Calcein Dye Leakage Research Articles

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.

Impact of the antimicrobial peptide Novicidin on membrane structure and integrity

We have studied the impact of an 18-residue cationic antimicrobial peptide Novicidin (Nc) on the structure and integrity of partially anionic lipid membranes using oriented circular dichroism (OCD), quartz crystal microbalance with dissipation (QCM-D), dual polarization interferometry (DPI), calcein dye leakage and fluorescence spectroscopy. OCD consistently showed that Nc is bound in an α-helical, surface bound state over a range of peptide to lipid (P/L) ratios up to ∼1:15. Realignment of Nc at higher P/L ratios correlates to loss of membrane integrity as shown by Laurdan fluorescence spectroscopy and by loss of lipid alignment in DPI analysis. Laurdan generalized polarity shows a decrease in water accessibility or mobility in the hydrophobic/hydrophilic interface of the lipid membrane, consistent with rearrangement of lipid packing. QCM-D studies on the interaction of Nc with lipid membranes emphasize the importance of including the dissipation factor in data analysis, revealing formation of a highly hydrated film after exposure to ⩾3 μM Nc. Our findings suggest a carpet mechanism of membrane disruption in which peptide binding first induces leakage at a critical surface concentration, probably through formation of transient pores or transient disruption of the membrane integrity, followed by more extensive membrane disintegration at higher P/L ratios.

Crystal Structure and Functional Studies Reveal that PAS Factor from Vibrio vulnificus is a Novel Member of the Saposin-fold Family

PAS factor is a novel putative bacterial secretion factor thought to induce secretion of periplasmic proteins. We solved the crystal structure of PAS factor from Vibrio vulnificus at 1.8A resolution and found it to be comprised of five alpha helices that form an antiparallel bundle with an up-and-down topology, and to adopt the saposin-fold characteristic of a family of proteins that bind to membranes and lipids. PAS factor lacks the disulfide bridge characteristic of mammalian saposin-fold proteins; in fact, it shows no sequence homology with mammalian proteins. Nevertheless, the molecular architectures are similar, and the shared propensity for membrane interaction suggests strongly that PAS factor is another member of the saposin-fold family. Analysis of the CD spectra showed that PAS factor binds to membranes directly, while measurement of calcein dye leakage showed that PAS factor interacts strongly with liposomes composed of anionic phospholipids, making them leaky, but binds very weakly with liposomes composed of zwitterionic phospholipids. Moreover, by analyzing tryptophan fluorescence emission from four single-tryptophan mutants (V10W, T22W, F35W, and L70W), we identified the putative phospholipid-binding site of PAS factor. The resultant membrane destabilization likely mediates secretion of periplasmic proteins required for the in vivo survival and pathogenesis of V.vulnificus.