Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix
ABSTRACT 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...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2019-06-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.01137-19 |
| _version_ | 1818826023427375104 |
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| author | Jeffrey S. Kavanaugh Caralyn E. Flack Jessica Lister Erica B. Ricker Carolyn B. Ibberson Christian Jenul Derek E. Moormeier Elizabeth A. Delmain Kenneth W. Bayles Alexander R. Horswill |
| author_facet | Jeffrey S. Kavanaugh Caralyn E. Flack Jessica Lister Erica B. Ricker Carolyn B. Ibberson Christian Jenul Derek E. Moormeier Elizabeth A. Delmain Kenneth W. Bayles Alexander R. Horswill |
| author_sort | Jeffrey S. Kavanaugh |
| collection | DOAJ |
| description | ABSTRACT 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. |
| first_indexed | 2024-12-19T00:21:04Z |
| format | Article |
| id | doaj.art-a8e208da8e384b019ca1c75e6293f2c3 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-19T00:21:04Z |
| publishDate | 2019-06-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-a8e208da8e384b019ca1c75e6293f2c32022-12-21T20:45:32ZengAmerican Society for MicrobiologymBio2150-75112019-06-0110310.1128/mBio.01137-19Identification of Extracellular DNA-Binding Proteins in the Biofilm MatrixJeffrey S. Kavanaugh0Caralyn E. Flack1Jessica Lister2Erica B. Ricker3Carolyn B. Ibberson4Christian Jenul5Derek E. Moormeier6Elizabeth A. Delmain7Kenneth W. Bayles8Alexander R. Horswill9Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USADepartment of Microbiology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USADepartment of Microbiology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USADepartment of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USADepartment of Microbiology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USADepartment of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USADepartment of Microbiology and Pathology, University of Nebraska Medical Center, Omaha, Nebraska, USADepartment of Microbiology and Pathology, University of Nebraska Medical Center, Omaha, Nebraska, USADepartment of Microbiology and Pathology, University of Nebraska Medical Center, Omaha, Nebraska, USADepartment of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mBio.01137-19MRSASouthwestern blottingStaphylococcus aureusbiofilmseDNAextracellular DNA |
| spellingShingle | Jeffrey S. Kavanaugh Caralyn E. Flack Jessica Lister Erica B. Ricker Carolyn B. Ibberson Christian Jenul Derek E. Moormeier Elizabeth A. Delmain Kenneth W. Bayles Alexander R. Horswill Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix mBio MRSA Southwestern blotting Staphylococcus aureus biofilms eDNA extracellular DNA |
| title | Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix |
| title_full | Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix |
| title_fullStr | Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix |
| title_full_unstemmed | Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix |
| title_short | Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix |
| title_sort | identification of extracellular dna binding proteins in the biofilm matrix |
| topic | MRSA Southwestern blotting Staphylococcus aureus biofilms eDNA extracellular DNA |
| url | https://journals.asm.org/doi/10.1128/mBio.01137-19 |
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