Abstract
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, and acetate because of oxygen limitation in biofilms. The strong positive charge of these proteins most likely mediates electrostatic interactions with anionic cell surface components, eDNA, and anionic metabolites. In consequence, this leads to strong cell aggregation and biofilm stabilization. Collectively, our study identified a new molecular mechanism during S. aureus biofilm formation and thus significantly widens the understanding of biofilm-associated S. aureus infections - an essential prerequisite for the development of novel antimicrobial therapies.
Highlights
Establishment of a flow system allowing multi-omics analysis of S. aureus biofilms. Biofilm proteome profiling plus metabolic footprint analysis
Establishing a flow system for highly reproducible cultivation of S. aureus biofilms enabled us to grow biofilms under conditions, which are relevant for different clinical scenarios, i.e. endocarditis or catheter-associated infections, thereby complementing studies employing static biofilm cultivation models
Using this multi-omics approach, we showed that S. aureus biofilms secrete high amounts of functional virulence factors like hemolysins, leukotoxins, and lipases, which are part of the extracellular matrix (ECM) but can be found in the biofilm FT
Summary
We comprehensively profiled intracellular and ECM proteomes of S. aureus flow biofilms and complemented these data by metabolic footprint analysis and phenotypic assays. The strong positive charge of these proteins most likely mediates electrostatic interactions with anionic cell surface components, eDNA, and anionic metabolites In consequence, this leads to strong cell aggregation and biofilm stabilization. The here presented study aims at a comprehensive characterization of the intra- and extracellular (ECM and flow-through (FT)) proteome, as well as the extracellular metabolome of S. aureus biofilms cultured in a physiologically highly relevant flow system [28] applying state-of-the-art omics technologies. We compared these biofilm protein and metabolite profiles to planktonic cells. Our analyses identified a so far nondescribed, molecular mechanism during biofilm formation, which uncovers moonlighting virulence factors and ribosomal proteins in the ECM as key players in mediating biofilm integrity
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