Influence of Adhesion Force on icaA and cidA Gene Expression and Production of Matrix Components in Staphylococcus aureus Biofilms.

The majority of human infections are caused by biofilms. The biofilm mode of growth enhances the pathogenicity of Staphylococcus spp. considerably, because once they adhere, staphylococci embed themselves in a protective, self-produced matrix of extracellular polymeric substances (EPSs). The aim of this study was to investigate the influence of forces of staphylococcal adhesion to different biomaterials on icaA (which regulates the production of EPS matrix components) and cidA (which is associated with cell lysis and extracellular DNA [eDNA] release) gene expression in Staphylococcus aureus biofilms. Experiments were performed with S. aureus ATCC 12600 and its isogenic mutant, S. aureus ATCC 12600 Δpbp4, deficient in peptidoglycan cross-linking. Deletion of pbp4 was associated with greater cell wall deformability, while it did not affect the planktonic growth rate, biofilm formation, cell surface hydrophobicity, or zeta potential of the strains. The adhesion forces of S. aureus ATCC 12600 were the strongest on polyethylene (4.9 ± 0.5 nN), intermediate on polymethylmethacrylate (3.1 ± 0.7 nN), and the weakest on stainless steel (1.3 ± 0.2 nN). The production of poly-N-acetylglucosamine, eDNA presence, and expression of icaA genes decreased with increasing adhesion forces. However, no relation between adhesion forces and cidA expression was observed. The adhesion forces of the isogenic mutant S. aureus ATCC 12600 Δpbp4 (deficient in peptidoglycan cross-linking) were much weaker than those of the parent strain and did not show any correlation with the production of poly-N-acetylglucosamine, eDNA presence, or expression of the icaA and cidA genes. This suggests that adhesion forces modulate the production of the matrix molecule poly-N-acetylglucosamine, eDNA presence, and icaA gene expression by inducing nanoscale cell wall deformation, with cross-linked peptidoglycan layers playing a pivotal role in this adhesion force sensing.

Harapanahalli AK ，Chen Y ，Li J ，Busscher HJ ，van der Mei HC ... -  《-》

Temporal expression of agrB, cidA, and alsS in the early development of Staphylococcus aureus UAMS-1 biofilm formation and the structural role of extracellular DNA and carbohydrates.

Extracellular DNA (eDNA) is an important component of the extracellular polymeric substance matrix and is important in the establishment and persistence of Staphylococcus aureus UAMS-1 biofilms. The aim of the study was to determine the temporal expression of genes involved in early biofilm formation and eDNA production. We used qPCR to investigate expression of agrB, which is associated with secreted virulence factors and biofilm dispersal, cidA, which is associated with biofilm adherence and genomic DNA release, and alsS, which is associated with cell lysis, eDNA release and acid tolerance. The contribution of eDNA to the stability of the biofilm matrix was assessed by digesting with DNase I (Pulmozyme) and quantifying structure by confocal microscopy and comstat image analysis. AgrB expression initially increased at 24 h but then dramatically decreased at 72 h in an inverse relationship to biomass, supporting its role in regulating biofilm dispersal. cidA and alsS expression steadily increased over 72 h, suggesting that eDNA was an important component of early biofilm development. DNase I had no effect on biomass, but did cause the biofilms to become more heterogeneous. Carbohydrates in the matrix appeared to play an important role in structural stability.

Grande R ，Nistico L ，Sambanthamoorthy K ，Longwell M ，Iannitelli A ，Cellini L ，Di Stefano A ，Hall Stoodley L ，Stoodley P ... -  《-》

[Comparison of genotypic and phenotypic characteristics in biofilm production of Staphylococcus aureus isolates].

Şahin R ，Kaleli İ 《MIKROBIYOLOJI BULTENI》

Emergent Properties in Streptococcus mutans Biofilms Are Controlled through Adhesion Force Sensing by Initial Colonizers.

Bacterial adhesion is accompanied by altered gene expression, leading to "emergent" properties of biofilm bacteria that are alien to planktonic ones. With the aim of revealing the role of environmental adhesion forces in emergent biofilm properties, genes in Streptococcus mutans UA159 and a quorum-sensing-deficient mutant were identified that become expressed after adhesion to substratum surfaces. Using atomic force microscopy, adhesion forces of initial S. mutans colonizers on four different substrata were determined and related to gene expression. Adhesion forces upon initial contact were similarly low across different substrata, ranging between 0.2 and 1.2 nN regardless of the strain considered. Bond maturation required up to 21 s, depending on the strain and substratum surface involved, but stationary adhesion forces also were similar in the parent and in the mutant strain. However, stationary adhesion forces were largest on hydrophobic silicone rubber (19 to 20 nN), while being smallest on hydrophilic glass (3 to 4 nN). brpA gene expression in thin (34 to 48 μm) 5-h S. mutans UA159 biofilms was most sensitive to adhesion forces, while expression of gbpB and comDE expressions was weakly sensitive. ftf, gtfB, vicR, and relA expression was insensitive to adhesion forces. In thicker (98 to 151 μm) 24-h biofilms, adhesion-force-induced gene expression and emergent extracellular polymeric substance (EPS) production were limited to the first 20 to 30 μm above a substratum surface. In the quorum-sensing-deficient S. mutans, adhesion-force-controlled gene expression was absent in both 5- and 24-h biofilms. Thus, initial colonizers of substratum surfaces sense adhesion forces that externally trigger emergent biofilm properties over a limited distance above a substratum surface through quorum sensing.IMPORTANCE A new concept in biofilm science is introduced: "adhesion force sensitivity of genes," defining the degree up to which expression of different genes in adhering bacteria is controlled by the environmental adhesion forces they experience. Analysis of gene expression as a function of height in a biofilm showed that the information about the substratum surface to which initially adhering bacteria adhere is passed up to a biofilm height of 20 to 30 μm above a substratum surface, highlighting the importance and limitations of cell-to-cell communication in a biofilm. Bacteria in a biofilm mode of growth, as opposed to planktonic growth, are responsible for the great majority of human infections, predicted to become the number one cause of death in 2050. The concept of adhesion force sensitivity of genes provides better understanding of bacterial adaptation in biofilms, direly needed for the design of improved therapeutic measures that evade the recalcitrance of biofilm bacteria to antimicrobials.

Wang C ，Hou J ，van der Mei HC ，Busscher HJ ，Ren Y ... -  《-》

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》