Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix.

We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms.

Kavanaugh JS ，Flack CE ，Lister J ，Ricker EB ，Ibberson CB ，Jenul C ，Moormeier DE ，Delmain EA ，Bayles KW ，Horswill AR ... -  《mBio》

Genetic and Biochemical Analysis of CodY-Mediated Cell Aggregation in Staphylococcus aureus Reveals an Interaction between Extracellular DNA and Polysaccharide in the Extracellular Matrix.

The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm.IMPORTANCEStaphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.

Mlynek KD ，Bulock LL ，Stone CJ ，Curran LJ ，Sadykov MR ，Bayles KW ，Brinsmade SR ... -  《-》

An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen that can form biofilms on various surfaces. These cell communities are protected from the environment by a self-produced extracellular matrix composed of proteins, DNA, and polysaccharide. The exact compositions and roles of the different components are not fully understood. In this study, we investigated the role of extracellular DNA (eDNA) and its interaction with the recently identified cytoplasmic proteins that have a moonlighting role in the biofilm matrix. These matrix proteins associate with the cell surface upon the drop in pH that naturally occurs during biofilm formation, and we found here that this association is independent of eDNA. Conversely, the association of eDNA with the matrix was dependent on matrix proteins. Both proteinase and DNase treatments severely reduced clumping of resuspended biofilms; highlighting the importance of both proteins and eDNA in connecting cells together. By adding an excess of exogenous DNA to DNase-treated biofilm, clumping was partially restored, confirming the crucial role of eDNA in the interconnection of cells. On the basis of our results, we propose that eDNA acts as an electrostatic net, interconnecting cells surrounded by positively charged matrix proteins at a low pH. Extracellular DNA (eDNA) is an important component of the biofilm matrix of diverse bacteria, but its role in biofilm formation is not well understood. Here we report that in Staphylococcus aureus, eDNA associates with cells in a manner that depends on matrix proteins and that eDNA is required to link cells together in the biofilm. These results confirm previous studies that showed that eDNA is an important component of the S. aureus biofilm matrix and also suggest that eDNA acts as an electrostatic net that tethers cells together via the proteinaceous layer of the biofilm matrix.

Dengler V ，Foulston L ，DeFrancesco AS ，Losick R ... -  《-》

Beyond the double helix: the multifaceted landscape of extracellular DNA in Staphylococcus aureus biofilms.

Staphylococcus aureus forms biofilms consisting of cells embedded in a matrix made of proteins, polysaccharides, lipids, and extracellular DNA (eDNA). Biofilm-associated infections are difficult to treat and can promote antibiotic resistance, resulting in negative healthcare outcomes. eDNA within the matrix contributes to the stability, growth, and immune-evasive properties of S. aureus biofilms. eDNA is released by autolysis, which is mediated by murein hydrolases that access the cell wall via membrane pores formed by holin-like proteins. The eDNA content of S. aureus biofilms varies among individual strains and is influenced by environmental conditions, including the presence of antibiotics. eDNA plays an important role in biofilm development and structure by acting as an electrostatic net that facilitates protein-cell and cell-cell interactions. Because of eDNA's structural importance in biofilms and its ubiquitous presence among S. aureus isolates, it is a potential target for therapeutics. Treatment of biofilms with DNase can eradicate or drastically reduce them in size. Additionally, antibodies that target DNABII proteins, which bind to and stabilize eDNA, can also disperse biofilms. This review discusses the recent literature on the release, structure, and function of eDNA in S. aureus biofilms, in addition to a discussion of potential avenues for targeting eDNA for biofilm eradication.

Bowden LC ，Finlinson J ，Jones B ，Berges BK ... -  《Frontiers in Cellular and Infection Microbiology》

Glycosyltransferase-Mediated Biofilm Matrix Dynamics and Virulence of Streptococcus mutans.

Streptococcus mutans is a key cariogenic bacterium responsible for the initiation of tooth decay. Biofilm formation is a crucial virulence property. We discovered a putative glycosyltransferase, SMU_833, in S. mutans capable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion of smu_833 decreases glucan and increases eDNA but maintains the overall biofilm biomass. The decrease in glucan is caused by a reduction in GtfB and GtfC, two key enzymes responsible for the synthesis of glucan. The increase in eDNA was accompanied by an elevated production of membrane vesicles, suggesting that SMU_833 modulates the release of eDNA via the membrane vesicles, thereby altering biofilm matrix constituents. Furthermore, glucan and eDNA were colocalized. The complete deletion of gtfBC from the smu_833 mutant significantly reduced the biofilm biomass despite the elevated eDNA, suggesting the requirement of minimal glucans as a binding substrate for eDNA within the biofilm. Despite no changes in overall biofilm biomass, the mutant biofilm was altered in biofilm architecture and was less acidic in vitro Concurrently, the mutant was less virulent in an in vivo rat model of dental caries, demonstrating that SMU_833 is a new virulence factor. Taken together, we conclude that SMU_833 is required for optimal biofilm development and virulence of S. mutans by modulating extracellular matrix components. Our study of SMU_833-modulated biofilm matrix dynamics uncovered a new target that can be used to develop potential therapeutics that prevent and treat dental caries.IMPORTANCE Tooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacterium Streptococcus mutans The bacteria utilize dietary sugars to build and strengthen biofilms, trapping acids onto the tooth's surface and causing demineralization and decay of teeth. As knowledge of our body's microbiomes increases, the need for developing therapeutics targeted to disease-causing bacteria has arisen. The significance of our research is in studying and identifying a novel therapeutic target, a dynamic biofilm matrix that is mediated by a new virulence factor and membrane vesicles. The study increases our understanding of S. mutans virulence and also offers a new opportunity to develop effective therapeutics targeting S. mutans In addition, the mechanisms of membrane vesicle-mediated biofilm matrix dynamics are also applicable to other biofilm-driven infectious diseases.

Rainey K ，Michalek SM ，Wen ZT ，Wu H ... -  《-》