Use of Microfluidic Technology To Analyze Gene Expression during Staphylococcus aureus Biofilm Formation Reveals Distinct Physiological Niches

Moormeier, Derek E.; Endres, Jennifer L.; Mann, Ethan E.; Sadykov, Marat R.; Horswill, Alexander R.; Rice, Kelly C.; Fey, Paul D.; Bayles, Kenneth W.

doi:10.1128/aem.00395-13

Cited by 88 publications

(94 citation statements)

References 50 publications

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“…For example, the circulation of blood around vascular catheters exerts shear flow forces on developing biofilms, replenishes nutrients, and removes bacterial waste products and signaling molecules-none of which are reproduced by the microtiter plate method. Therefore, we developed a biofilm model using a flow-based live cell imaging system (BioFlux 1000; Fluxion) (13,14) and performed quantitative comparisons among new and previously described targets using time-lapse video microscopy for longitudinal monitoring of the complete biofilm development cycle. This flow-based assay was further adapted to assess potential host-pathogen interactions via the introduction of freshly isolated human neutrophils to the system.…”

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confidence: 99%

Antibodies to PhnD Inhibit Staphylococcal Biofilms

et al. 2014

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Biofilm formation on central lines or peripheral catheters is a serious threat to patient well-being. Contaminated vascular devices can act as a nidus for bloodstream infection and systemic pathogen dissemination. Staphylococcal biofilms are the most common cause of central-line-associated bloodstream infections, and antibiotic resistance makes them difficult to treat. As an alternative to antibiotic intervention, we sought to identify anti-staphylococcal biofilm targets for the development of a vaccine or antibody prophylactic. A screening strategy was devised using a microfluidic system to test antibody-mediated biofilm inhibition under biologically relevant conditions of shear flow. Affinity-purified polyclonal antibodies to target antigen PhnD inhibited both Staphylococcus epidermidis and S. aureus biofilms. PhnD-specific antibodies blocked biofilm development at the initial attachment and aggregation stages, and deletion of phnD inhibited normal biofilm formation. We further adapted our microfluidic biofilm system to monitor the interaction of human neutrophils with staphylococcal biofilms and demonstrated that PhnD-specific antibodies also serve as opsonins to enhance neutrophil binding, motility, and biofilm engulfment. These data support the identification of PhnD as a lead target for biofilm intervention strategies performed either by vaccination or through passive administration of antibodies.

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confidence: 99%

Antibodies to PhnD Inhibit Staphylococcal Biofilms

et al. 2014

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“…Cultures were diluted to an OD 600 of 0.1 in TSB and added to a 96-well black-wall, clear-bottom plate (Corning, Inc., Corning, NY), where growth (OD 600 ), GFP fluorescence (Ex, 488 nm; emission [Em], 518 nm), and DsRed fluorescence (Ex, 560 nm; Em, 590 nm) were monitored in real-time by using (40,41). Cyclic di-AMP measurements.…”

Section: Methodsmentioning

confidence: 99%

Cyclic di-AMP Released from Staphylococcus aureus Biofilm Induces a Macrophage Type I Interferon Response

et al. 2016

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Staphylococcus aureus is a leading cause of community-and nosocomial-acquired infections, with a propensity for biofilm formation. S. aureus biofilms actively skew the host immune response toward an anti-inflammatory state; however, the biofilm effector molecules and the mechanism(s) of action responsible for this phenomenon remain to be fully defined. The essential bacterial second messenger cyclic diadenylate monophosphate (c-di-AMP) is an emerging pathogen-associated molecular pattern during intracellular bacterial infections, as c-di-AMP secretion into the infected host cytosol induces a robust type I interferon (IFN) response. Type I IFNs have the potential to exacerbate infectious outcomes by promoting anti-inflammatory effects; however, the type I IFN response to S. aureus biofilms is unknown. Additionally, while several intracellular proteins function as c-di-AMP receptors in S. aureus, it has yet to be determined if any extracellular role for c-di-AMP exists and its release during biofilm formation has not yet been demonstrated. This study examined the possibility that c-di-AMP released during S. aureus biofilm growth polarizes macrophages toward an anti-inflammatory phenotype via type I interferon signaling. DacA, the enzyme responsible for c-di-AMP synthesis in S. aureus, was highly expressed during biofilm growth, and 30 to 50% of total c-di-AMP produced from S. aureus biofilm was released extracellularly due to autolytic activity. S. aureus biofilm c-di-AMP release induced macrophage type I IFN expression via a STING-dependent pathway and promoted S. aureus intracellular survival in macrophages. These findings identify c-di-AMP as another mechanism for how S. aureus biofilms promote macrophage anti-inflammatory activity, which likely contributes to biofilm persistence. Staphylococcus aureus is a leading cause of prosthetic joint infections (1, 2), whereupon adherence to the implant surface facilitates biofilm formation. These infections are particularly challenging to treat and typically require a two-stage surgical process for insertion of a new device (1). Moreover, biofilms actively skew the host immune response toward an anti-inflammatory state, thereby contributing to the chronic nature of biofilm-mediated infections (3-5). This is evident by macrophage polarization toward an alternatively activated phenotype and the recruitment of myeloid-derived suppressor cells (MDSCs) (3,(6)(7)(8). While this immune deviation appears to be driven by S. aureus biofilm products, the effectors and their mechanism(s) of action remain to be fully identified.To further understand mechanisms of immune modification by S. aureus biofilms, we investigated the role of the essential bacterial second messenger cyclic diadenylate monophosphate (c-di-AMP), since it has been identified as an emerging pathogen-associated molecular pattern (PAMP) that influences immune responsiveness (9, 10). While c-di-AMP has been found to regulate many important functions in bacteria, including cell wall stress and peptidoglycan homeostas...

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“…Several reports have led to the conclusion that holins probably play important roles in biofilm formation. For example, the Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix (62,63). Evidence has been presented suggesting that the cidA and lrgA genes encode holin-and antiholin-like proteins which function to regulate processes similarly to bacteriophage-induced death and lysis.…”

Section: Involvement Of Holins In Bacterial Biofilm Formationmentioning

confidence: 99%

Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

Saier

Reddy

2015

J Bacteriol

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bHolins form pores in the cytoplasmic membranes of bacteria for the primary purpose of releasing endolysins that hydrolyze the cell wall and induce cell death. Holins are encoded within bacteriophage genomes, where they promote cell lysis for virion release, and within bacterial genomes, where they serve a diversity of potential or established functions. These include (i) release of gene transfer agents, (ii) facilitation of programs of differentiation such as those that allow sporulation and spore germination, (iii) contribution to biofilm formation, (iv) promotion of responses to stress conditions, and (v) release of toxins and other proteins. There are currently 58 recognized families of holins and putative holins with members exhibiting between 1 and 4 transmembrane ␣-helical spanners, but many more families have yet to be discovered. Programmed cell death in animals involves holin-like proteins such as Bax and Bak that may have evolved from bacterial holins. Holin homologues have also been identified in archaea, suggesting that these proteins are ubiquitous throughout the three domains of life. Phage-mediated cell lysis of dualmembrane Gram-negative bacteria also depends on outer membrane-disrupting "spanins" that function independently of, but in conjunction with, holins and endolysins. In this minireview, we provide an overview of their modes of action and the first comprehensive summary of the many currently recognized and postulated functions and uses of these cell lysis systems. It is anticipated that future studies will result in the elucidation of many more such functions and the development of additional applications.

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Use of Microfluidic Technology To Analyze Gene Expression during Staphylococcus aureus Biofilm Formation Reveals Distinct Physiological Niches

Cited by 88 publications

References 50 publications

Antibodies to PhnD Inhibit Staphylococcal Biofilms

Antibodies to PhnD Inhibit Staphylococcal Biofilms

Cyclic di-AMP Released from Staphylococcus aureus Biofilm Induces a Macrophage Type I Interferon Response

Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

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