Biosensor-guided detection of outer membrane-specific antimicrobial activity against Pseudomonas aeruginosa from fungal cultures and medicinal plant extracts

Pseudomonas aeruginosa responds to sub-lethal antimicrobial exposure by inducing the expression of lipopolysaccharide (LPS) surface modifications that mask antibiotic binding sites and contribute to repair and resistance of the outer membrane (OM). We exploit these membrane damage-responsive operons in a biosensor approach used to discover new antimicrobials that specifically target the OM. Chromosomal transcriptional luxCDABE reporters from the pmr (polymyxin resistance; aminoarabinose LPS modification) and speD2E2 (spermidine synthesis) operons are induced by validated outer membrane-acting agents including cationic antimicrobial peptides, cation chelators, ascorbic acid, detergents, and cell wall synthesis inhibitors cycloserine and bacitracin. To identify novel sources of OM-disrupting antimicrobials, we used these OM damage-responsive biosensors to screen a panel of fungal culture supernatants for novel antimicrobial and biosensor activity. Biosensor activity was used to determine the optimal time point of antimicrobial production from fungal supernatants and to guide the purification of active fractions after size-exclusion chromatography. Water and ethanol extracts of Chinese medicinal plants also proved to be a source of biosensor activity. The pathogen box is a 400-member drug library of potential antimicrobials, but none of these compounds induced our OM damage biosensors. This novel, sensitive, cell-based screening assay has potential for future discovery of lead compounds that specifically target the outer membrane, which is a significant barrier to antibiotic entry into Gram-negative bacteria.