Abstract
Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition.
Highlights
Virulence can be defined as the capability of microorganisms to infect a host by releasing virulence factors or by activating a mechanism which can cause damage
Luciferase in the presence of luciferin, adenosine -triphosphate (ATP), and oxygen catalyzes the production of oxyluciferin, AMP, PPi, and light
The amount of light is directly proportional to the ATP concentration in the mixture and inversely proportional to the enzymatic activity in the kinase reaction
Summary
Virulence can be defined as the capability of microorganisms to infect a host by releasing virulence factors or by activating a mechanism which can cause damage. Antivirulence agents are molecules which can interfere with virulence, preventing the infection establishment [1,2]. Since virulence is not essential for bacterial growth and survival, antivirulence agents are considered less prone to resistance development than antibiotics, representing a new frontier to fight bacterial infections [4,5]. Bacteria can communicate by releasing and detecting chemical signal molecules called autoinducers (AI). This process, known as quorum sensing (QS), has been linked e.g., to virulence factor production and biofilm formation [6]. Due to the complexity of its regulation and the variety of molecules involved, QS offers a plethora of approaches for developing antivirulence strategies [7,8]
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