Phloretin, an Apple Phytoalexin, Affects the Virulence and Fitness of Pectobacterium brasiliense by Interfering With Quorum-Sensing

Abstract

The effects of phloretin a phytoalexin from apple, was tested on Pectobacterium brasiliense (Pb1692), an emerging soft-rot pathogen of potato. Exposure of Pb1692 to 0.2 mM phloretin a concentration that does not affect growth, or to 0.4 mM a 50% growth inhibiting concentration (50% MIC), reduced motility, biofilm formation, secretion of plant cell wall-degrading enzymes, production of acyl–homoserine lactone (AHL) signaling molecules and infection, phenotypes that are associated with bacterial population density-dependent system known as quorum sensing (QS). To analyze the effect of growth inhibition on QS, the activity of ciprofloxacin, an antibiotic that impairs cell division, was compared to that of phloretin at 50% MIC. Unlike phloretin, the antibiotic hardly affected the tested phenotypes. The use of DH5α, a QS-negative Escherichia coli strain, transformed with an AHL synthase (ExpI) from Pb1692, allowed to validate direct inhibition of AHL production by phloretin, as demonstrated by two biosensor strains, Chromobacterium violaceaum (CV026) and E. coli (pSB401). Expression analysis of virulence-related genes revealed downregulation of QS-regulated genes (expI, expR, luxS, rsmB), plant cell wall degrading enzymes genes (pel, peh and prt) and motility genes (motA, fim, fliA, flhC and flhD) following exposure to both phloretin concentrations. The results support the inhibition of ExpI activity by phloretin. Docking simulations were used to predict the molecular associations between phloretin and the active site of ExpI, to suggest a likely mode of action for the compound’s inhibition of virulence.