Correspondence: Dimitri Margetis - dimargetis@free.fr
Annals of Intensive Care 2016, 6(Suppl 1):P167
Introduction The cranberry fruit (Vaccinium macrocarpon) is particularly rich in polyphenols (tannins), and among these, the primary active compound is the A-type proanthocyanidin (cPAC), which exhibit potent antibacterial properties. In a previous study, we have made evidence of the strong inhibitory effect of cPAC on growth, adhesion and virulence of oropharyngeal and lung isolates of E. coli [1].
The present study was undertaken to establish a baseline of knowledge of the molecular responses of the highly virulent E. coli strain 536 during growth in the presence of cPAC. We employed proteomic analysis and made complementary experiences to characterize the global response of exponential and stationary phases E. coli 536 grown in a nutrient-rich broth medium supplemented with 1 mg/mL, cPAC as compared with a reference culture grown without cPAC.
Materials and methodsE. coli 536 exposed or not to 1 mg/ml of cPAC proteome was obtained in exponential and stationary growth phase by mass spectrometry proteomic analysis LC–MS/MS.
Evaluation of the intracellular ROS level by the dichlorofluorescein diacetate DCFH-DA method was evaluated using a spectrofluorimeter. Membrane potential, cell size and cell viability were evaluated using a flow cytometer (Guava EasyCyte Plus, Millipore). To examine the changes of membrane potential after cPAC exposure, the cells were stained with DiOC6(3) (3,3′-dihexyloxacarbocyanine iodide) and then immediately analyzed using a flow cytometer. The fluorescence-based LIVE/DEAD kit was used to examine the cell viability.
Results A total of 641 proteins were detected, and among these, 483 were in both exponential and stationary growth phases, whereas 506 and 618 proteins were detected only during the exponential and stationary growth phases, respectively. Proteins were manually grouped into 25 metabolic pathway categories. Among the 641 identified proteins, 117 had a significant effect, and among these, 11 were in both exponential and stationary growth phases, whereas 68 and 60 proteins were detected only during the exponential and stationary growth phases, respectively. Considerable differences were also observed in protein abundances between the untreated and treated cells. During the exponential and stationary growth phases, many proteins were over-abundant (31 and 29, respectively) and under-abundant (37 and 31, respectively).
The characterization of 117 proteins with a significant effect revealed that more proteins were over-abundant in different functional categories, especially those involved in glycolyse, fermentation, iron metabolism and detoxification. In contrast, the under-abundant proteins were mainly implicated in transport, TCA cycle and respiration.
cPAC exposure mainly affected the pathways of iron metabolism with strong evidence of iron privation, cellular detoxification as response to the oxidative stress exerted by cPAC and a redirection of respiratory metabolism through the fermentative pathways. cPAC treatment also alters many membrane functions by the strong inhibition of membrane proteins transport and secretion, affecting the respiratory chains, fimbrial expression, cell division and proton-motrice force.
Supplementary experiments showed no effect of cPAC on bacterial viability, a size increase as result of cellular division inhibition and a drastic reduction of the proton-motrice force.
ROS dosage showed a significant decrease during stationary phase of growth in cPAC treated bacteria as response to the oxidative stress exerted by proanthocyanidins.
Conclusion Proteomic analysis indicates the metabolic pathways affected by cPAC and E. coli adaptation. Treated bacteria are in a state of iron limitation, energy metabolism is redirected to fermentation and membrane proteins secretion and transport systems strongly inhibited. As a result, we observe a fimbriation inhibition affecting bacterial mobility, adhesion and virulence and alteration of the proton-motrice force and cellular division in E. coli. Our results highlight strategy adapted by E. coli 536 to resist the toxicity of tannins. During exposure to cranberry, E. coli is subjected to complex metabolic adaptations that aim to reduce the rate of intracellular ROS. The lack of production of fimbriae caused by cranberry contributes to the permanent activation of the oxidative stress response.
Competing interests None.
Reference
1. Margetis, D., D. Roux, et al. (2015). “Effects of Proanthocyanidins on Adhesion, Growth, and Virulence of Highly Virulent Extraintestinal Pathogenic Escherichia coli Argue for Its Use to Treat Oropharyngeal Colonization and Prevent Ventilator-Associated Pneumonia.” Crit Care Med 43(6): e170–8.