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.
Highlights
Phytopathogenic pectobacteria are responsible for economically significant yield losses caused by soft rot and blackleg disease in a wide range of commercial crops, including potato (Solanum tuberosum), vegetables and ornamentals (Ma et al, 2007; Mansfield et al, 2012; Charkowski, 2018)
The lowest concentration of phloretin that completely inhibited bacterial growth was 1 mM; whereas 0.4 mM caused less than 50% growth inhibition (Supplementary Figure 1)
Intercellular communication that is based on quorum-sensing is activated when bacteria reach a certain population density. It is mediated by signaling molecules synthesized by an acylhomoserine lactone (AHL) synthase, of which acyl-homoserine lactones (AHLs) are the most common in Gram-negative bacteria (Gutierrez-Pacheco et al, 2019)
Summary
Phytopathogenic pectobacteria are responsible for economically significant yield losses caused by soft rot and blackleg disease in a wide range of commercial crops, including potato (Solanum tuberosum), vegetables and ornamentals (Ma et al, 2007; Mansfield et al, 2012; Charkowski, 2018). In Pectobacteria, a genus in the family Pectobacteriaceae (Adeolu et al, 2016), virulence regulation is under the strict control of quorum-sensing (QS), a mechanism that regulates a complex set of transcriptional factors and posttranscriptional regulators according to the level of cell density (Fuqua et al, 1994; Chatterjee et al, 1995; Koiv and Mae, 2001). This cell–cell communication system allows bacteria to sense small diffusible communication signals known as auto-inducers. In Pectobacterium, QS mainly involves the production, detection and responses to the signal molecule N-acylhomoserine lactone (AHL), of which the most common in Pectobacterium are 3oxo-C6-HSL and 3-oxo-C8-HSL (3-oxo-hexanonyl homoserine lactone and 3-oxo-octanonyl homoserine lactone, respectively) (Pollumaa et al, 2012)
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