Fluorogenic Substrate for Monitoring Activity of Muralytic Enzymes

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.