Inorganic polyphosphate (polyP) and its metabolic enzymes are important in several cellular processes related with virulence and antibiotic susceptibility. Accordingly, bacterial polyP synthesis has been proposed as a good target for designing novel antivirulence molecules as alternative to conventional antibiotics. In most pathogenic bacteria, polyphosphate kinase 1 (PPK1), in charge of polyP synthesis from ATP, is widely conserved. Current colorimetric and radioactive polyP synthesis enzymatic assays are not suitable for high-throughput screening of PPK1 inhibitors. Given the ability of polyP to modify the excitation-emission spectra of DAPI (4'-6-diamidino-2-phenylindole), a fluorescence assay was previously developed by using a purified recombinant PPK1 enzyme from Escherichia coli. In this work we have developed a suitable methodology for high-throughput measurement of E. coli PPK1 activity. This platform can be used for the screening putative antimicrobial molecules for related enteropathogenic bacteria.
Keywords: DAPI; affinity purification; antimicrobial; antivirulence; fluorescence enzymatic activity; polyphosphate kinase.