Ramakrishnan Nagaraj

Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity.

Species right across the evolutionary scale from insects to mammals use peptides as part of their host-defense system to counter microbial infection. The primary structures of a large number of these host-defense peptides have been determined. While there is no primary structure homology, the peptides are characterized by a preponderance of cationic and hydrophobic amino acids. The secondary structures of many of the host-defense peptides have been determined by a variety of techniques. The acyclic peptides tend to adopt helical conformation, especially in media of low dielectric constant, whereas peptides with more than one disulfide bridge adopt beta-structures. Detailed investigations have indicated that a majority of these host-defense peptides exert their action by permeabilizing microbial membranes. In this review, we discuss structural and charge requirements for the interaction of endogenous antimicrobial peptides and short peptides that have been derived from them, with membranes.

Cell-lytic and antibacterial peptides that act by perturbing the barrier function of membranes: facets of their conformational features, structure-function correlations and membrane-perturbing abilities

Almost all hemolytic and antimicrobial peptides form part of the defense mechanism of species widely distributed across the evolutionary scale. Although these peptides are of varying lengths and composition, they form amphiphilic structures in a hydrophobic environment. They also have the ability to form channels in natural and model membranes. Hemolytic peptides have proven to be very useful in studying the mechanism of hemolysis and the permeability properties of red blood cells. Preliminary investigations indicate that these peptides may also be useful in the investigation of complex cellular phenomena like exocytosis and neurotransmission. Although molecules like vancomycin, bacitracin and penicillins have been extensively used as antibiotics for therapeutic purposes, most species throughout the evolutionary scale use peptides as antimicrobial agents. These peptides exert their activity by altering the permeability properties of the bacterial plasma membrane and do not interfere with macro molecular synthesis like the other antibiotics that are presently used in therapies. Hence it is likely that resistance to peptide antibacterial agents may not develop easily. Since the problem of antibiotic resistance is presently a particularly severe one, peptide antibiotics may be the drugs of choice in the future.

Induction of autophagic cell death in Leishmania donovani by antimicrobial peptides

We demonstrate that antimicrobial peptides induce an autophagic cell death in the protozoan pathogen, Leishmania donovani. In our study, three antimicrobial peptides, Indolicidin, and two peptides derived from Seminalplasmin exhibit antileishmanial activity with a 50% lethal dose of 3.5 x 10(-5), 3.8 x 10(-4) and 1.7 x 10(-8) microM, respectively. The action of these antimicrobial peptides on the Leishmania cell involves ionic interactions, which are modulated by lipophosphoglycan on the parasites surface. Peptide treatment caused dissipation of membrane potential and equilibration of intracellular pH with extracellular environment. However, there was no release of intracellular GFP molecules upon peptide treatment of a GFP expressing Leishmania clone. Transmission electron microscopic studies show extensive intracellular damage including cytoplasmic vacuolization and degeneration of cellular organization without disruption of the plasma membrane. These peptides induce cell death via a non-apoptotic process as shown by lack of nuclear fragmentation or DNA laddering and independent of caspase-like activity. Instead, Monodansylcadaverine (MDC), a biochemical marker of autophagy specifically labels the vacuoles induced by peptides. Collectively, these results indicate that in addition to their effects on the leishmanial membrane, these antimicrobial peptides induce pathway(s) for autophagic cell death in L. donovani.

Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin

The antimicrobial and hemolytic activities of the 13‐residue peptide indolicidin (ILPWKWPWWPWRR‐NH2), present in bovine neutrophils, and its analogs have been determined with a view to gaining insight into the structural roles of tryptophan and proline. Peptides where proline was replaced by alanine and tryptophan by phenylalanine showed antibacterial activities comparable to that of indolicidin. The peptides do not exhibit a strong propensity to occur in either helical or β‐sheet conformation. The peptides also do not appear to exert their activity by permeabilizing the bacterial plasma membrane unlike other endogenous antibacterial peptides. The presence of tryptophan appears to be essential for hemolytic activity as the phenylalanine analog does not exhibit any hemolytic activity.

Host-defense antimicrobial peptides: importance of structure for activity.

Antimicrobial peptides are important components of innate immunity in species across the evolutionary scale. Unlike therapeutically used antibiotics, this class of peptides exert their activity by permeabilizing bacterial membranes. Despite the seemingly common mechanism of action, there is considerable variation in their primary structures, length and number of positive charges. Host-defense antimicrobial peptides have been the subject of extensive biophysical studies with a view at delineate structural requirements for activity. In this article, the structures of host defence antibacterial peptides and the structural requirements for activity are reviewed.

Antifungal Activities of Human Beta-Defensins HBD-1 to HBD-3 and Their C-Terminal Analogs Phd1 to Phd3

ABSTRACT The activities of defensins HBD-1, HBD-2, and HBD-3 and their C-terminal analogs Phd1, Phd2, and Phd3 against Candida albicans were investigated. Phd1 to Phd3 showed lower-level activities than HBD-1 to HBD-3, although metabolic inhibitors did not render Phd1 to Phd3 inactive. Their activities were also less salt sensitive than those of HBD-1 to HBD-3. Confocal microscope images indicated that the initial site of action was the fungal membrane.

Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity

Melittin, the 26‐residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C‐terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5–7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been trans‐positioned to the N‐terminal region from the C‐terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.

Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines

ABSTRACT The antibacterial activity of human neutrophil defensin HNP-1 analogs without cysteines has been investigated. A peptide corresponding to the HNP-1 sequence without the six cysteines (HNP-1ΔC) exhibited antibacterial activity toward gram-negative and gram-positive bacteria. Truncated analogs wherein the nine N-terminal residues of HNP-1 and the remaining three cysteines were deleted (HNP-1ΔC18) or the G was replaced with A (HNP-1ΔC18A) also exhibited antibacterial activity. Substantial activity was observed for HNP-1ΔC and HNP-1ΔC18 in the presence of 100 mM NaCl, except in the case of Pseudomonas aeruginosa. The linear peptides were active in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that proton motive force was not essential for killing of bacteria by the peptides. In fact, in the presence of CCCP, the peptides were active against P. aeruginosa even in the presence of 100 mM NaCl. The antibacterial activity of HNP-1ΔC, but not that of the shorter, 18-residue peptides, was attenuated in the presence of serum. The generation of defensins without cysteines would be easier than that of disulfide-linked defensins. Hence, linear defensins could have potential as therapeutic agents.

Antibacterial activities of synthetic peptides corresponding to the carboxy-terminal region of human β-defensins 1–3

The antibacterial activities of synthetic human beta-defensin analogs, constrained by a single disulfide bridge and in the reduced form, have been investigated. The peptides span the carboxy-terminal region of human beta-defensins (HBD-1-3), which have a majority of cationic residues present in the native defensins. The disulfide constrained peptides exhibited activity against Escherichia coli and Staphylococcus aureus whereas the reduced forms were active only against E. coli. The antibacterial activities were attenuated in the presence of increasing concentrations of NaCl and divalent cations such as Ca(2+) and Mg(2+). The site of action was the bacterial membrane. Peptides spanning the carboxy-terminal region of human beta-defensins could be of help in understanding facets of antimicrobial activity of beta-defensins such as salt sensitivity and mechanisms of bacterial membrane damage.

Antibacterial activities and conformations of bovine β-defensin BNBD-12 and analogs:structural and disulfide bridge requirements for activity

Structure and biological activities of synthetic peptides corresponding to bovine neutrophil beta-defensin BNBD-12, GPLSC(1)GRNGGVC(2)IPIRC(3) PVPMRQIGTC(4) FGRPVKC(5) C(6)RSW with disulfide connectivities C(1)-C(5), C(2)-C(4) and C(3)-C(6) and its variants with one, two and three disulfide bridges have been investigated. Selective protection of cysteine thiols was necessary in the four and six cysteine containing peptides for the formation of disulfide connectivities as observed in BNBD-12. Circular dichroism (CD) spectra indicate that in aqueous medium, only a small fraction of molecules populate turn-like conformations. In the presence of micelles and lipid vesicles, the single, two and three disulfide containing peptides adopt beta-hairpin or beta-sheet structures. Antibacterial activity was observed for all the peptides, irrespective of the number of disulfide bridges or how they were connected. Our results suggest that a rigid beta-sheet structure or the presence of three disulfide bridges does not appear to be stringent requirements for antibacterial activity in beta-defensins.