Spatiotemporal Control of Staphylococcus aureus Biofilm Development

Chronic biofilm-related infections caused by the human pathogen Staphylococcus aureus often lead to significant increases in morbidity and mortality in both hospital- and community-associated settings. Typically, S. aureus biofilm development occurs in three stages: 1) attachment, 2) tower maturation, and 3) dispersal. Here, utilizing BioFlux1000 time-lapse microscopy we have expanded upon these fundamental stages of biofilm development and also unveiled and characterized two additional stages (multiplication and exodus). The attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Following multiplication, an exodus of the biofilm population that followed the transition of the biofilm to DNase I sensitivity was demonstrated. Furthermore, disruption of the gene encoding staphylococcal nuclease (nuc) abrogated this exodus event, causing hyper-proliferation of the biofilm and disrupting tower development. Prior to exodus, cells carrying a Pnuc::gfp promoter fusion demonstrated Sae-dependent expression, but only in a subpopulation of cells. Additionally, we also determined that other Sae-regulated genes demonstrated unique Sae-dependent stochastic expression patterns. Collectively, these results suggest the presence of a Sae-controlled nuclease-mediated exodus of a biofilm subpopulation that is required for tower development as well as controlling the stochastic expression of Sae-regulated factors. The cidABC and lrgAB operons have previously been shown to play specific roles in controlled cell death and release of extracellular DNA (eDNA) during biofilm maturation. Although the exact mechanisms controlling the cid and lrg operons have yet to be completely elucidated, the expression of the operons is dependent on altered metabolic cues as a result of overflow metabolism. We hypothesized that the differential expression of the cid and lrg operons within a biofilm is a function of the metabolic heterogeneity found within different biofilm microenvironments. Time-lapse epifluorescent images indicate that expression of these operons is specific to distinct regions of a growing biofilm. Additionally, these results revealed the existence of different tower types, possibly reflecting their different functional roles in development. Altogether, nuclease-mediated eDNA degradation modulates the biofilm to produce two distinct towers that there are both spatially and temporally different between compared to the rest of the biofilm

Temporal and stochastic control of Staphylococcus aureus biofilm development.

Biofilm communities contain distinct microniches that result in metabolic heterogeneity and variability in gene expression. Previously, these niches were visualized within Staphylococcus aureus biofilms by observing differential expression of the cid and lrg operons during tower formation. In the present study, we examined early biofilm development and identified two new stages (designated multiplication and exodus ) that were associated with changes in matrix composition and a distinct reorganization of the cells as the biofilm matured. The initial attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Interestingly, after 6 h of growth, an exodus of the biofilm population that followed the transition of the biofilm to DNase I sensitivity was demonstrated. Furthermore, disruption of the gene encoding staphylococcal nuclease (nuc) abrogated this exodus event, causing hyperproliferation of the biofilm and disrupting normal tower development. Immediately prior to the exodus event, S. aureus cells carrying a nuc::gfp promoter fusion demonstrated Sae-dependent expression but only in an apparently random subpopulation of cells. In contrast to the existing model for tower development in S. aureus, the results of this study suggest the presence of a Sae-controlled nuclease-mediated exodus of biofilm cells that is required for the development of tower structures. Furthermore, these studies indicate that the differential expression of nuc during biofilm development is subject to stochastic regulatory mechanisms that are independent of the formation of metabolic microniches. Importance: In this study, we provide a novel view of four early stages of biofilm formation by the human pathogen Staphylococcus aureus. We identified an initial nucleoprotein matrix during biofilm development that is DNase I insensitive until a critical point when a nuclease-mediated exodus of the population is induced prior to tower formation. Unlike the previously described dispersal of cells that occurs after tower development, we found that the mechanism controlling this exodus event is dependent on the Sae regulatory system and independent of Agr. In addition, we revealed that the gene encoding the secreted staphylococcal nuclease was expressed in only a subpopulation of cells, consistent with a model in which biofilms exhibit multicellular characteristics, including the presence of specialized cells and a division of labor that imparts functional consequences to the remainder of the population

Observations of Shear Stress Effects on Staphylococcus aureus Biofilm Formation

Staphylococcus aureus bacteria form biofilms and distinctive microcolony or tower structures that facilitate their ability to tolerate antibiotic treatment and to spread within the human body. The formation of microcolonies, which break off, get carried downstream, and serve to initiate biofilms in other parts of the body, is of particular interest here. It is known that flow conditions play a role in the development, dispersion, and propagation of biofilms in general. The influence of flow on microcolony formation and, ultimately, what factors lead to microcolony development are, however, not well understood. The hypothesis being examined is that microcolony structures form within a specific range of levels of shear stress. In this study, laminar shear flow over a range of 0.15 to 1.5 dynes/cm2 was examined. It was found that microcolony structures form in a narrow range of shear stresses around 0.6 dynes/cm2 Further, measurements of cell density as a function of space and time showed that shear dependence can be observed hours before microcolonies form. This is significant because, among other physiologic flows, this is the same shear stress found in large veins in the human vasculature, which, along with catheters of similar diameters and flow rates, may therefore play a critical role in biofilm development and subsequent spreading of infections throughout the body.IMPORTANCE It is well known that flow plays an important role in the formation, transportation, and dispersion of Staphylococcus aureus biofilms. What was heretofore not known was that the formation of tower structures in these biofilms is strongly shear stress dependent; there is, in fact, a narrow range of shear stresses in which the phenomenon occurs. This work quantifies the observed shear dependence in terms of cell growth, distribution, and fluid mechanics. It represents an important first step in opening up a line of questioning as to the interaction of fluid forces and their influence on the dynamics of tower formation, break-off, and transportation in biofilms by identifying the parameter space in which this phenomenon occurs. We have also introduced state-of-the-art flow measurement techniques to address this problem

Stochastic Expression of Sae-Dependent Virulence Genes during Staphylococcus aureus Biofilm Development Is Dependent on SaeS

The intricate process of biofilm formation in the human pathogen Staphylococcus aureus involves distinct stages during which a complex mixture of matrix molecules is produced and modified throughout the developmental cycle. Early in biofilm development, a subpopulation of cells detaches from its substrate in an event termed “exodus” that is mediated by SaePQRS-dependent stochastic expression of a secreted staphylococcal nuclease, which degrades extracellular DNA within the matrix, causing the release of cells and subsequently allowing for the formation of metabolically heterogenous microcolonies. Since the SaePQRS regulatory system is involved in the transcriptional control of multiple S. aureus virulence factors, the expression of several additional virulence genes was examined within a developing biofilm by introducing fluorescent gene reporter plasmids into wild-type S. aureus and isogenic regulatory mutants and growing these strains in a microfluidic system that supplies the bacteria with a constant flow of media while simultaneously imaging developing biofilms in 5-min intervals. This study demonstrated that multiple virulence genes, including nuc, were expressed stochastically within a specialized subpopulation of cells in nascent biofilms. We demonstrated that virulence genes regulated by SaePQRS were stochastically expressed in nearly all strains examined whereas Agr-regulated genes were expressed more homogenously within maturing microcolonies. The commonly used Newman strain contains a variant of SaeS (SaeSP) that confers constitutive kinase activity to the protein and caused this strain to lack the stochastic expression pattern observed in other strain backgrounds. Importantly, repair of the SaeSP allele resulting in reversion to the well-conserved SaeSL allele found in other strains restored stochastic expression in this strain

Impact of Vancomycin on sarA-Mediated Biofilm Formation: Role in Persistent Endovascular Infections Due to Methicillin-Resistant Staphylococcus aureus

Background. Staphylococcus aureus is the most common cause of endovascular infections. The staphylococcal accessory regulator A locus (sarA) is a major virulence determinant that may potentially impact methicillin-resistant S. aureus (MRSA) persistence in such infections via its influence on biofilm formation. Methods. Two healthcare-associated MRSA isolates from patients with persistent bacteremia and 2 prototypical community-acquired MRSA strains, as well as their respective isogenic sarA mutants, were studied for in vitro biofilm formation, fibronectin-binding capacity, autolysis, and protease and nuclease activities. These assays were done in the presence or absence of sub-minimum inhibitory concentrations (MICs) of vancomycin. In addition, these strain pairs were compared for intrinsic virulence and responses to vancomycin therapy in experimental infective endocarditis, a prototypical biofilm model. Results. All sarA mutants displayed significantly reduced biofilm formation and binding to fibronectin but increased protease production in vitro, compared with their respective parental strains. Interestingly, exposure to sub-MICs of vancomycin significantly promoted biofilm formation and fibronectin-binding in parental strains but not in sarA mutants. In addition, all sarA mutants became exquisitely susceptible to vancomycin therapy, compared with their respective parental strains, in the infective endocarditis model. Conclusions. These observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formatio

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

Inactivation of the Pta-AckA Pathway Causes Cell Death in \u3ci\u3eStaphylococcus aureus\u3c/i\u3e

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD+, and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism

Stochastic Expression of Sae-Dependent Virulence Genes during Staphylococcus aureus Biofilm Development Is Dependent on SaeS

Staphylococcus aureus is an important human pathogen capable of colonizing diverse tissue types and inducing severe disease in both immunocompromised and otherwise healthy individuals. Biofilm infections caused by this bacterial species are of particular concern because of their persistence, even in the face of intensive therapeutic intervention. The results of the current study demonstrate the stochastic nature of Sae-mediated virulence gene expression in S. aureus and indicate that this regulatory system may function as a “bistable switch” in a manner similar to that seen with regulators controlling competence gene expression in Bacillus subtilis and persister cell formation in Escherichia coli. The results of this study provide a new perspective on the complex mechanisms utilized by S. aureus during the establishment of infections.The intricate process of biofilm formation in the human pathogen Staphylococcus aureus involves distinct stages during which a complex mixture of matrix molecules is produced and modified throughout the developmental cycle. Early in biofilm development, a subpopulation of cells detaches from its substrate in an event termed “exodus” that is mediated by SaePQRS-dependent stochastic expression of a secreted staphylococcal nuclease, which degrades extracellular DNA within the matrix, causing the release of cells and subsequently allowing for the formation of metabolically heterogenous microcolonies. Since the SaePQRS regulatory system is involved in the transcriptional control of multiple S. aureus virulence factors, the expression of several additional virulence genes was examined within a developing biofilm by introducing fluorescent gene reporter plasmids into wild-type S. aureus and isogenic regulatory mutants and growing these strains in a microfluidic system that supplies the bacteria with a constant flow of media while simultaneously imaging developing biofilms in 5-min intervals. This study demonstrated that multiple virulence genes, including nuc, were expressed stochastically within a specialized subpopulation of cells in nascent biofilms. We demonstrated that virulence genes regulated by SaePQRS were stochastically expressed in nearly all strains examined whereas Agr-regulated genes were expressed more homogenously within maturing microcolonies. The commonly used Newman strain contains a variant of SaeS (SaeSP) that confers constitutive kinase activity to the protein and caused this strain to lack the stochastic expression pattern observed in other strain backgrounds. Importantly, repair of the SaeSP allele resulting in reversion to the well-conserved SaeSL allele found in other strains restored stochastic expression in this strain

Impact of Vancomycin on sarA-Mediated Biofilm Formation: Role in Persistent Endovascular Infections Due to Methicillin-Resistant Staphylococcus aureus

BackgroundStaphylococcus aureus is the most common cause of endovascular infections. The staphylococcal accessory regulator A locus (sarA) is a major virulence determinant that may potentially impact methicillin-resistant S. aureus (MRSA) persistence in such infections via its influence on biofilm formation.MethodsTwo healthcare-associated MRSA isolates from patients with persistent bacteremia and 2 prototypical community-acquired MRSA strains, as well as their respective isogenic sarA mutants, were studied for in vitro biofilm formation, fibronectin-binding capacity, autolysis, and protease and nuclease activities. These assays were done in the presence or absence of sub-minimum inhibitory concentrations (MICs) of vancomycin. In addition, these strain pairs were compared for intrinsic virulence and responses to vancomycin therapy in experimental infective endocarditis, a prototypical biofilm model.ResultsAll sarA mutants displayed significantly reduced biofilm formation and binding to fibronectin but increased protease production in vitro, compared with their respective parental strains. Interestingly, exposure to sub-MICs of vancomycin significantly promoted biofilm formation and fibronectin-binding in parental strains but not in sarA mutants. In addition, all sarA mutants became exquisitely susceptible to vancomycin therapy, compared with their respective parental strains, in the infective endocarditis model.ConclusionsThese observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formation

Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix

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.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