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Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays
Published on: October 1, 2013
Fluorogenic Substrate for Monitoring Activity of Muralytic Enzymes
Marcell Simon1, Amber L B Mogg1, Chloë F Klein1
1Department of Natural Sciences of Pitzer and Scripps Colleges, Claremont, California 91711, United States.
Abstract:
Antibiotic-resistant infections are one of the most common causes of morbidity and mortality around the world. The accelerated evolutionary rate of bacteria makes finding effective antibiotics to treat infections difficult. A promising alternative to traditional antibiotics is muralytic enzymes, which are effective at killing bacteria with little evidence of bacterial defense mechanisms. However, there are a lack of tools available to assess their activity, and their engineered variants, in a high-throughput manner, making it difficult to develop effective therapeutics. In this work, we report a new internally quenched substrate that can report on the activity of enzymes that lyse Gram-positive bacteria. As a proof of study, the substrate was "turned on" by the catalytic domains of both LytM and lysostaphin in bacterial lysate, with a robust signal over background. Additionally, the synthetic substrate was able to report on the activity differences between the native enzymes and their mutants. A mock screen study showed that the substrate could be used to assess the activity of multiple samples in a high-throughput manner. This work provides additional tools that can be used to characterize existing and new enzyme therapeutics and lays the groundwork for tools that can be expanded to other lytic enzymes.
Insights
Researchers developed a novel substrate to easily measure the activity of muralytic enzymes, which are promising alternatives to antibiotics for fighting bacterial infections. This tool aids in developing new enzyme therapeutics against antibiotic-resistant bacteria.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- Antibiotic-resistant infections pose a significant global health threat, driving the need for novel therapeutic strategies.
- Muralytic enzymes offer a promising alternative to traditional antibiotics due to their potent bactericidal activity and limited bacterial resistance.
- Current limitations in high-throughput assays hinder the development and characterization of muralytic enzyme therapeutics.
Purpose of the Study:
- To develop and validate a novel internally quenched substrate for high-throughput assessment of muralytic enzyme activity.
- To demonstrate the substrate's utility in characterizing enzymes targeting Gram-positive bacteria.
- To facilitate the development of new enzyme-based antibacterial therapies.
Main Methods:
- Design and synthesis of an internally quenched substrate.
- Enzyme activity assays using the substrate with bacterial lysates containing LytM and lysostaphin catalytic domains.
- Evaluation of substrate performance with native enzymes and their mutants.
- Mock screening to assess high-throughput capability.
Main Results:
- The internally quenched substrate successfully detected activity of LytM and lysostaphin catalytic domains in bacterial lysate.
- A robust signal-to-background ratio was achieved, confirming substrate sensitivity.
- The substrate differentiated activity levels between native enzymes and their engineered mutants.
- Mock screening demonstrated the substrate's suitability for high-throughput analysis.
Conclusions:
- A novel, sensitive substrate has been developed for assaying muralytic enzyme activity against Gram-positive bacteria.
- This tool enables high-throughput characterization of existing and engineered muralytic enzymes.
- The developed substrate lays the foundation for broader applications in enzyme therapeutic development.
