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Flipping the switch: tools for detecting small molecule inhibitors of staphylococcal virulence.

Quave CL, Horswill AR - Front Microbiol (2014)

Bottom Line: Many have argued for the discovery and development of quorum sensing inhibitors (QSIs) to augment existing antibiotics as adjuvant therapies.Examples include fluorescent reporters, MS-detection of autoinducing peptide production, agar plate methods for detection of hemolysins and lipase, High performance liquid chromatography-detection of hemolysins from supernatants, and cell-toxicity assays for detecting damage (or relief thereof) against human keratinocyte cells.In addition to providing a description of these various approaches, we also discuss their amenability to low-, medium-, and high-throughput screening efforts for the identification of novel QSIs.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Emory University School of Medicine Atlanta, GA, USA ; Center for the Study of Human Health, Emory University College of Arts and Sciences Atlanta, GA, USA.

ABSTRACT
Through the expression of the accessory gene regulator quorum sensing cascade, Staphylococcus aureus is able to produce an extensive array of enzymes, hemolysins and immunomodulators essential to its ability to spread through the host tissues and cause disease. Many have argued for the discovery and development of quorum sensing inhibitors (QSIs) to augment existing antibiotics as adjuvant therapies. Here, we discuss the state-of-the-art tools that can be used to conduct screens for the identification of such QSIs. Examples include fluorescent reporters, MS-detection of autoinducing peptide production, agar plate methods for detection of hemolysins and lipase, High performance liquid chromatography-detection of hemolysins from supernatants, and cell-toxicity assays for detecting damage (or relief thereof) against human keratinocyte cells. In addition to providing a description of these various approaches, we also discuss their amenability to low-, medium-, and high-throughput screening efforts for the identification of novel QSIs.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the phospholipase plate assay for the identification of quorum sensing inhibitor (QSI) activity. In this test, 18 h cultures are grown either in the presence or absence of QSI, the supernatants harvested and sterile filtered, and then added to the punch holes made in the phospholipid plate. Following an additional 18 h incubation period, the plates are exposed to UV irradiation and photographed. The orange halos indicate the lytic activity of untreated supernatants. Supernatants from QSI-treated cultures would exhibit limited to no lytic effects and no orange halo.
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Figure 2: Schematic representation of the phospholipase plate assay for the identification of quorum sensing inhibitor (QSI) activity. In this test, 18 h cultures are grown either in the presence or absence of QSI, the supernatants harvested and sterile filtered, and then added to the punch holes made in the phospholipid plate. Following an additional 18 h incubation period, the plates are exposed to UV irradiation and photographed. The orange halos indicate the lytic activity of untreated supernatants. Supernatants from QSI-treated cultures would exhibit limited to no lytic effects and no orange halo.

Mentions: In addition to the production of toxins, S. aureus also produces a number of enzymes, some of which exhibit lipolytic activity as (phospho)lipases (Laabei et al., 2014). A new version of the lipase plate assay involves a modification of the original method (Kouker and Jaeger, 1987) that incorporates olive oil (1%) and rhodamine B (0.001%) into the substrate added to the agar medium (Laabei et al., 2014). Holes are then punched into the agar and supernatant is added, the plate incubated, irradiated with UV light, and images captured for analysis. An orange halo emerging around the supernatant indicates the presence of lipase (Figure 2). Treatment of S. aureus with a successful QSI will not yield the halo zone in the agar dish, indicating the lack of lipolytic enzymes present in the supernatant.


Flipping the switch: tools for detecting small molecule inhibitors of staphylococcal virulence.

Quave CL, Horswill AR - Front Microbiol (2014)

Schematic representation of the phospholipase plate assay for the identification of quorum sensing inhibitor (QSI) activity. In this test, 18 h cultures are grown either in the presence or absence of QSI, the supernatants harvested and sterile filtered, and then added to the punch holes made in the phospholipid plate. Following an additional 18 h incubation period, the plates are exposed to UV irradiation and photographed. The orange halos indicate the lytic activity of untreated supernatants. Supernatants from QSI-treated cultures would exhibit limited to no lytic effects and no orange halo.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264471&req=5

Figure 2: Schematic representation of the phospholipase plate assay for the identification of quorum sensing inhibitor (QSI) activity. In this test, 18 h cultures are grown either in the presence or absence of QSI, the supernatants harvested and sterile filtered, and then added to the punch holes made in the phospholipid plate. Following an additional 18 h incubation period, the plates are exposed to UV irradiation and photographed. The orange halos indicate the lytic activity of untreated supernatants. Supernatants from QSI-treated cultures would exhibit limited to no lytic effects and no orange halo.
Mentions: In addition to the production of toxins, S. aureus also produces a number of enzymes, some of which exhibit lipolytic activity as (phospho)lipases (Laabei et al., 2014). A new version of the lipase plate assay involves a modification of the original method (Kouker and Jaeger, 1987) that incorporates olive oil (1%) and rhodamine B (0.001%) into the substrate added to the agar medium (Laabei et al., 2014). Holes are then punched into the agar and supernatant is added, the plate incubated, irradiated with UV light, and images captured for analysis. An orange halo emerging around the supernatant indicates the presence of lipase (Figure 2). Treatment of S. aureus with a successful QSI will not yield the halo zone in the agar dish, indicating the lack of lipolytic enzymes present in the supernatant.

Bottom Line: Many have argued for the discovery and development of quorum sensing inhibitors (QSIs) to augment existing antibiotics as adjuvant therapies.Examples include fluorescent reporters, MS-detection of autoinducing peptide production, agar plate methods for detection of hemolysins and lipase, High performance liquid chromatography-detection of hemolysins from supernatants, and cell-toxicity assays for detecting damage (or relief thereof) against human keratinocyte cells.In addition to providing a description of these various approaches, we also discuss their amenability to low-, medium-, and high-throughput screening efforts for the identification of novel QSIs.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Emory University School of Medicine Atlanta, GA, USA ; Center for the Study of Human Health, Emory University College of Arts and Sciences Atlanta, GA, USA.

ABSTRACT
Through the expression of the accessory gene regulator quorum sensing cascade, Staphylococcus aureus is able to produce an extensive array of enzymes, hemolysins and immunomodulators essential to its ability to spread through the host tissues and cause disease. Many have argued for the discovery and development of quorum sensing inhibitors (QSIs) to augment existing antibiotics as adjuvant therapies. Here, we discuss the state-of-the-art tools that can be used to conduct screens for the identification of such QSIs. Examples include fluorescent reporters, MS-detection of autoinducing peptide production, agar plate methods for detection of hemolysins and lipase, High performance liquid chromatography-detection of hemolysins from supernatants, and cell-toxicity assays for detecting damage (or relief thereof) against human keratinocyte cells. In addition to providing a description of these various approaches, we also discuss their amenability to low-, medium-, and high-throughput screening efforts for the identification of novel QSIs.

No MeSH data available.


Related in: MedlinePlus