The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation

Eisenbeis, Janina; Saffarzadeh, Mona; Peisker, Henrik; Jung, Philipp; Thewes, Nicolas; Preissner, Klaus T.; Herrmann, Mathias; Molle, Virginie; Geisbrecht, Brian V.; Jacobs, Karin; Bischoff, Markus

doi:10.3389/fcimb.2018.00235

Cited by 51 publications

(35 citation statements)

References 52 publications

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“…Four of the ribosomal proteins and an additional two nonribosomal cytoplasmic proteins in the list (see the Foulston et al column in Table S1) were previously identified as being part of the biofilm matrix by the studies that led to the development of the electrostatic net model (33, 34), indicating that the proteins identified by the SW technique also corroborated approximately 20% of the cytoplasmic proteins identified in this study. Importantly, a number of proteins known to bind DNA were identified, including Atl (bands 1, 8, and 9) (36), SarA (band 5) (37), IsaB (band 5) (38), Eap (band 1) (39), and the phenol-soluble modulins (PSMs; band 11) (40). Interestingly, in the case of PSMs, it was further shown that the binding of PSMs to DNA protected the DNA from digestion by DNase (40), and in the case of Eap, it was found that this protein can condense extended DNA strands (39), raising the possibility that Eap might also afford protection from host DNase.…”

Section: Resultsmentioning

confidence: 99%

“…Importantly, a number of proteins known to bind DNA were identified, including Atl (bands 1, 8, and 9) (36), SarA (band 5) (37), IsaB (band 5) (38), Eap (band 1) (39), and the phenol-soluble modulins (PSMs; band 11) (40). Interestingly, in the case of PSMs, it was further shown that the binding of PSMs to DNA protected the DNA from digestion by DNase (40), and in the case of Eap, it was found that this protein can condense extended DNA strands (39), raising the possibility that Eap might also afford protection from host DNase.…”

Section: Resultsmentioning

confidence: 99%

“…We examined two of the better-characterized proteins identified in more detail, starting with the extracellular adherence protein (Eap) and immunodominant surface protein B (IsaB). Both Eap and IsaB are characterized, secreted proteins, and both are known to bind DNA (38, 39). Coupled with our SW analysis results (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix

Kavanaugh

Flack

Lister

et al. 2019

mBio

135

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix

Kavanaugh

Flack

Lister

et al. 2019

mBio

135

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“…Recently, it has been demonstrated that S. aureus extracellular protein Eap shows a neutrophil serine protease inhibitor activity in association with NET functions (49) and binds and aggregates DNA, thus blocking neutrophil extracellular trap formation (50). With this study, FnBPB is added to the list of potential factors that inhibit this important mechanism of host innate defense.…”

Section: Interactions Of Histones With S Aureus Fnbpbmentioning

confidence: 99%

Fibronectin-binding protein B (FnBPB) from Staphylococcus aureus protects against the antimicrobial activity of histones

Pietrocola

Nobile

Alfeo

et al. 2019

Journal of Biological Chemistry

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Staphylococcus aureus is a Gram-positive bacterium that can cause both superficial and deep-seated infections. Histones released by neutrophils kill bacteria by binding to the bacterial cell surface and causing membrane damage. We postulated that cell wall-anchored proteins protect S. aureus from the bactericidal effects of histones by binding to and sequestering histones away from the cell envelope. Here, we focused on S. aureus strain LAC and by using an array of biochemical assays, including surface plasmon resonance and ELISA, discovered that fibronectin-binding protein B (FnBPB) is the main histone receptor. FnBPB bound all types of histones, but histone H3 displayed the highest affinity and bactericidal activity and was therefore investigated further. H3 bound specifically to the A domain of recombinant FnBPB with a K D of 86 nM, ϳ20-fold lower than that for fibrinogen. Binding apparently occurred by the same mechanism by which FnBPB binds to fibrinogen, because FnBPB variants defective in fibrinogen binding also did not bind H3. An FnBPB-deletion mutant of S. aureus LAC bound less H3 and was more susceptible to its bactericidal activity and to neutrophil extracellular traps, whereas an FnBPBoverexpressing mutant bound more H3 and was more resistant than the WT. FnBPB bound simultaneously to H3 and plasminogen, which after activation by tissue plasminogen activator cleaved the bound histone. We conclude that FnBPB provides a dual immune-evasion function that captures histones and prevents them from reaching the bacterial membrane and simultaneously binds plasminogen, thereby promoting its conversion to plasmin to destroy the bound histone. Staphylococcus aureus is a leading cause of diverse infections ranging from mild skin diseases such as impetigo, cellulitis, and skin abscesses to serious invasive diseases, including sepsis, endocarditis, osteomyelitis, toxic shock syndrome, and necro-tizing pneumoniae (1, 2). Strains that are resistant to multiple antibiotics are a major problem in healthcare settings in developed countries (3). These are referred to as hospital-associated methicillin-resistant S. aureus (MRSA) 3 and occur in individuals with pre-disposing risk factors, such as surgical wounds and indwelling medical devices (4, 5). Recently, there has been a dramatic increase in the incidence of community-associated MRSA infections that occur in otherwise healthy individuals (6, 7). Community-associated MRSA strains, exemplified by the USA300 clone (8), express a low level of resistance to ␤-lactam antibiotics and cause serious skin and soft tissue infections (9-11). S. aureus expresses a plethora of virulence factors, including both secreted and cell wall-anchored (CWA) proteins. The latter mediate adherence to the extracellular matrix, promote invasion of and survival within host cells, neutralize phagocytes, and modulate the immune response (12). CWA proteins are covalently anchored to peptidoglycan via a conserved C-terminal sorting signal mediated by the membrane-associated sortase A (13). Fibronec...

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“…S1), suggesting two possibilities, that Eap and SasG interact with and protect eDNA from nucleases and that the importance of eDNA is masked by these proteins. Recently, it was reported that Eap can bind to DNA and is capable of blocking neutrophil extracellular trap (NET) formation (44). However, the DNA binding capacity of SasG has not yet been elucidated.…”

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confidence: 99%

Redundant and Distinct Roles of Secreted Protein Eap and Cell Wall-Anchored Protein SasG in Biofilm Formation and Pathogenicity of Staphylococcus aureus

et al. 2019

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Chronic and fatal infections caused by Staphylococcus aureus are sometimes associated with biofilm formation. Secreted proteins and cell wall-anchored proteins (CWAPs) are important for the development of polysaccharide-independent biofilms, but functional relationships between these proteins are unclear. In the present study, we report the roles of the extracellular adherence protein Eap and the surface CWAP SasG in S. aureus MR23, a clinical methicillin-resistant isolate that forms a robust protein-dependent biofilm and accumulates a large amount of Eap in the extracellular matrix. Double deletion of eap and sasG, but not single eap or sasG deletion, reduced the biomass of the formed biofilm. Mutational analysis demonstrated that cell wall anchorage is essential for the role of SasG in biofilm formation. Confocal laser scanning microscopy revealed that MR23 formed a rugged and thick biofilm; deletion of both eap and sasG reduced biofilm ruggedness and thickness. Although sasG deletion did not affect either of these features, eap deletion reduced the ruggedness but not the thickness of the biofilm. This indicated that Eap contributes to the rough irregular surface structure of the MR23 biofilm and that both Eap and SasG play roles in biofilm thickness. The level of pathogenicity of the Δeap ΔsasG strain in a silkworm larval infection model was significantly lower (P Ͻ 0.05) than those of the wild type and single-deletion mutants. Collectively, these findings highlight the redundant and distinct roles of a secreted protein and a CWAP in biofilm formation and pathogenicity of S. aureus and may inform new strategies to control staphylococcal biofilm infections.

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The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation

Cited by 51 publications

References 52 publications

Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix

Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix

Fibronectin-binding protein B (FnBPB) from Staphylococcus aureus protects against the antimicrobial activity of histones

Redundant and Distinct Roles of Secreted Protein Eap and Cell Wall-Anchored Protein SasG in Biofilm Formation and Pathogenicity of Staphylococcus aureus

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