Membrane lysis by the antibacterial peptide, cecropin B and its analogs cecropins B1 and B3 on lipid bilayers
by Wei Wang
xvi, 191 leaves : ill. (some col.) ; 30 cm
The natural antibacterial peptide, cecropin B (CB), and it analogs cecropin B1 (CB1), cecropin B2 (CB2) and cecropin B3 (CB3) were synthesized. These peptides were used to investigate their liposome lysis, anti-bacterial and anti-cancer activities. The strength of liposome lysis activity and the potency of these analogs on bacteria are reduced two- to three-fold when the analogs are used instead of natural CB based on the DL50 analysis and lethal concentration (LC) measurements, respectively. In contrast, on leukemia cells, the potency of CB1 and CB2 is about two- to three-fold greater than that of natural CB based on the IC50 measurements. These results indicate that the designed cationic lytic peptides, having extra cationic residues, are less effective in breaking liposomes and killi...[ Read more ]
The natural antibacterial peptide, cecropin B (CB), and it analogs cecropin B1 (CB1), cecropin B2 (CB2) and cecropin B3 (CB3) were synthesized. These peptides were used to investigate their liposome lysis, anti-bacterial and anti-cancer activities. The strength of liposome lysis activity and the potency of these analogs on bacteria are reduced two- to three-fold when the analogs are used instead of natural CB based on the DL50 analysis and lethal concentration (LC) measurements, respectively. In contrast, on leukemia cells, the potency of CB1 and CB2 is about two- to three-fold greater than that of natural CB based on the IC50 measurements. These results indicate that the designed cationic lytic peptides, having extra cationic residues, are less effective in breaking liposomes and killing bacteria but more effective in lysing cancer cells.
The different characteristics of these peptides, CB, CB1, CB2, and CB3, were used to study the morphological changes in the bacterial cell, Klebsiella pneumoniae, and the leukemia cancer cell, HL-60, by scanning electron microscopy and transmission electron microscopy. The natural and analog peptides have comparable secondary structures as shown by circular dichroism measurements. This indicates that the potency of the peptides on cell membranes is dependent of the helical characteristics rather than the helical strength. The microscopic results show that the morphological changes of the cells treated with CB are distinguishably different from those treated with CBl and CB2, which are designed to have enhanced anti-cancer properties by having an extra amphipathic α-helix. The morphological differences may be due to their different modes of action on the cell membranes resulting in the different potencies with lower LC and higher IC50 of CB on bacterium and cancer cell, respectively, as compared with CBl and CB2. These results provide microscopic evidence that different killing pathways are involved with the peptides.
The differences of the characteristics of CB, CB1 and CB3 were further used as the rationale for a study of their efficacy in breaking liposomes with different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC). Biosensor binding measurements and encapsulating dye leakage studies showed that the higher binding affinity of CB and CB1 to the polar heads of lipids is not necessary for the peptides to be more effective in lysing lipid bilayers, especially when liposomes have a higher PA content. Kinetic studies, by intrinsic and extrinsic fluorescence stopped-flow measurements, revealed two transitional steps in liposome breakage by CB and CB1, although only one kinetic step was found for CB3. Circular dichroism stopped-flow measurements, monitoring the formation of secondary structure in the peptides, revealed one kinetic step for the interaction of all of the peptides with the liposomes. Also, the α-helical motif of the peptides was maintained after interacting with the liposomes.
For spin labeled lipids, 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC and 16'SL-PC, the ESR spectra showed that when compared with CB3, CBl gives rise to larger outer hyperfine splitting (2Amax) when interacting with liposomes of different compositions. This corresponds to a larger restriction of the motion of spin-labeled chains in the presence of CB1 . Investigations of the effective order parameter (chain flexibility gradient) and relationship of 2Amax versus the peptide/lipid ratio further indicate that the lysis action of CB1 is dependent on its capacity to bind to the lipid bilayers, whereas, there is no evidence of binding for CB3. Four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3 and C30SL-CB3, were used to examine the binding and the state of aggregation of the peptides. Association isotherms of the peptides were measured for liposomes of β=0.25 and β=0.75 (β: fraction of PA, β=PA/(PA+PC)). The membrane binding of the CB1 peptides exhibited a cooperative behavior, while the association isotherm of CB3 showed binding to the lipid only for β=0.75 liposomes. To further identify the location of CB1 in lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggest that the NH2-terminal α-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal α-helix of CB1 may be inside the surface of the lipid bilayers. These outcomes were further supported by the observed relationship between the partition distribution of peptides bound to liposomes with different PA/PC ratios and the amount of free peptides.
A method of fluorescence quenching is used to explore the kinetics of liposome lysis by CB, CB1 and CB3 at molecular level. By the study of the interaction between fluorophore, NBD-labeled lipids and quencher, DN, the mode of membrane lysis of CB/CB1 and CB3 on liposomes of different compositions (β = 0.25 and β = 0.75) was determined as all-or-none. The kinetic observations show two different permeabilization pathways: multimeric pore formation of CB/CB1 and gross membrane destabilization of CB3. The data are consistent with our former kinetic results from dye-leakage and Trp fluorescence. Detailed kinetic analysis also show that tetramers of CB/CB1 in the membrane are formed during the liposome (β = 0.25) lysis.
To elucidate the pathways of membrane lysis, CB, CB1 and CB3 have been used to measure the real time interaction with liposomes. The liposomes of different compositions immobilized on the surface of biosensor chip are established and their permeabilization has been studied by CB, CB1 and CB3. At higher peptide concentration (2.5 to 100 μM), the results show- that CB and CBl bound to the liposomes (but lysis does not occur) since the response unit (RU) is proportionally increased (δRU [is more than] 0) as the concentration of peptide is increased. Whereas, the negative RU (δRU [is less than] 0) is observed if the concentrations of CB/CB1 are decreased below 2.5 μM. This indicates that CB/CB1 peptides not only bound to the liposomes but subsequently lyse liposomes under the conditions of limited peptide quantity. The dose dependence of CB/CB1 on the permeabilization of liposomes is therefore discussed. Addition of CB3 to the liposomes does not show any capacity of bindings (δRU ≤ 0). This implies that the lysis of liposomes by CB3 occurs, but the peptide does not go through the bindings.
Based on these results, the possible mechanisms of liposome lysis by CB, CB1 and CB3 are proposed.