Biomedical subjects

Sathiah Thennarasu

Publications and source records attributed to Sathiah Thennarasu.

7 recordsLinked to original sources

PubMed2006

Deletion of all cysteines in tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and lipopolysaccharide selective binding.

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.

Anti-Infective Agents↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

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.

Antimicrobial Cationic Peptides↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2005

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 alpha-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.

Anti-Bacterial Agents↗

PubMed2005

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.

Bacterial Proteins↗

PubMed2003

Spectrophotometric and fluorimetric determination of hexamine in pure form and its pharmaceutical formulation.

Ethyl acetoacetate and methyl acetoacetate have been tested for the determination of hiprex (methenamine hippurate). Both the esters react with hexamine and hiprex forming fluorescent yellow Hantzsch esters. The products show absorption maximum at 370 nm and a fluorescence emission maximum at 465 nm in aqueous n-propanol. Important parameters such as pH, temperature, mole ratio and time of reaction have been optimized. The co-efficient of variation for inter and intra-day precisions were less than 1%. The chromogens show linearity in the concentration ranges of 2.0-35.0 microg x ml(-1) and 0.6-40.0 micro-1) for the spectrometric and fluorimetric determinations of hiprex, respectively.

Ascorbic Acid↗