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Staphopains modulate Staphylococcus aureus biofilm integrity.

ABSTRACT: Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (?sigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ?sigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ?sigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture.

SUBMITTER: Mootz JM

PROVIDER: S-EPMC3754231 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Staphopains modulate Staphylococcus aureus biofilm integrity.

Mootz Joe M JM Malone Cheryl L CL Shaw Lindsey N LN Horswill Alexander R AR

Infection and immunity 20130624 9

Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address ...[more]

PMID: 23798534

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Project description: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.

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Staphopains modulate Staphylococcus aureus biofilm integrity.

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Staphopains modulate Staphylococcus aureus biofilm integrity.

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