Antimicrobial activity,bactericidal mechanism and LPS‐neutralizing activity of the cell‐penetrating peptide pVEC and its analogs

pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Design,synthesis and biological activity of cell‐penetrating peptide‐modified octreotide analogs

Four novel octreotide analogs with cell‐penetrating peptides (CPPs) at the N‐terminus or C‐terminus were synthesized by a stepwise Fmoc solid‐phase synthesis strategy. The synthesized peptides were analyzed and characterized using reverse phase HPLC and MALDI‐TOF mass spectrometry. The antiproliferative activity of the analogs was tested in vitro on human gastric (SGC‐7901) and hepatocellular cancer (BEL7402) cell lines using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Interestingly, these analogs showed a higher anticancer activities than the parent octreotide except CMTPT03 analog. The results demonstrate that the designed octreotide analogs enhance their anticancer activity after linking together the CPPs to octreotide at the N‐terminus, and are potential molecules for future use in cancer therapy and drug targeting. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine,a cell‐penetrating peptide

Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra‐, hexa‐, or octapeptides) cell‐penetrating peptides on the cytostatic activity in vitro and in vivo. Br‐Vindoline‐(l )‐Trp‐OH attached to the N‐terminus of octaarginine was the most effective compound in vitro on HL‐60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br‐vindoline‐(l )‐Trp‐Arg₈ and Br‐vindoline‐(d )‐Trp‐Arg₈ suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l )‐Trp was more active than conjugate with the (d )‐isomer. In contrast, conjugates had very similar effect on both the HL‐60 and MDA‐MB‐231 cells. In preliminary experiments, conjugate Br‐vindoline‐(l )‐Trp‐Arg₈ exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor‐bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.     

Screening and characterization of a novel high‐efficiency tumor‐homing cell‐penetrating peptide from the buffalo cathelicidin family

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell‐penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo‐derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell‐penetrating antimicrobial peptides, including the well‐known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC‐7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC‐5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia‐derived type‐I ribosome‐inactivating protein MAP 30, and the cytotoxicity of the MAP 30‐CAT fusion protein in the tumor cell line SMMC‐7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30‐CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC‐5 was approximately twofold higher than the value for SMMC‐7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38‐fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor‐homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.     

Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.     

Cellular uptake and biological activity of peptide nucleic acids conjugated with peptides with and without cell‐penetrating ability

A 12‐mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell‐penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell‐penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA‐peptide conjugates to be not primarily related to the cell‐penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA‐peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Cyclization of a cell‐penetrating peptide via click‐chemistry increases proteolytic resistance and improves drug delivery

In this work we report synthesis and biological evaluation of a cell‐penetrating peptide (CPP), that is partly cyclized via a triazole bridge. Recently, beneficious properties have been reported for cyclized peptides concerning their metabolic stability and intracellular uptake. A CPP based on human calcitonin was used in this study, and side chain cyclization was achieved via copper catalyzed alkyne‐azide click reaction. Cell viability studies in several cell‐lines revealed no cytotoxic effects. Furthermore, efficient uptake in breast cancer MCF‐7 cells could be determined. Moreover, preliminary studies using this novel peptide as drug transporter for daunorubicin were performed. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The heparin‐binding domain of HB‐EGF as an efficient cell‐penetrating peptide for drug delivery

Cell‐penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human‐derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin‐binding domain of HB‐EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30‐HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C‐terminus of MAP30 promoted the cellular uptake of recombinant MAP30‐HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30‐induced apoptosis through the activation of the mitochondrial‐ and death receptor‐mediated signaling pathways. In addition, the MAP30‐HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30‐HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB‐EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Antifungal effect and action mechanism of antimicrobial peptide polybia‐CP

The incidence of life‐threatening invasive fungal infections increased significantly in recent years. However, the antifungal therapeutic options are very limited. Antimicrobial peptides are a class of potential lead chemical for the development of novel antifungal agents. Antimicrobial peptide polybia‐CP was purified from the venom of the social wasp Polybia paulista. In this study, we synthesized polybia‐CP and determined its antifungal effects against a series of Candidian species. Our results showed that polybia‐CP has potent antifungal activity and fungicidal activity against the tested fungal cells with a proposed membrane‐active action mode. In addition, polybia‐CP could induce the increase of cellular reactive oxygen species production, which would attribute to its antifungal activity. In conclusion, the present study suggests that polybia‐CP has potential as an antifungal agent or may offer a new strategy for antifungal therapeutic option. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Design of a bioactive cell‐penetrating peptide: when a transduction domain does more than transduce

The discovery of cell‐penetrating peptides (CPPs) has facilitated delivery of peptides into cells to affect cellular behavior. Previously, we were successful at developing a phosphopeptide mimetic of the small heat shock‐like protein HSP20 . Building on this success we developed a cell‐permeant peptide inhibitor of mitogen‐activated protein kinase‐activated protein kinase 2 (MK2). It is well documented that inhibition of MK2 may be beneficial for a myriad of human diseases including those involving inflammation and fibrosis. During the optimization of the activity and specificity of the MK2 inhibitor (MK2i) we closely examined the effect of cell‐penetrating peptide identity. Surprisingly, the identity of the CPP dictated kinase specificity and functional activity to an extent that rivaled that of the therapeutic peptide. The results reported herein have wide implications for delivering therapeutics with CPPs and indicate that judicious choice of CPP is crucial to the ultimate therapeutic success. Published in 2009 by John Wiley & Sons, Ltd.     

Cholesterol prevents interaction of the cell‐penetrating peptide transportan with model lipid membranes

Interaction of the cell‐penetrating peptide (CPP) cysteine‐transportan (Cys‐TP) with model lipid membranes was examined by spin‐label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys‐TP on membrane structure was studied. The influence of Cys‐TP on membrane permeability was monitored by the reduction of a liposome‐trapped water‐soluble spin probe. Cys‐TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys‐TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so‐called liquid‐ordered phase, no effect of Cys‐TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys‐TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP–lipid interaction in laterally heterogeneous membranes, while it implies that the CPP–lipid interaction can be different at different points along the membrane. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Candidacidal mechanism of a Leu/Lys‐rich α‐helical amphipathic model antimicrobial peptide and its diastereomer composed of D,L‐amino acids

We investigated the mechanism of candidacidal action of a Lys/Leu‐rich α‐helical model antimicrobial peptide (K₉L₈W) and its diastereomeric peptide (D₉‐K₉L₈W) composed of D ,L ‐amino acids. K₉L₈W killed completely Candida albicans within 30 min, but D₉‐K₉L₈W killed only 72% of C. albicans even after 100 min. Tryptophan fluorescence spectroscopy indicated that the fungal cell selectivity of D₉‐K₉L₈W is closely correlated with a selective interaction with the negatively charged PC/PE/PI/ergosterol (5:2.5:2.5:1, w/w/w/w) phospholipids, which mimic the outer leaflet of the plasma membrane of C. albicans. K₉L₈W was able to induce almost 100% calcein leakage from PC/PE/PI/ergosterol (5:2.5:2.5:1, w/w/w/w) liposomes at a peptide:lipid molar ratio of 1:16, whereas D₉‐K₉L₈W caused only 25% dye leakage even at a peptide:lipid molar ratio of 1:2. Confocal laser‐scanning microscopy revealed that FITC‐labeled D₉‐K₉L₈W penetrated the cell wall and cell membrane and accumulated inside the cells, whereas FITC‐labeled K₉L₈W did not penetrate but associated with the membranes. Collectively, our results demonstrated that the candidacidal activity of K₉L₈ W and D₉‐K₉L₈W may be due to the transmembrane pore/channel formation or perturbation of the fungal cytoplasmic membranes and the inhibition of intracellular functions, respectively. Finally, D₉‐K₉L₈W with potent anti‐Candida activity but no hemolytic activity may be potentially a useful lead compound for the development of novel antifungal agents. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Membrane damage as first and DNA as the secondary target for anti‐candidal activity of antimicrobial peptide P7 derived from cell‐penetrating peptide ppTG20 against Candida albicans

P7, a peptide analogue derived from cell‐penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti‐Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l ‐phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin‐treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC‐P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Analogues of the frog skin peptide alyteserin‐2a with enhanced antimicrobial activities against Gram‐negative bacteria

The emergence of strains of multidrug‐resistant Gram‐negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin‐2a (ILGKLLSTAAGLLSNL.NH₂) is a cationic, α‐helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin‐2a while maintaining amphipathicity by the substitution Gly¹¹→ Lys enhanced the potency against both Gram‐negative and Gram‐positive bacteria by between fourfold and 16‐fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC₅₀ = 24 µm ). Antimicrobial potency was increased further by the additional substitution Ser⁷→Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC₅₀ = 38 µm ). However, the peptide containing d ‐lysine at positions 7 and 11 showed high potency against a range of Gram‐negative bacteria, including multidrug‐resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µm ) but appreciably lower haemolytic activity (LC₅₀ = 185 µm ) and cytotoxicity against A549 human alveolar basal epithelial cells (LC₅₀ = 65 µm ). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Immobilization and orientation‐dependent activity of a naturally occurring antimicrobial peptide

A naturally occurring antimicrobial peptide, SMAP‐29, was synthesized with an n‐terminal or c‐terminal cysteine, termed c_SMAP and SMAP_c, respectively, for site‐directed immobilization to superparamagnetic beads. Immobilized SMAP orientation‐dependent activity was probed against multiple bacteria of clinical interest including Acinetobacter baumannii, Pseudomonas aeruginosa, Bacillus anthracis sterne and Staphylococcus aureus. A kinetic microplate assay was employed to reveal both concentration and time‐dependent activity for elucidation of minimum bactericidal concentration (MBC) and sub‐lethal effects. Immobilized SMAP activity was equivalent or reduced compared with soluble SMAP_c and c_SMAP regardless of immobilization orientation, with only one exception. A comparison of immobilized SMAP_c and c_SMAP activity revealed a bacteria‐specific potency dependent on immobilization orientation, which was contrary to that seen in solution, wherein SMAP_c was more potent against all bacteria than c_SMAP. Sub‐MBC kinetic studies displayed the influence of peptide exposure to the cells with multiple bacteria exhibiting increased susceptibility and efficacy at lower concentrations upon extended exposure (i.e. MBC enhancement). For instances in which complete killing was not achieved, two predominant effects were evident: retardation of growth rate and an increased lag phase. Both effects, seen independently and concomitantly, indicate some degree of induced cellular damage that can serve as a predictor toward eventual cell death. SMAP_c immobilized on glass through standard silanization chemistry was also investigated to ascertain the influence of substrate on activity against select bacteria. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.     

Tat cell-penetrating peptide has the characteristics of a poly(proline) II helix in aqueous solution and in SDS micelles.

Tat cell-penetrating peptide (GRKKRRQRRRPPQG) is able to translocate and carry molecules across cell membranes. Using CD spectroscopy the conformation of this synthetic peptide was studied in aqueous and membrane-mimicking, micellar SDS solutions at different temperatures. The CD spectrum of the Tat cell-penetrating peptide in SDS micellar solution was virtually unchanged from that in aqueous solution, and at low temperature it was close to that of a poly(proline) II helix.     

Prokaryotic expression and antimicrobial mechanism of XPF‐St7‐derived α‐helical peptides

XPF‐St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α‐helical segment of XPF‐St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram‐negative and Gram‐positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6‐fold and 6.7‐fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Secondary structure of cell‐penetrating peptides during interaction with fungal cells

Cell‐penetrating peptides (CPPs) are peptides that cross cell membranes, either alone or while carrying molecular cargo. Although their interactions with mammalian cells have been widely studied, much less is known about their interactions with fungal cells, particularly at the biophysical level. We analyzed the interactions of seven CPPs (penetratin, Pep‐1, MPG, pVEC, TP‐10, MAP, and cecropin B) with the fungal pathogen Candida albicans using experiments and molecular simulations. Circular dichroism (CD) of the peptides revealed a structural transition from a random coil or weak helix to an α‐helix occurs for all peptides when the solvent is changed from aqueous to hydrophobic. However, CD performed in the presence of C. albicans cells showed that proximity to the cell membrane is not necessarily sufficient to induce this structural transition, as penetratin, Pep‐1, and MPG did not display a structural shift in the presence of cells. Monte Carlo simulations were performed to further probe the molecular‐level interaction with the cell membrane, and these simulations suggested that pVEC, TP‐10, MAP, and cecropin B strongly penetrate into the hydrophobic domain of the membrane lipid bilayer, inducing a transition to an α‐helical conformation. In contrast, penetratin, Pep‐1 and MPG remained in the hydrophilic region without a shift in conformation. The experimental data and MC simulations combine to explain how peptide structure affects their interaction with cells and their mechanism of translocation into cells (direct translocation vs. endocytosis). Our work also highlights the utility of combining biophysical experiments, biological experiments, and molecular modeling to understand biological phenomena.     

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

The yeast‐like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP‐28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP‐28 sterilized Prototheca cultures within 30–60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3–6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70–90% killing, suggesting they act via non‐lytic mechanisms. In circular dichroism studies, the conformation of BMAP‐28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP‐28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non‐lytic mechanisms may be exploited for the development of target‐selective drugs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin

To investigate the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin (IN), a series of IN analogs with Trp→Lys substitution were synthesized. All IN analogs displayed an approximately 7- to 18-fold higher cell selectivity, compared with IN. IN, IN-1 and IN-2 depolarized (50−90%) the cytoplasmic membrane potential of Staphylococcus aureus close to minimal inhibitory concentration (5–10 μg mL⁻¹). However, other IN analogs (IN-3 and IN-4) displayed very low ability in membrane depolarization even at 40 μg mL⁻¹. Confocal laser-scanning microscopy revealed that IN-3 and IN-4 penetrated the Escherichia coli cell membrane, whereas IN, IN-1 and IN-2 did not enter the cell membrane. In the gel retardation assay, IN-3 and IN-4 bound more strongly to DNA compared with IN, IN-1 and IN-2. These findings suggest that the mechanism of antimicrobial action of IN-3 and IN-4 may be involved in the inhibition of intracellular functions via interference with DNA/RNA synthesis. Unlike IN, all IN analogs did not inhibit nitric oxide production or inducible nitric oxide synthase mRNA expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells, indicating that the hydrophobicity of IN is more important for anti-inflammatory activity in lipopolysaccharide-treated macrophage cells than the positive charge.