanism. Detachment is protease-mediated, as established biofilms are sensitive to a non-specific protease and quorum-sensing activation increases the production of extracellular proteases. Using mutations in the protease genes, we show that these secreted enzymes are required for the detachment mechanism. These findings denote that S. aureus quorum-sensing can function as a dispersal mechanism to colonize new sites, and our results suggest this mechanism could be modulated to treat recalcitrant biofilms.