Fluorescence enzymatic assay for bacterial polyphosphate kinase 1 (PPK1) as a platform for screening antivirulence molecules

Francisca Campos,1 Javiera A Álvarez,1 Javiera Ortiz-Severín,1 Macarena A Varas,1 Carlos F Lagos,2 Ricardo Cabrera,3 Sergio A Álvarez,4 Francisco P Chávez11Laboratorio de Microbiología de Sistemas, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago de Chile, Chile; 2Chemical Biology & Drug Discovery Laboratory, Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago de Chile, Chile; 3Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago de Chile, Chile; 4Laboratorio de Microbiología, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas Y Farmacéuticas, Universidad de Chile, Santiago de Chile, ChileAbstract: 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 (4ʹ-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: antivirulence, antimicrobial, affinity purification, polyphosphate kinase, DAPI, fluorescence enzymatic activity