Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and ³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. ³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

Limiting an antimicrobial peptide to the lipid-water interface enhances its bacterial membrane selectivity: a case study of MSI-367

In a minimalist design approach, a synthetic peptide MSI-367 [(KFAKKFA)(3)-NH(2)] was designed and synthesized with the objective of generating cell-selective nonlytic peptides, which have a significant bearing on cell targeting. The peptide exhibited potent activity against both bacteria and fungi, but no toxicity to human cells at micromolar concentrations. Bacterial versus human cell membrane selectivity of the peptide was determined via membrane permeabilization assays. Circular dichroism investigations revealed the intrinsic helix propensity of the peptide, β-turn structure in aqueous buffer and extended and turn conformations upon binding to lipid vesicles. Differential scanning calorimetry experiments with 1,2-dipalmitoleoyl-sn-glycero-3-phosphatidylethanolamine bilayers indicated the induction of positive curvature strain and repression of the fluid lamellar to inverted hexagonal phase transition by MSI-367. Results of isothermal titration calorimetry (ITC) experiments suggested the possibility of formation of specific lipid-peptide complexes leading to aggregation. (2)H nuclear magnetic resonance (NMR) of deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) multilamellar vesicles confirmed the limited effect of the membrane-embedded peptide at the lipid-water interface. (31)P NMR data indicated changes in the lipid headgroup orientation of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine lipid bilayers upon peptide binding. Membrane-embedded and membrane-inserted states of the peptide were observed via sum frequency generation vibrational spectroscopy. Circular dichroism, ITC, and (31)P NMR data for Escherichia coli lipids agree with the hypothesis that strong electrostatic lipid-peptide interactions embrace the peptide at the lipid-water interface and provide the basis for bacterial cell selectivity.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Effects of salt and denaturant on structure of the amino terminal alpha-helical segment of an antibacterial peptide dermaseptin and its binding to model membranes.

The amino terminal 1-18 domain of dermaseptin s is an important determinant of its structure as well as the antibacterial activity. A thorough investigation on the structure of the 18-residue peptide (D18) and its binding to model membranes in presence of salt and denaturant guanidinium chloride has been carried out. In presence of salt, there is an increase in the fraction of peptide molecules in helical conformation. In presence of the denaturant, D18 is unordered, but addition of the structure-promoting solvent trifluoroethanol results in a transition to the helical conformation. In presence of denaturant, the peptide is unordered, but binding to lipid vesicles is not abolished. Investigation of model membrane permeabilizing ability of the peptide in solutions containing various proportions of sodium chloride and guanidinium chloride indicates that vesicle permeabilization parallels extent of binding. The peptide thus binds to lipid vesicles in an unfolded state. Since the peptide has propensity to fold into a helical conformation, lipid induced transition to a helical structure occurs, followed by membrane permeabilization as a result of pore formation.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers, determined by NMR spectroscopy

Magainins are antimicrobial peptides that selectively disrupt bacterial cell membranes. In an effort to determine the propensity for oligomerization of specific highly active magainin analogues in membrane mimetic systems, we studied the structures and lipid interactions of two synthetic variants of magainins (MSI-78 and MSI-594) originally designed by Genaera Corp. Using NMR experiments on these peptides solubilized in dodecylphosphocholine (DPC) micelles, we found that the first analogue, MSI-78, forms an antiparallel dimer with a "phenylalanine zipper" holding together two highly helical protomers, whereas the second analogue, MSI-594, whose phenylalanines 12 and 16 were changed into glycine and valine, respectively, does not dimerize under our experimental conditions. In addition, magic angle spinning solid-state NMR experiments carried out on multilamellar vesicles were used to corroborate the helical conformation of the peptides found in detergent micelles and support the existence of a more compact structure for MSI-78 and a pronounced conformational heterogeneity for MSI-594. Since magainin activity is modulated by oligomerization within the membrane bilayers, this study represents a step forward in understanding the role of self-association in determining magainin function.     

Solid-state NMR investigation of the membrane-disrupting mechanism of antimicrobial peptides MSI-78 and MSI-594 derived from magainin 2 and melittin

The mechanism of membrane interaction of two amphipathic antimicrobial peptides, MSI-78 and MSI-594, derived from magainin-2 and melittin, is presented. Both the peptides show excellent antimicrobial activity. The 8-anilinonaphthalene-1-sulfonic acid uptake experiment using Escherichia coli cells suggests that the outer membrane permeabilization is mainly due to electrostatic interactions. The interaction of MSI-78 and MSI-594 with lipid membranes was studied using 31P and 2H solid-state NMR, circular dichroism, and differential scanning calorimetry techniques. The binding of MSI-78 and MSI-594 to the lipid membrane is associated with a random coil to alpha-helix structural transition. MSI-78 and MSI-594 also induce the release of entrapped dye from POPC/POPG (3:1) vesicles. Measurement of the phase-transition temperature of peptide-DiPoPE dispersions shows that both MSI-78 and MSI-594 repress the lamellar-to-inverted hexagonal phase transition by inducing positive curvature strain. 15N NMR data suggest that both the peptides are oriented nearly perpendicular to the bilayer normal, which infers that the peptides most likely do not function via a barrel-stave mechanism of membrane-disruption. Data obtained from 31P NMR measurements using peptide-incorporated POPC and POPG oriented lamellar bilayers show a disorder in the orientation of lipids up to a peptide/lipid ratio of 1:20, and the formation of nonbilayer structures at peptide/lipid ratio>1:8. 2H-NMR experiments with selectively deuterated lipids reveal peptide-induced disorder in the methylene units of the lipid acyl chains. These results are discussed in light of lipid-peptide interactions leading to the disruption of membrane via either a carpet or a toroidal-type mechanism.     

Cell selectivity correlates with membrane-specific interactions: a case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and 31P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. 31P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

A Fourteen Gene GBM Prognostic Signature Identifies Association of Immune Response Pathway and Mesenchymal Subtype with High Risk Group

Background

Recent research on glioblastoma (GBM) has focused on deducing gene signatures predicting prognosis. The present study evaluated the mRNA expression of selected genes and correlated with outcome to arrive at a prognostic gene signature.

Methods

Patients with GBM (n = 123) were prospectively recruited, treated with a uniform protocol and followed up. Expression of 175 genes in GBM tissue was determined using qRT-PCR. A supervised principal component analysis followed by derivation of gene signature was performed. Independent validation of the signature was done using TCGA data. Gene Ontology and KEGG pathway analysis was carried out among patients from TCGA cohort.

Results

A 14 gene signature was identified that predicted outcome in GBM. A weighted gene (WG) score was found to be an independent predictor of survival in multivariate analysis in the present cohort (HR = 2^.507; B = 0^.919; p<0^.001) and in TCGA cohort. Risk stratification by standardized WG score classified patients into low and high risk predicting survival both in our cohort (p = <0^.001) and TCGA cohort (p = 0^.001). Pathway analysis using the most differentially regulated genes (n = 76) between the low and high risk groups revealed association of activated inflammatory/immune response pathways and mesenchymal subtype in the high risk group.

Conclusion

We have identified a 14 gene expression signature that can predict survival in GBM patients. A network analysis revealed activation of inflammatory response pathway specifically in high risk group. These findings may have implications in understanding of gliomagenesis, development of targeted therapies and selection of high risk cancer patients for alternate adjuvant therapies.     

Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A

Subtilosin A is an antimicrobial peptide produced by the soil bacterium Bacillus subtilis that possesses bactericidal activity against a diverse range of bacteria, including Listeria monocytogenes. Recent structural studies have found that subtilosin A is posttranslationally modified in a unique way, placing it in a new class of bacteriocins. In this study, in order to understand the mechanism of membrane-disruption by subtilosin A, the interaction of the peptide with model phospholipid bilayers is characterized using fluorescence, solid-state NMR and differential scanning calorimetry (DSC) experiments. Our results in this study show that subtilosin A interacts with the lipid head group region of bilayer membranes in a concentration dependent manner. Fluorescence experiments reveal the interaction of subtilosin A with small unilamellar vesicles (SUVs) composed of POPC, POPG and E. coli total lipids, and that at least one edge of the molecule is buried in membrane bilayers. At high concentrations, it induces leakage from SUVs of POPC and POPE/POPG (7:3) mixture. (15)N solid-state NMR data suggests that the cyclic peptide is partially inserted into bilayers, which is in agreement with the fluorescence data. (31)P and (2)H NMR experiments and DSC data support the hypothesis that subtilosin A adopts a partially buried orientation in lipid bilayers, by showing that it induces a conformational change in the lipid headgroup and disordering in the hydrophobic region of bilayers. These results suggest that the lipid perturbation observed in this study may be one of the consequences of subtilosin A binding to lipid bilayers, which results in membrane permeabilization at high peptide concentrations.