An extensively validated whole-cell biosensor for specific, sensitive and high-throughput detection of antibacterial inhibitors targeting cell-wall biosynthesis

Background Whole-cell biosensor strains are powerful tools for antibacterial drug discovery, in principle allowing the identification of inhibitors acting on specific, high-value target pathways. Whilst a variety of biosensors have been described for detecting cell-wall biosynthesis inhibitors (CWBIs), these strains typically lack specificity and/or sensitivity, and have for the most part not been rigorously evaluated as primary screening tools. Here, we describe several Staphylococcus aureus CWBI biosensors and show that specific and sensitive biosensor-based discovery of CWBIs is achievable. Methods Biosensors comprised lacZ reporter fusions with S. aureus promoters (P-gltB, P-ilvD, P-murZ, P-oppB, P-ORF2768, P-sgtB) that are subject to up-regulation following inhibition of cell-wall biosynthesis. Induction of biosensors was detected by measuring expression of beta-galactosidase using fluorogenic or luminogenic substrates. Results Three of the six biosensors tested (those based on P-gltB, P-murZ, P-sgtB) exhibited apparently specific induction of beta-galactosidase expression in the presence of CWBIs. Further validation of one of these (P-murZ) using an extensive array of positive and negative control compounds and conditional mutants established that it responded appropriately and uniquely to inhibition of cell-wall biosynthesis. Using this biosensor, we established, validated and deployed a high-throughput assay that identified a potentially novel CWBI from a screen of >9000 natural product extracts. Conclusions Our extensively validated P-murZ biosensor strain offers specific and sensitive detection of CWBIs, and is well-suited for high-throughput screening; it therefore represents a valuable tool for antibacterial drug discovery.

Automated summary

This study described several Staphylococcus aureus whole-cell biosensors for cell-wall biosynthesis inhibitors. One of the validated biosensors, P-murZ, showed specific and sensitive detection of these inhibitors, making it a valuable tool for high-throughput screening in antibacterial drug discovery.