Exploring molecular targets and pathways involved in the antibiofilm activity of zosteric acid related structure by a multi-strategy proteomic approach

The worldwide safety is seriously affected by the emergence and spread of microorganisms in form of biofilm that are resistant to traditional antimicrobial treatment. An innovative approach is based on the use of sub-lethal concentrations of natural compounds that interfere with specific key-steps of biofilm formation disarming microorganisms without affecting their existence. Zosteric acid (ZA) from the seagrass Zostera marina was shown to be suitable for implementation as a preventive or integrative biocide-free approach against both bacterial and fungal biofilms1. Our comparative proteomic study on E. coli surface-exposed planktonic cells suggested that ZA modulates the threshold level of the extracellular signalling molecule autoinducer-2 and it induces a hypermotile phenotype unable to firmly adhere on surfaces2. To elucidate the molecular targets and pathways involved in the antibiofilm activity of molecules with ZA-backbone structure, different strategies were explored. Affinity purification of protein(s) from E. coli extracts on immobilized ZA moieties should allow the identification of targets directly interacting with these antibiofilm compounds. According to a biological screening of a small library of molecules, some with ZA-related structure, p-aminocinnamic acid and p-aminosalicylic acid were selected and their covalent coupling to Sepharose 4B-6-aminohexanoic acid was optimized. Functionalization of solid support was successfully demonstrated by fluorescence analysis, while the optimization of the affinity chromatography procedure is still in progress. Proteins differently expressed in Candida albicans biofilm upon ZA treatment were investigated. Conditions for fungal biofilm protein extraction were set up and 2D-PAGE protein profiles of more than 100 spots were obtained for both control and treated samples. Proteins with altered response to the antibiofilm agent have been established and the corresponding spots identified by MS.