Mechanisms of alpha-defensin bactericidal action: comparative membrane disruption by Cryptdin-4 and its disulfide-null analogue

Abstract

Mammalian alpha-defensins all have a conserved triple-stranded beta-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse alpha-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has bactericidal activity equal to or greater than that of the native peptide, providing a rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live Escherichia coli ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage in a manner similar to that of Crp4 but had less overall membrane permeabilizing activity. Crp4 induction of the leakage of the fluorophore from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid/polydiacetylene vesicles showed that Crp4 inserts more pronouncedly into membranes containing a high fraction of electronegative lipids or cardiolipin than (6C/A)-Crp4 does, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C/A)-Crp4, native, beta-sheet-containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration.

FIGURE 1

Panel A: Permeabilization of E. coli ML35 by Crp4 and (6C/A)-Crp4. E. coli ML35 growing in log-phase were exposed to 3 μM peptide concentrations in the presence of ONPG at 37 °C. ONPG hydrolysis was measured to determine the amount of permeabilization caused by the experimental peptides. Symbols: Crp4 (-●-), (6C/A)-Crp4 (-○-), and proCrp4, (-▼-). Panel B: Potassium Efflux from E. coli ML35. E. coli ML35 cells were resuspended at 2.5 × 10⁸ CFU/ml in a final volume of 250 μl. After a 90 sec equilibration period, 2 μl of peptide solution were added to a final concentration of 25 μg peptide per ml (6.66 μM Crp4, 6.65 μM (6C/A)-Crp4, and 3.04 μM proCrp4) K+ efflux was monitored as described (Materials and Methods). Although the proCrp4 concentration was approximately half that of the Crp4 peptides, 6 μM proCrp4 does not induce E. coli cell permeabilization (25) or potassium efflux (not shown) Symbols: Crp4 (-●-), (6C/A)-Crp4 (-▼-), proCrp4 (-○-), no peptide addition (-▽-).

FIGURE 2

Time course of dye leakage from (A) PE/PG (3:7, 70% charge), (B) PE/PG (7:3, 30% charge), (C) E. coli (∼35% charge) and (D) PE/PG/CL (7:1:1, 30% charge) liposomes induced in the presence of (▲) melittin (2 μM), (◆) (6C/A)-Crp4 (5.9 μM) and (○) native Crp4 (5.9 μM).. The leakage is measured over the entire 90 min course of the assay (usually a plateau was reached within the first 15 min) with peptides and liposomes mixed at t = 2 min. An increase in fluorescence corresponds to the release of dye and is indicative of liposome leakage or lysis. A result average of three independent experiments is shown for each liposome preparation.

FIGURE 3

CF leakage from (○) PE/PG (3:7, 70% charge), (■) PE/PG (7:3, 30% charge), (●) PE/PG/CL (7:1:1, 30% charge) and (▲) E. coli (∼35% charge) liposomes as a function of concentration of native Crp4 (top panel), (6C/A)-Crp4 (middle panel), and melittin (bottom panel). Liposomes containing carboxyfluoerescein as a fluorescent marker were incubated with various concentrations of peptides for 90 min and the percentage of dye released was determined by measuring the fluorescence as described in “Materials and methods”. Averages of three experiments and standard deviations are shown.

FIGURE 4

%CR transitions induced by Crp4, (6C/A)-Crp4 and melittin in vesicles of different lipid composition. Vesicle solutions containing (A) POPG/DOPE/PDA (7:3:15, 70% charge) and (B) CL/POPG/DOPE/PDA (1:1:7:14, 30% charge) were exposed to Crp4 (-○-), (6C/A)-Crp4 (-◆-) or melittin (--▲--). The net charge of the lipid assemblies pertains exclusively to the phospholipid components of the phospholipid/PDA mixed vesicles. Higher %CR indicates more pronounced interfacial peptide binding.

FIGURE 5

Distinctive sensitivity of Crp4 and (6C/A)-Crp4 membrane interactions to lipid composition. The degree of blue-to-red transitions (%CR) induced by Crp4 and (6C/A)-Crp4 was measured in mixed vesicles composed of: POPG/DOPE/PDA (3:7:15) and CL/POPG/DOPE/PDA (1:1:7:14). Both types of vesicles possess 30% of surface negative charge (accounting only to the phospholipid components of the mixed vesicles).

FIGURE 6

Detection of peptide translocation by selective digestion of G1W-Crp4 or (6C/A)G1W-Crp4 on the outer leaflet. Dansyl-labeled vesicles were injected into the peptide solution at the time indicated by the arrow and the fluorescence intensity of the dansyl group was recorded at 528nm (excitation at 280nm). Binding of the peptide increased fluorescence due to FRET. Addition of chymotrypsin (0.125 μg/mL) at various incubation intervals (0, 1, 5, 10 or 15 min, traces 2-6 respectively) resulted in digestion of the peptide molecules from the outer leaflet in a time-dependent manner, indicating that prolonged incubation protected the peptide from enzyme attack. Pre-incubation of the peptides with the enzyme for 5 min (trace 1 in A, B and dotted line in C, D) before addition of liposomes was used as a control. (A) G1W-Crp4 and PE/PG/CL liposomes with 30% negative charge (B) G1W-Crp4 and PE/PG liposomes with 70% negative charge (C) (6C/A)G1W-Crp4 and PE/PG/CL liposomes with 30% negative charge (D) (6C/A)G1W-Crp4 and PE/PG liposomes with 70% negative charge. The final peptide and lipid concentrations were 5.9 μM and 100 μM, respectively. Each trace is the average of three to four experiments.

FIGURE 6

Detection of peptide translocation by selective digestion of G1W-Crp4 or (6C/A)G1W-Crp4 on the outer leaflet. Dansyl-labeled vesicles were injected into the peptide solution at the time indicated by the arrow and the fluorescence intensity of the dansyl group was recorded at 528nm (excitation at 280nm). Binding of the peptide increased fluorescence due to FRET. Addition of chymotrypsin (0.125 μg/mL) at various incubation intervals (0, 1, 5, 10 or 15 min, traces 2-6 respectively) resulted in digestion of the peptide molecules from the outer leaflet in a time-dependent manner, indicating that prolonged incubation protected the peptide from enzyme attack. Pre-incubation of the peptides with the enzyme for 5 min (trace 1 in A, B and dotted line in C, D) before addition of liposomes was used as a control. (A) G1W-Crp4 and PE/PG/CL liposomes with 30% negative charge (B) G1W-Crp4 and PE/PG liposomes with 70% negative charge (C) (6C/A)G1W-Crp4 and PE/PG/CL liposomes with 30% negative charge (D) (6C/A)G1W-Crp4 and PE/PG liposomes with 70% negative charge. The final peptide and lipid concentrations were 5.9 μM and 100 μM, respectively. Each trace is the average of three to four experiments.

FIGURE 7

Relationship between Crp4 translocation and membrane leakage. The time courses of carboxyfluorescein leakage and peptide translocation are shown by solid curves and symbols, respectively: (A) trace and ▲, PE/PG/CL liposomes with 30% negative charge; (B) trace and ●, PE/PG liposomes with 70% negative charge. Percent translocation was estimated as 100× (ΔF/F₀) from Figs. 7A & 7B. The peptide and lipid concentrations were 5.9 μM and 100 μM respectively.