Lipid coated liquid crystal droplets for the on-chip detection of antimicrobial peptides.

We describe a novel biosensor based on phospholipid-coated nematic liquid crystal (LC) droplets and demonstrate the detection of Smp43, a model antimicrobial peptide (AMP) from the venom of North African scorpion Scorpio maurus palmatus. Mono-disperse lipid-coated LC droplets of diameter 16.7 ± 0.2 µm were generated using PDMS microfluidic devices with a flow-focusing configuration and were the target for AMPs. The droplets were trapped in a bespoke microfluidic trap structure and were simultaneously treated with Smp43 at gradient concentrations in six different chambers. The disruption of the lipid monolayer by the Smp43 was detected (<6 µM) at concentrations well within its biologically active range, indicated by a dramatic change in the appearance of the droplets associated with the transition from a typical radial configuration to a bipolar configuration, which is readily observed by polarizing microscopy. This suggests the system has feasibility as a drug-discovery screening tool. Further, compared to previously reported LC droplet biosensors, this LC droplet biosensor with a lipid coating is more biologically relevant and its ease of use in detecting membrane-related biological processes and interactions has the potential for development as a reliable, low-cost and disposable point of care diagnostic tool.

Visualization of diffusion limited antimicrobial peptide attack on supported lipid membranes.

Understanding the mechanism of action of antimicrobial peptides (AMP) is fundamental to the development and design of peptide based antimicrobials. Utilizing fast-scan atomic force microscopy (AFM) we detail the attack of an AMP on both prototypical prokaryotic (DOPC:DOPG) and eukaryotic (DOPC:DOPE) model lipid membranes on the nanoscale and in real time. Previously shown to have a favourable therapeutic index, we study Smp43, an AMP with a helical-hinge-helical topology isolated from the venom of the North African scorpion Scorpio maurus palmatus. We observe the dynamic formation of highly branched defects being supported by 2D diffusion models and further experimental data from liposome leakage assays and quartz crystal microbalance-dissipation (QCM-D) analysis, we propose that Smp43 disrupts these membranes via a common mechanism, which we have termed 'diffusion limited disruption' that encompasses elements of both the carpet model and the expanding pore mechanism.

Therapeutic Potential of a Scorpion Venom-Derived Antimicrobial Peptide and Its Homologs Against Antibiotic-Resistant Gram-Positive Bacteria.

The alarming rise in the prevalence of antibiotic resistance among pathogenic bacteria poses a unique challenge for the development of effective therapeutic agents. Antimicrobial peptides (AMPs) have attracted a great deal of attention as a possible solution to the increasing problem of antibiotic-resistant bacteria. Marcin-18 was identified from the scorpion Mesobuthus martensii at both DNA and protein levels. The genomic sequence revealed that the marcin-18 coding gene contains a phase-I intron with a GT-AG splice junction located in the DNA region encoding the N-terminal part of signal peptide. The peptide marcin-18 was also isolated from scorpion venom. A protein sequence homology search revealed that marcin-18 shares extremely high sequence identity to the AMPs meucin-18 and megicin-18. In vitro, chemically synthetic marcin-18 and its homologs (meucin-18 and megicin-18) showed highly potent inhibitory activity against Gram-positive bacteria, including some clinical antibiotic-resistant strains. Importantly, in a mouse acute peritonitis model, these peptides significantly decreased the bacterial load in ascites and rescued nearly all mice heavily infected with clinical methicillin-resistant Staphylococcus aureus from lethal bacteremia. Peptides exerted antimicrobial activity via a bactericidal mechanism and killed bacteria through membrane disruption. Taken together, marcin-18 and its homologs have potential for development as therapeutic agents for treating antibiotic-resistant, Gram-positive bacterial infections.

Phospholipid dependent mechanism of smp24, an α-helical antimicrobial peptide from scorpion venom.

Determining the mechanism of action of antimicrobial peptides (AMPs) is critical if they are to be developed into the clinical setting. In recent years high resolution techniques such as atomic force microscopy (AFM) have increasingly been utilised to determine AMP mechanism of action on planar lipid bilayers and live bacteria. Here we present the biophysical characterisation of a prototypical AMP from the venom of the North African scorpion Scorpio maurus palmatus termed Smp24. Smp24 is an amphipathic helical peptide containing 24 residues with a charge of +3 and exhibits both antimicrobial and cytotoxic activity and we aim to elucidate the mechanism of action of this peptide on both membrane systems. Using AFM, quartz crystal microbalance-dissipation (QCM-D) and liposomal leakage assays the effect of Smp24 on prototypical synthetic prokaryotic (DOPG:DOPC) and eukaryotic (DOPE:DOPC) membranes has been determined. Our data points to a toroidal pore mechanism against the prokaryotic like membrane whilst the formation of hexagonal phase non-lamellar phase structures is seen in eukaryotic like membrane. Also, phase segregation is observed against the eukaryotic membrane and this study provides direct evidence of the same peptide having multiple mechanisms of action depending on the membrane lipid composition.

Characterisation of three alpha-helical antimicrobial peptides from the venom of Scorpio maurus palmatus.

Scorpion venoms provide a rich source of anti-microbial peptides. Here we characterise three from the venom of Scorpion maurus palmatus. Smp13 is biologically inactive, despite sharing homology with other antimicrobial peptides, probably because it lacks a typically charged structure. Both Smp-24 and Smp-43 have broad spectrum antimicrobial activity, disrupting bacterial membranes. In addition, there is evidence that Smp24 may inhibit DNA synthesis in Bacillus subtilis. Smp24 haemolysed red blood cells but in contrast, Smp43 was non-haemolytic. The introduction of a flexible Gly-Val-Gly hinge into the middle of Smp24 did not alter the haemolytic activity of Smp24 (as might have been predicted from earlier studies with Pandinin2 (Pin2), although C-terminal truncation of Smp-24 reduced its haemolytic activity, in agreement with earlier Pin 2 studies. Smp24 and its derivatives, as well as Smp-43, were all cytotoxic (ATP release assay) toward mammalian HepG2 liver cells. Our results highlight the beneficial effect of helical-hinge-helical conformation on promoting prokaryotic selectivity of long chain scorpion AMPs, as well as the importance of examining a wide range of mammalian cell types in cytotoxicity testing.

Temocillin: a new candidate antibiotic for local antimicrobial delivery in orthopaedic surgery?

OBJECTIVES: To assess the performance of the Gram-negative-specific antibiotic temocillin in polymethylmethacrylate bone cement pre-loaded with gentamicin, as a strategy for local antibiotic delivery. METHODS: Temocillin was added at varying concentrations to commercial gentamicin-loaded bone cement. The elution of the antibiotic from cement samples over a 2 week period was quantified by LC-MS. The eluted temocillin was purified by fast protein liquid chromatography and the MICs for a number of antibiotic-resistant Escherichia coli were determined. The impact strength of antibiotic-loaded samples was determined using a Charpy-type impact testing apparatus. RESULTS: LC-MS data showed temocillin eluted to clinically significant concentrations within 1 h in this laboratory system and the eluted temocillin retained antimicrobial activity against all organisms tested. Impact strength analysis showed no significant difference between cement samples with or without temocillin. CONCLUSIONS: Temocillin can be added to bone cement and retains its antimicrobial activity after elution. The addition of up to 10% temocillin did not affect the impact strength of the cement. The results show that temocillin is a promising candidate for use in antibiotic-loaded bone cement.

Antimicrobial peptides from scorpion venoms.

The need for new antimicrobial agents is becoming one of the most urgent requirements in modern medicine. The venoms of many different species are rich sources of biologically active components and various therapeutic agents have been characterized including antimicrobial peptides (AMPs). Due to their potent activity, low resistance rates and unique mode of action, AMPs have recently received much attention. This review focuses on AMPs from the venoms of scorpions and examines all classes of AMPs found to date. It gives details of their biological activities with reference to peptide structure. The review examines the mechanism of action of AMPs and with this information, suggests possible mechanisms of action of less well characterised peptides. Finally, the review examines current and future trends of scorpion AMP research, by discussing recent successes obtained through proteomic and transcriptomic approaches.