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 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.

Extracellular DNA promotes efficient extracellular electron transfer by pyocyanin in Pseudomonas aeruginosa biofilms

“…P. aeruginosa eDNA originates from the genomic DNA of dead cells and is therefore high molecular weight and may be bound by other biomolecules (e.g. proteins) (Kavanaugh et al, 2019). Therefore, DNase treatment was likely only partially effective because it could not cleave ds DNA in the presence of other bound matrix components, and/or because it did not have enough activity to degrade the eDNA completely to eliminate phenazine binding sites.…”

The Accessory Gene saeP of the SaeR/S Two-Component Gene Regulatory System Impacts Staphylococcus aureus Virulence During Neutrophil Interaction

“…Previously published in vitro studies suggest these proteins form a complex with SaeS that deactivates SaeR (Jeong et al, 2012). It has also been shown that increased expression of saeP impacts biofilm formation by increasing retention of high molecular weight DNA on the biofilm surface (Kavanaugh et al, 2019). The same study also demonstrated increases in saeP gene expression correlated with decreases in nuclease activity during biofilm development (Kavanaugh et al, 2019).…”

“…It has also been shown that increased expression of saeP impacts biofilm formation by increasing retention of high molecular weight DNA on the biofilm surface (Kavanaugh et al, 2019). The same study also demonstrated increases in saeP gene expression correlated with decreases in nuclease activity during biofilm development (Kavanaugh et al, 2019). Considering the importance of Sae during neutrophil interactions, we investigated the importance of saeP and saeQ during challenge with human neutrophils and in vivo using murine models of invasive disease and skin and softtissue infection.…”

“…Published data indicate that saeP encodes a lipoprotein anchored to the exterior surface of the plasma membrane, whereas saeQ encodes a transmembrane protein (Jeong et al, 2012;Kavanaugh et al, 2019). To investigate the roles of these genes, we utilized a saeP deletion mutant (USA300 saeP) generated previously in Kavanaugh et al (2019), and deleted the first third of saeQ (164 bp) in USA300 LAC using allelic replacement to create an isogenic saeQ deletion mutant (USA300 saeQ) that preserves the P3 promoter (Bae and Schneewind, 2006;Jeong et al, 2011). Absence of saeP and saeQ genes were verified by PCR ( Figure 1A).…”

Gene expression profiles based flux balance model to predict the carbon source for Bacillus subtilis

“…To achieve a novel treatment strategy, a good model is required. As mentioned, although 13 C metabolic data is very detailed, it is difficult to achieve and interpret results from 13 C isotopic tracer analysis; particularly, under this complicated environment (24,28) .…”