Membrane damage-responsive biosensors for the discovery of antimicrobials from Lenzites betulina and Haploporus odorus

Abstract

Antibiotic resistance of pathogens to antibiotics is a growing concern in medical treatment of infections. Conventional methods for antimicrobial screening relies on using disc diffusion to observe zones of inhibition.

However, this method is not sensitive enough to possibly detect sub-lethal activities. We have developed biosensors in Pseudomonas aeruginosa that produce light from transcriptional lux fusions in response to compounds that damage the outer membrane. Sub-lethal exposure to antimicrobial peptides, the antibiotic cycloserine, and cation chelators induces the expression of arnC::lux (PA3553) and speE2::lux (PA4774). These genes encode outer surface modifications that ultimately protect the P. aeruginosa outer membrane from exposure to sub-lethal concentrations of membrane-active compounds. We used high throughput screening in 96 well micro plate format to test the culture supernatants from a panel of fungal species isolated in Alberta. Preliminary results showed a significant induction of both strains of lux biosensors in 23 of the 29 fungal species screened. Due to the significant amount of biosensor induction induced by the supernatants from Lenzites betulina and Haploporus odorus, efforts have been directed to the purification, isolation and classification of the active component of the supernatant. Whole genome sequencing has become necessary in order to properly analyze the mass spectrometry results that have already been obtained. In a short period of time, it has been possible to quickly and more accurately detect sub-lethal concentrations of antimicrobial compounds that might have been over looked if tradition methods, such as disc diffusion, had been employed.

* Indicates faculty mentor