Materials

1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) was purchased from Avanti Polar Lipids (Alabaster, USA). UV/Vis Spectroscopy grade Chloroform and Methanol were purchased from Merck (Darmstadt, Germany). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 99% purity) was purchased from Acros Organics (Geel, Belgium). The fluorescently-labelled melittin derivative, melittin K-14 AlexaFluor-430, was synthesized on a Fmoc-PAL-EG-PS resin, as detailed by Rapson et al25. The AlexaFluor-430 fluorescent label used for the melittin derivative was purchased from Molecular Probes (Carlsbad, USA). All materials were used without further purification. High purity water used in the preparation of all aqueous solutions was generated by a Milli-Q™ Ultra Pure Water system or a Milli-Q™ Academic system (Millipore Corp., Bedford, USA) and had a resistivity of 18.2 Ω.m.

The GFP expressing plasmid used in this study is a derivative of the vector pGFP (Clontech). It was constructed by taking the NcoI/EcoRI fragment from plasmid pGLO (Bio-Rad) and replacing the equivalent fragment containing the native GFP gene in pGFP. It is believed that the plasmid pGLO is the same as the plasmid pBAD-GFPuv which has a "fast-folding" mutant form of GFP39. The new plasmid was transformed into competent E. coli JM109. The bacteria were grown overnight at 37°C in LB broth prior to use.

Methods

All glassware was cleaned immediately prior to use by first sonicating for one hour in 10% Dextran surfactant solution, rinsing with Milli-Q™ water followed by soaking for 4 hours in concentrated (~5 M) sodium hydroxide solution then finally rinsing with Milli-Q™ water.

All aqueous solutions were10 mM HEPES that were pH adjusted to 7.4 ± 0.05 and filtered through a 0.22 μm Teflon membrane. Melittin derivative solutions were made at a concentration of 9 μM in HEPES. All solutions were stored at 4°C for no longer than 2 weeks.

Giant Unilamellar Vesicles (GUVs) composed of pure DPPC phospholipid were prepared following the Solvent Evaporation Method as reported by Moscho et al40. Briefly, a 0.1 M solution of DPPC lipid dissolved in chloroform was added to chloroform: methanol mixture (9.8:1 ratio). Filtered HEPES buffer solution was then carefully added to the organic phase. The organic phase removed via vacuum rotary evaporation using a Büchi V-500 Diaphragm Pump operating at 40 rpm, 40°C and 100 mBar minimum pressure. The remaining aqueous GUV suspension was centrifuged at 14,000 rpm (15,300 g) for 10 minutes to remove multilamellar vesicles and lipid debris. The supernatant was collected and stored in glass vials at room temperature (23 – 26°C) for no more than two weeks.

A GUV sample was prepared for FLIM by aliquoting 100 μL of the GUV supernatant onto a clean glass cover-slip mounted in a Sykes and Moore chamber. The solution was allowed to equilibrate for 45–60 minutes, after which transmitted light microscopy was used to identify a suitable GUV of interest. A sufficient amount of the melittin derivative solution was injected into the sample solution for a global lipid-to-peptide (L:P) ratio of 27:1. Injections were made as close to the point of interest under the wide field lens as possible to minimize diffusion effects of the fluorescent peptide, since mixing might dislodge the vesicles from their identified locations.

The bacterial samples for FLIM were prepared by depositing 200 μL of bacterial suspension onto a clean glass cover-slip mounted in a Sykes and Moore chamber. The bacteria were allowed to adhere for 2 hours. The suspension and any loosely-adhered bacteria were replaced with PBS solution by gentle rinsing. This ensures that the remaining bacteria were surface-immobilized for the duration of the experiment. Melittin derivative solution was added to give a final concentration of 9 μM, which is 2 times the published MIC for melittin/E. coli.

Fluorescence lifetime imaging microscopy (FLIM)

All fluorescence lifetime imaging experiments were performed using a LIFA instrument (Lambert Instruments, Leutingwolde, The Netherlands) attached to an inverted microscope (TE2000U, Nikon Inc., Japan). Glass cover slips were used for sample preparation and observed through a 100 × NA 1.2 oil objective (Nikon Plan-Fluor, Nikon Inc, Japan). The fluorescence excitation source was a 470 nm LED with a sinusoidal modulation frequency of 40 MHz (Lambert Instruments, Leutingwolde, The Netherlands). Phase and modulation lifetimes were determined by taking a series of 12 phase images of differing phase shift (fluorescence lifetime image stack), utilizing the LI-FLIM software package (Version 1.2.3.11) supporting the LIFA instrument. Photobleaching was corrected through the use of pseudo-random phase ordering in all experiments. The reference used for all lifetime determinations was Rhodamine 6G (lifetime R6G: 4.1 ns). Fluorescence lifetime image stacks were recorded as often as possible after introduction of the peptide for up to 15 minutes (typically 1–4 minute intervals) and then every 15-minute interval beyond that for up to 3 hours. A transmitted light microscopy image was recorded immediately after each fluorescence lifetime image stack acquisition.

The FLIM data were transformed into phasor space, where x = mcosφ and y = msinφ, m is the modulation and φ is the phase. To further decompose the fluorescence into fractional states, the fractional fluorescence from free peptide and peptide pore states were computed. For a given phasor, r(x,y), the fractional fluorescence from the peptide pore state, f pore , is given by:

where r(x,y) free is the (constant) phasor for free melittin derivative and r(x,y) pore is the (constant) phasor for peptide pore. These phasors are fixed and used as a point of reference.

For the bacteria transfected with GFP, there are three states to decompose, GFP fluorescence as well as free peptide and peptide pore. In this case, the observed phasor (r(x,y,)) is the sum of the free peptide, peptide pore and GFP fluorescence phasors, weighted by their respective fractional fluorescence contributions f free , f pore, and f GFP , viz:

where

Substitution of equation (3) into equation (2) and inversion then yields the three desired fractions: f free , f pore, and f GFP .

Because GFP fluorescence can potentially swamp contributions from free and pore-peptide states, it is useful to show that the fraction free and fraction pore states in the absence of GFP fluorescence can be extracted from the data. These values can be determined from the GFP-bacteria using the formulae from equations (4)–(6):

where I is the relative fluorescence intensity for each fluorescent species as specified by the subscript. Combining Equations (4) and (5) to eliminate I GFP gives:

Equation (6) shows that the ratio of the fractional fluorescence for pore and free states is the same in the absence and presence of GFP.