Virulence Factors Produced by Staphylococcus aureus Biofilms Have a Moonlighting Function Contributing to Biofilm Integrity[S]

We comprehensively profiled intracellular and ECM proteomes of S. aureus flow biofilms and complemented these data by metabolic footprint analysis and phenotypic assays. We show that moonlighting, secreted virulence factors and ribosomal proteins within the ECM contribute to biofilm stabilization. Mechanistically, we propose that these alkaline proteins get protonated in an acidified ECM (because of the release of acids upon fermentation) mediating electrostatic interactions with anionic cell surface components, eDNA, and metabolites, which leads to cell aggregation and ECM stabilization. [Display omitted] Highlights •Establishment of a flow system allowing multi-omics analysis of S. aureus biofilms.•Biofilm proteome profiling (intracellular and ECM) plus metabolic footprint analysis.•Virulence factors and ribosomal proteins stabilize the ECM as moonlighting proteins.•They act as electrostatic bridges between anionic cell surfaces, eDNA and metabolites. Staphylococcus aureus is the causative agent of various biofilm-associated infections in humans causing major healthcare problems worldwide. This type of infection is inherently difficult to treat because of a reduced metabolic activity of biofilm-embedded cells and the protective nature of a surrounding extracellular matrix (ECM). However, little is known about S. aureus biofilm physiology and the proteinaceous composition of the ECM. Thus, we cultivated S. aureus biofilms in a flow system and comprehensively profiled intracellular and extracellular (ECM and flow-through (FT)) biofilm proteomes, as well as the extracellular metabolome compared with planktonic cultures. Our analyses revealed the expression of many pathogenicity factors within S. aureus biofilms as indicated by a high abundance of capsule biosynthesis proteins along with various secreted virulence factors, including hemolysins, leukotoxins, and lipases as a part of the ECM. The activity of ECM virulence factors was confirmed in a hemolysis assay and a Galleria mellonella pathogenicity model. In addition, we uncovered a so far unacknowledged moonlighting function of secreted virulence factors and ribosomal proteins trapped in the ECM: namely their contribution to biofilm integrity. Mechanistically, it was revealed that this stabilizing effect is mediated by the strong positive charge of alkaline virulence factors and ribosomal proteins in an acidic ECM environment, which is caused by the release of fermentation products like formate, lactate,