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High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin-antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.

Biofilm formation by Staphylococcus aureus involves the formation of an extracellular matrix, but the composition of this matrix has been uncertain. Here we report that the matrix is largely composed of cytoplasmic proteins that reversibly associate with the cell surface in a manner that depends on pH. We propose a model for biofilm formation in which cytoplasmic proteins are released from cells in stationary phase. These proteins associate with the cell surface in response to decreasing pH during biofilm formation. Rather than utilizing a dedicated matrix protein, S. aureus appears to recycle cytoplasmic proteins that moonlight as components of the extracellular matrix. IMPORTANCE Staphylococcus aureus is a leading cause of multiantibiotic-resistant nosocomial infections and is often found growing as a biofilm in catheters and chronic wounds. Biofilm formation is an important pathogenicity strategy that enhances resistance to antimicrobials, thereby limiting treatment options and ultimately contributing to increased morbidity and mortality. Cells in a biofilm are held together by an extracellular matrix that consists in whole or in part of protein, but the nature of the proteins in the S. aureus matrix is not well understood. Here we postulate that S. aureus recycles proteins from the cytoplasm to form the extracellular matrix. This strategy, of cytoplasmic proteins moonlighting as matrix proteins, could allow enhanced flexibility and adaptability for S. aureus in forming biofilms under infection conditions and could promote the formation of mixed-species biofilms in chronic wounds. Staphylococcus aureus is a leading cause of multiantibiotic-resistant nosocomial infections and is often found growing as a biofilm in catheters and chronic wounds. Biofilm formation is an important pathogenicity strategy that enhances resistance to antimicrobials, thereby limiting treatment options and ultimately contributing to increased morbidity and mortality. Cells in a biofilm are held together by an extracellular matrix that consists in whole or in part of protein, but the nature of the proteins in the S. aureus matrix is not well understood. Here we postulate that S. aureus recycles proteins from the cytoplasm to form the extracellular matrix. This strategy, of cytoplasmic proteins moonlighting as matrix proteins, could allow enhanced flexibility and adaptability for S. aureus in forming biofilms under infection conditions and could promote the formation of mixed-species biofilms in chronic wounds.

Bacteria naturally form communities of cells known as biofilms. However the physiological roles of biofilms produced by non-pathogenic microbiota remain largely unknown. To assess the impact of a biofilm on host physiology we explored the effect of several non-pathogenic biofilm-forming bacteria on Caenorhabditis elegans . We show that biofilm formation by Bacillus subtilis, Lactobacillus rhamnosus and Pseudomonas fluorescens induces C. elegans stress resistance. Biofilm also protects against pathogenic infection and prolongs lifespan. Total mRNA analysis identified a set of host genes that are upregulated in response to biofilm formation by B. subtilis . We further demonstrate that mtl-1 is responsible for the biofilm-mediated increase in oxidative stress resistance and lifespan extension. Induction of mtl-1 and hsp-70 promotes biofilm-mediated thermotolerance. ilys-2 activity accounts for biofilm-mediated resistance to Pseudomonas aeruginosa killing. These results reveal the importance of non-pathogenic biofilms for host physiology and provide a framework to study commensal biofilms in higher organisms.

Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered. Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam(3)CSK(4), are dependent upon an integrin, α(3)β(1). The mechanism for integrin α(3)β(1) involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam(3)CSK(4) is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane. Here we identify integrin α(3)β(1) as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α(3)β(1)-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α(3)β(1) serves as a mechanism for modulating inflammatory responses.

The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance.

Staphylococcus aureus is a leading cause of both nosocomial and community-acquired infection. Biofilm formation at the site of infection reduces antimicrobial susceptibility and can lead to chronic infection. During biofilm formation, a subset of cells liberate cytoplasmic proteins and DNA, which are repurposed to form the extracellular matrix that binds the remaining cells together in large clusters. Using a strain that forms robust biofilms in vitro during growth under glucose supplementation,we carried out a genome-wide screen for genes involved in the release of extracellular DNA (eDNA). A high-density transposon insertion library was grown under biofilm-inducing conditions, and the relative frequency of insertions was compared between genomic DNA (gDNA) collected from cells in the biofilm and eDNA from the matrix. Transposon insertions into genes encoding functions necessary for eDNA release were identified by reduced representation in the eDNA. On direct testing, mutants of some of these genes exhibited markedly reduced levels of eDNA and a concomitant reduction in cell clustering. Among the genes with robust mutant phenotypes were gdpP, which encodes a phosphodiesterase that degrades the second messenger cyclic-di-AMP, and xdrA, the gene for a transcription factor that, as revealed by RNA-sequencing analysis, influences the expression of multiple genes, including many involved in cell wall homeostasis. Finally, we report that growth in biofilm-inducing medium lowers cyclic-di-AMP levels and does so in a manner that depends on the gdpP phosphodiesterase gene.

Background Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered. Methodology/Principal Findings Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam3CSK4, are dependent upon an integrin, α3β1. The mechanism for integrin α3β1 involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam3CSK4 is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane. Conclusion/Significance Here we identify integrin α3β1 as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α3β1-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α3β1 serves as a mechanism for modulating inflammatory responses.

Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered. Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam.sub.3 CSK.sub.4, are dependent upon an integrin, [alpha].sub.3 [beta].sub.1 . The mechanism for integrin [alpha].sub.3 [beta].sub.1 involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam.sub.3 CSK.sub.4 is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane. Here we identify integrin [alpha].sub.3 [beta].sub.1 as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin [alpha].sub.3 [beta].sub.1 -mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin [alpha].sub.3 [beta].sub.1 serves as a mechanism for modulating inflammatory responses.

Staphylococcus aureus is a leading cause of nosocomial infections in the United States, and isolates of this pathogen are capable of forming biofilms through different mechanisms. Biofilm formation reduces susceptibility to antimicrobials, and can lead to chronic infections. Therefore, understanding the matrix components and genetic regulation of S. aureus biofilms is an important step towards combatting the rise in illness and deaths caused by S. aureus. My work has focused on the HG003 strain which forms robust biofilms in vitro. Here I present a model for biofilm formation in S. aureus that is dependent on a drop in pH over time driven by glucose fermentation, and on release of cytoplasmic proteins and DNA from a subset of cells that are recycled as matrix components and bind the remaining cells together in large clusters. Furthermore, though many clinical isolates of S. aureus rely on polysaccharide intercellular adhesin for matrix production, I describe how a mutant strain deleted for the genes encoding this component form biofilms in a polysaccharide-independent fashion. Following the development of the initial model, I examine the role of the major autolysins in biofilm production. Next, I describe a new unbiased genome-wide screening method for identification of genes involved in the release of extracellular DNA (eDNA) that leverages the ability to separate eDNA from biofilm cells and relies on transposon sequencing. I identify a set of genes likely to be required for eDNA release, and characterize their biofilm and eDNA phenotypes in deletion mutants. In addition to measuring biofilm-associated eDNA and biofilm biomass, I use microscopy to visualize how strains deficient in eDNA production have severe defects in cell-cell adhesion resulting in reduced clustering. I further evaluated mutant strains using a Congo red sensitivity assay to identify strains with enhanced cell wall strength. Finally, I characterize the regulon of a novel biofilm regulator using RNA sequencing. My research represents an advancement in understanding of staphylococcal biofilm formation and regulation, and presents a novel method for identifying biofilm-related genes that could serve as targets for future therapies designed to dissociate or prevent biofilms.