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ABSTRACTStaphylococcus aureusis a leading cause of community- and nosocomial-acquired infections, with a propensity for biofilm formation. S. aureusbiofilms actively skew the host immune response toward an anti-inflammatory state; however, the biofilm effector molecules and the mechanism(s) of action responsible for this phenomenon remain to be fully defined. The essential bacterial second messenger cyclic diadenylate monophosphate (c-di-AMP) is an emerging pathogen-associated molecular pattern during intracellular bacterial infections, as c-di-AMP secretion into the infected host cytosol induces a robust type I interferon (IFN) response. Type I IFNs have the potential to exacerbate infectious outcomes by promoting anti-inflammatory effects; however, the type I IFN response to S. aureusbiofilms is unknown. Additionally, while several intracellular proteins function as c-di-AMP receptors in S. aureus, it has yet to be determined if any extracellular role for c-di-AMP exists and its release during biofilm formation has not yet been demonstrated. This study examined the possibility that c-di-AMP released during S. aureusbiofilm growth polarizes macrophages toward an anti-inflammatory phenotype via type I interferon signaling. DacA, the enzyme responsible for c-di-AMP synthesis in S. aureus, was highly expressed during biofilm growth, and 30 to 50% of total c-di-AMP produced from S. aureusbiofilm was released extracellularly due to autolytic activity. S. aureusbiofilm c-di-AMP release induced macrophage type I IFN expression via a STING-dependent pathway and promoted S. aureusintracellular survival in macrophages. These findings identify c-di-AMP as another mechanism for how S. aureusbiofilms promote macrophage anti-inflammatory activity, which likely contributes to biofilm persistence.

Staphylococcus aureusis an important human pathogen capable of colonizing diverse tissue types and inducing severe disease in both immunocompromised and otherwise healthy individuals. Biofilm infections caused by this bacterial species are of particular concern because of their persistence, even in the face of intensive therapeutic intervention. The results of the current study demonstrate the stochastic nature of Sae-mediated virulence gene expression in S. aureusand indicate that this regulatory system may function as a “bistable switch” in a manner similar to that seen with regulators controlling competence gene expression in Bacillus subtilisand persister cell formation in Escherichia coli. The results of this study provide a new perspective on the complex mechanisms utilized by S. aureusduring the establishment of infections.

ABSTRACTBiofilm communities contain distinct microniches that result in metabolic heterogeneity and variability in gene expression. Previously, these niches were visualized within Staphylococcus aureusbiofilms by observing differential expression of the cidand lrgoperons during tower formation. In the present study, we examined early biofilm development and identified two new stages (designated “multiplication” and “exodus”) that were associated with changes in matrix composition and a distinct reorganization of the cells as the biofilm matured. The initial attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Interestingly, after 6 h of growth, an exodus of the biofilm population that followed the transition of the biofilm to DNase I sensitivity was demonstrated. Furthermore, disruption of the gene encoding staphylococcal nuclease (nuc) abrogated this exodus event, causing hyperproliferation of the biofilm and disrupting normal tower development. Immediately prior to the exodus event, S. aureuscells carrying a nuc::gfppromoter fusion demonstrated Sae-dependent expression but only in an apparently random subpopulation of cells. In contrast to the existing model for tower development in S. aureus, the results of this study suggest the presence of a Sae-controlled nuclease-mediated exodus of biofilm cells that is required for the development of tower structures. Furthermore, these studies indicate that the differential expression of nucduring biofilm development is subject to stochastic regulatory mechanisms that are independent of the formation of metabolic microniches.IMPORTANCEIn this study, we provide a novel view of four early stages of biofilm formation by the human pathogen Staphylococcus aureus. We identified an initial nucleoprotein matrix during biofilm development that is DNase I insensitive until a critical point when a nuclease-mediated exodus of the population is induced prior to tower formation. Unlike the previously described dispersal of cells that occurs after tower development, we found that the mechanism controlling this exodus event is dependent on the Sae regulatory system and independent of Agr. In addition, we revealed that the gene encoding the secreted staphylococcal nuclease was expressed in only a subpopulation of cells, consistent with a model in which biofilms exhibit multicellular characteristics, including the presence of specialized cells and a division of labor that imparts functional consequences to the remainder of the population.