Cellular K+ response of E coli to manuka honey exposure

Potassium-binding benzofuran isopthalate (PBFI) is an insoluble, monovalent cation-binding fluorescent compound (Fig. 7.1). The dye is cell permeable (MP 01262, Molecular Probes), so can either measure the total K+ _{content of a culture plus broth, or separately measure the} culture supernatant and cell pellet to determine leakage vs retention of K+_{, and thus indicate} (i) the membrane permeability of the cell, or (ii) the cell’s current metabolic state regarding potassium ions.

Figure 7.1 PBFI structure (non-ester form): two benzofuran isophthalate fluorophores linked to the nitrogens of a diazacrown ether with a cavity size that confers selectivity for K+ _(affinity 1.5 times that for Na+_{)(Meuwis et al., 1995).}

The hypothesis being tested was that manuka honey may perturb membranes, either directly, or as a consequence of other mechanisms resulting in cell death. Membrane perturbation will also affect K+ _{leakage, transport or retention. In addition, the enterobacterial} response to MGO is to pump K+ _{out of the cell with the KefB and KefC proton antiporter} system, thus acidifying the cytoplasm to protect from MGO’s effects. The response to osmotic pressure/aw is to import K+ into the cell using the Kdp transporter and also alkalinise the cytoplasm in response to the K+ _{charge to retain electroneutrality and thus maintain} membrane polarization. Thus a change should be observed in K+ _{levels in the cells and} culture supernatant, depending upon membrane damage and the priority/severity of the cells’ response to MGO and osmotic pressure.

129 7.2.1 Methods

A culture of E. coli Nissle (108 _{cfu/mL) was centrifuged, and washed three times by} centrifugation and resuspension in water to remove culture media components before being resuspended in water (to minimise excess background K+ _{present in the media) containing} manuka honey or artificial honey (12.5% (w/v)), or MGO at manuka honey equivalent concentration (1 mM). A culture resuspended in water was used as a control. After a 30 min incubation period, samples were rapidly prepared to ensure that changing the osmotic state upon removal of the humectants (honey solutions) minimised the impact on intracellular K+ concentration. Cultures were briefly centrifuged at maximum g in an eppendorf microfuge, and cells were resuspended in water. Aliquots (0.025 mL) were further diluted into water (0.075 mL) in a microplate and 0.01 mL of PBFI (100 µM in 80% DMSO) was added to give a final concentration of approximately 10 µM in 8% DMSO. Fluorescence was measured after 3 min (Fig. 7.2), and again at 5 min, using a Bio-TEK Synergy HT fluorescence plate reader set to an excitation wavelength of 340 nm and emission wavelength of 520 nm.

7.2.2 Results and discussion

Figure 7.2. PBFI determination of intracellular K+ _{measured in E. coli cells treated with} manuka honey (12.5% (w/v)), artificial honey (12.5% (w/v)), or MGO (1.0 mM, equivalent to 12.5% (w/v) manuka honey) solutions and compared to unsupplemented (control) cells. Data are expressed as fluorescence units (Ex. 340 nm; Em. 520 nm) after 3 min and are the mean of 3 replicates. SEM shown. Data which are not significantly (P<0.05) different are marked with the same letter.

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There was no difference between fluorescence readings after 3 (fig. 7.2) or 5 min (data not shown). All three treatments led to significantly (P<0.05) lower K+ _{levels in the cells than in} the control group. Artificial honey and MGO treatments led to approximately the same K+ content, less than the control. The manuka honey treatment resulted in the lowest K+ _levels. In addition, the K+ levels in the cells treated with manuka honey were significantly lower than in those treated with artificial honey or MGO. The latter two treatments were not significantly different from each other. The decrease in intracellular K+ _{in the MGO- and} manuka honey-treated cells was expected. However, the decrease in K+ _{in the artificial} honey-treated cells was not the expected osmotic response. This may indicate that earlier comments on alternative modes of action of the osmotically active solutes glucose and fructose may be responsible for the inhibition of cells grown under these conditions. Alternatively, all three treatments may have compromised cells to the extent that they all lost K+ _{ions through damaged membranes, and that manuka honey caused the greatest loss by} virtue of being more antimicrobial than either the artificial honey or MGO alone. This hypothesis would have to be further tested by inclusion of controls containing a mixture of artificial honey and MGO. In addition, whilst the washing and incubation steps were conducted as rapidly as possible to minimise changes in cytoplasmic K+ _{levels due to} changes in the osmotic state of the cells, the impact of this must be considered.

Separation of cells from the growth medium has been performed by centrifugation through silicon oil (Bakker and Mangerich, 1981) to minimise cation or other solute loss from the cytoplasm due to changes in the osmotic environment. However, given that K+ _{levels were} lower than the controls, which must be considered the normal state, and that returning the treated cells to the same state as the controls would, at worst, reduce the difference between the control and treated cells, then still observing changes induced by the treatment indicates that the experiment was successful in terms of indicating divergent K+ _{levels. Certainly,} making claims on the actual value in terms of molar concentration of intracellular K+ could not be determined by the method used. Finally, levels of cytoplasmic water have been shown to be unlikely to change within the time period used to wash the samples (~1 min)(Cayley et al., 2000).

7.2.3 Summary

Treatment of a washed cell suspension (108 _{cells/mL) with manuka honey, artificial honey or} MGO all resulted in decreased levels of cytoplasmic K+ _{compared to untreated controls. This}

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may indicate that the MGO-tolerance response (decrease in K+ with commensurate increase in H+_{) is the more likely mechanism than the osmotic response (increase K}+_{, decrease H}+_). This non-osmotic response, even upon artificial honey treatment, either reinforces earlier suggestions that these sugars exert a non-osmotic effect to account for their inhibition, or suggests that all the treated cells have perturbed membranes causing leakage of cytoplasmic K+_.