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Mel4 is a novel cationic peptide with potent activity against Gram-positive bacteria. The current study examined the anti-staphylococcal mechanism of action of Mel4 and its precursor peptide melimine. The interaction of peptides with lipoteichoic acid (LTA) and with the cytoplasmic membrane using DiSC(3)-5, Sytox green, Syto-9 and PI dyes were studied. Release of ATP and DNA/RNA from cells exposed to the peptides were determined. Bacteriolysis and autolysin-activated cell death were determined by measuring decreases in OD620nm and killing of Micrococcus lysodeikticus cells by cell-free media. Both peptides bound to LTA and rapidly dissipated the membrane potential (within 30 seconds) without affecting bacterial viability. Disturbance of the membrane potential was followed by the release of ATP (50% of total cellular ATP) by melimine and by Mel4 (20%) after 2 minutes exposure (p<0.001). Mel4 resulted in staphylococcal cells taking up PI with 3.9% cells predominantly stained after 150 min exposure, whereas melimine showed 34% staining. Unlike melimine, Mel4 did not release DNA/RNA. Cell-free media from Mel4 treated cells hydrolysed peptidoglycan and produced greater zones of inhibition against M. lysodeikticus lawn than melimine treated samples. These findings suggest that pore formation is unlikely to be involved in Mel4-mediated membrane destabilization for staphylococci, since there was no significant Mel4-induced PI staining and DNA/RNA leakage. It is likely that the S. aureus killing mechanism of Mel4 involves the release of autolysins followed by cell death. Whereas, membrane interaction is the primary bactericidal activity of melimine, which includes membrane depolarization, pore formation, release of cellular contents leading to cell death.

Wound infections are still problematic in many cases and demand new alternatives for current treatment strategies. In recent years, biomaterials-based wound dressings have received much attention due to their potentials and many studies have been performed based on them. Accordingly, in this study, we fabricated and optimized an antibacterial chitosan/silk fibroin (CS/SF) electrospun nanofiber bilayer containing different concentrations of a cationic antimicrobial peptide (AMP) for wound dressing applications. The fabricated CS/SF nanofiber was fully characterized and compared to the electrospun silk fibroin and electrospun chitosan alone in vitro. Then, the release rate of different concentrations of peptide (16, 32, and 64 µg/ml) from peptide-loaded CS/SF nanofiber was investigated. Finally, based on cytotoxic activity, the antibacterial activity of scaffolds containing 16 and 32 µg/ml of the peptide was evaluated against standard and multi-drug resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa isolated from burn patients. The peptide-loaded CS/SF nanofiber displayed appropriate mechanical properties, high water uptake, suitable biodegradation rate, a controlled release without cytotoxicity on Hu02 human foreskin fibroblast cells at the 16 and 32 µg/ml concentrations of peptide. The optimized CS/SF containing 32 μg/ml peptide showed strong antibacterial activity against all experimental strains from standard to resistance. The results showed that the fabricated antimicrobial nanofiber has the potential to be applied as a wound dressing for infected wound healing, although further studies are needed in vivo.

Intracellular delivery of antimicrobial agents by nanoparticles, such as mesoporous silica particles (MSPs), offers an interesting strategy to treat intracellular infections. In tuberculosis (TB), Mycobacterium tuberculosis avoids components of the immune system by residing primarily inside alveolar macrophages, which are the desired target for TB therapy. We have previously identified a peptide, called NZX, capable of inhibiting both clinical and multi-drug resistant strains of M. tuberculosis at therapeutic concentrations. In this study we analysed the potential of MSPs containing NZX for the treatment of tuberculosis. The MSPs released functional NZX gradually into simulated lung fluid and the peptide filled MSPs were easily taken up by primary macrophages. In an intracellular infection model, the peptide containing particles showed increased mycobacterial killing compared to free peptide. The therapeutic potential of peptide containing MSPs was investigated in a murine infection model, showing that MSPs preserved the effect to eliminate M. tuberculosis in vivo. In this study we found that loading the antimicrobial peptide NZX into MSPs increased the inhibition of intracellular mycobacteria in primary macrophages and preserved the ability to eliminate M. tuberculosis in vivo in a murine model. Our studies provide evidence for the feasibility of using MSPs for treatment of tuberculosis.

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.

Antimicrobial cationic peptides are of interest because they can combat multi-drug-resistant microbes. Most peptides form α-helices or β-sheet-like structures that can insert into and subsequently disintegrate negatively charged bacterial cell surfaces. Here, we show that a novel class of core–shell nanoparticles formed by self-assembly of an amphiphilic peptide have strong antimicrobial properties against a range of bacteria, yeasts and fungi. The nanoparticles show a high therapeutic index against Staphylococcus aureus infection in mice and are more potent than their unassembled peptide counterparts. Using Staphylococcus aureus -infected meningitis rabbits, we show that the nanoparticles can cross the blood–brain barrier and suppress bacterial growth in infected brains. Taken together, these nanoparticles are promising antimicrobial agents that can be used to treat brain infections and other infectious diseases. A class of core–shell nanoparticles self-assembled from amphiphilic peptides can kill a range of bacteria, yeast and fungus. They are more potent than their unassembled peptide counterparts and can suppress bacterial growth in the brains of rabbits infected with meningitis. These particles, which carry a high number of positive charges, are promising antimicrobial agents.

Superresolution, single-particle tracking reveals effects of the cationic antimicrobial peptide LL-37 on the Escherichia coli cytoplasm. Seconds after LL-37 penetrates the cytoplasmic membrane, the chromosomal DNA becomes rigidified on a length scale of ∼30 nm, evidenced by the loss of jiggling motion of specific DNA markers. The diffusive motion of a subset of ribosomes is also frozen. The mean diffusion coefficients of the DNA-binding protein HU and the nonendogenous protein Kaede decrease twofold. Roughly 10⁸ LL-37 copies flood the cell (mean concentration ∼90 mM). Much of the LL-37 remains bound within the cell after extensive rinsing with fresh growth medium. Growth never recovers. The results suggest that the high concentration of adsorbed polycationic peptides forms a dense network of noncovalent, electrostatic linkages within the chromosomal DNA and among 70S-polysomes. The bacterial cytoplasm comprises a concentrated collection of biopolymers that are predominantly polyanionic (e.g., DNA, ribosomes, RNA, and most globular proteins). In normal cells, this provides a kind of electrostatic lubrication, enabling facile diffusion despite high biopolymer volume fraction. However, this same polyanionic nature renders the cytoplasm susceptible to massive adsorption of polycationic agents once penetration of the membranes occurs. If this phenomenon proves widespread across cationic agents and bacterial species, it will help explain why resistance to antimicrobial peptides develops only slowly. The results suggest two design criteria for polycationic peptides that efficiently kill gram-negative bacteria: facile penetration of the outer membrane and the ability to alter the cytoplasm by electrostatically linking double-stranded DNA and 70S-polysomes.

Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10-8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.

is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary. In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against . Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against in vitro. The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations. This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.

Observations of real-time changes in living cells have contributed much to the field of cellular biology. The ability to image whole, living cells with nanometre resolution on a timescale that is relevant to dynamic cellular processes has so far been elusive 1 , 2 . Here, we investigate the kinetics of individual bacterial cell death using a novel high-speed atomic force microscope optimized for imaging live cells in real time. The increased time resolution (13 s per image) allows the characterization of the initial stages of the action of the antimicrobial peptide CM15 on individual Escherichia coli cells with nanometre resolution. Our results indicate that the killing process is a combination of a time-variable incubation phase (which takes seconds to minutes to complete) and a more rapid execution phase. High-speed atomic force microscopy imaging in liquid reveals the variability of the onset of cell death induced by antimicrobial peptides on a cell-by-cell basis.

Since the discovery of magainins, cecropins and defensins 30 years ago, antimicrobial peptides (AMPs) have been hailed as a potential solution to the dearth of novel antibiotic development. AMPs have shown robust activity against a wide variety of pathogens, including drug-resistant bacteria. Unlike small-molecule antibiotics, however, AMPs have failed to translate this success to the clinic. Only the polymyxins, gramicidins, nisin and daptomycin are currently approved for medical use; the latter is the only example to have been developed in the last several decades. Nonetheless, researchers continue to isolate, modify and develop novel AMPs for therapeutic applications. Efforts have focused on increasing stability, reducing cytotoxicity, improving antimicrobial activity and incorporating AMPs in novel formulations, including nanoscale particles. As peptide synthesis and recombinant production methodologies improve, and more relevant bioassays become available, it becomes increasingly likely that AMPs will break the regulatory barrier and enter the marketplace as valuable antimicrobial weapons in the next 10 years.