RESUMO
Introduction: The eons-long co-evolvement of plants and bacteria led to a plethora of interactions between the two kingdoms, in which bacterial pathogenicity is counteracted by plant-derived antimicrobial defense molecules. In return, efflux pumps (EP) form part of the resistance mechanism employed by bacteria to permit their survival in this hostile chemical environment. In this work we study the effect of combinations of efflux pump inhibitors (EPIs) and plant-derived phytochemicals on bacterial activity using Pectobacteriun brasiliense 1692 (Pb1692) as a model system. Methods: We measured the minimal inhibitory concentration (MIC) of two phytochemicals, phloretin (Pht) and naringenin (Nar), and of one common antibiotic ciprofloxacin (Cip), either alone or in combinations with two known inhibitors of the AcrB EP of Escherichia coli, a close homolog of the AcrAB-TolC EP of Pb1692. In addition, we also measured the expression of genes encoding for the EP, under similar conditions. Results: Using the FICI equation, we observed synergism between the EPIs and the phytochemicals, but not between the EPIs and the antibiotic, suggesting that EP inhibition potentiated the antimicrobial activity of the plant derived compounds, but not of Cip. Docking simulations were successfully used to rationalize these experimental results. Discussion: Our findings suggest that AcrAB-TolC plays an important role in survival and fitness of Pb1692 in the plant environment and that its inhibition is a viable strategy for controlling bacterial pathogenicity.
RESUMO
Bis(2-carboxyphenyl) succinate (disalicylic acid; DSA) is composed of two salicylic acids connected by a succinyl linker. Here, we propose its use as a new, synthetic plant-protection agent. DSA was shown to control Pectobacterium brasiliense, an emerging soft-rot pathogen of potato and ornamental crops, at minimal inhibitory concentrations (MIC) lower than those of salicylic acid. Our computational-docking analysis predicted that DSA would inhibit the quorum-sensing (QS) synthase of P. brasiliense ExpI more strongly than SA would. In fact, applying DSA to P. brasiliense inhibited its biofilm formation, secretion of plant cell wall-degrading enzymes, motility and production of acyl-homoserine lactones (AHL) and, subsequently, impaired its virulence. DSA also inhibited the production of AHL by a QS-negative Escherichia coli strain (DH5α) that had been transformed with P. brasiliense AHL synthase, as demonstrated by the biosensors Chromobacterium violaceaum CV026 and E. coli pSB401. Inhibition of the QS machinery appears to be one of the mechanisms by which DSA inhibits specific virulence determinants. A new route is proposed for the synthesis of DSA, which holds greater potential for use as an anti-virulence agent than its precursor SA. Based on these findings, DSA is an excellent candidate for repurposing for new applications.
RESUMO
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.