A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria

Background: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole Staphylococcus aureus and Staphylococcus epidermidis. Methods: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (diSC3(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. Results: The ion channel-forming gramicidin D induced a rapid increase of diSC3(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. Conclusions: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs

Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils.

Cytoplasmic granules of neutrophils store a variety of cationic polypeptides, which exert in vitro a potent antibacterial action and are potentially involved in host defense mechanisms. From an acid extract of bovine neutrophil granules we have purified over 2,000-fold a dodecapeptide exhibiting bactericidal activity against both Escherichia coli and Staphylococcus aureus at 10(-7)-10(-5) M concentration. The purification procedure involved only two steps of ion-exchange and reversed-phase chromatography. The peptide, named bactenecin, has the amino acid sequence, Arg-Leu-Cys-Arg-Ile-Val-Val-Ile-Arg-Val-Cys-Arg, maintained in a cyclic structure by a disulfide bond between the two cysteine residues. Computer modeling of the dodecapeptide resulted in a conformation in which the chain adopts an antiparallel extended structure forming a gamma turn at residue 7

Exploring the biological properties and therapeutic potential of antimicrobial peptides

The researchers involved in the Trans2Care project at the Department of Medical and Biological Sciences of the University of Udine investigate the biological properties of the antimicrobial peptides (AMPs) of the immune system and their therapeutic potential for human and veterinary application. In addition to potent and broad-spectrum antimicrobial activities, some AMPs display anti-inflammatory and immunomodulatory effects and hold promise as novel antiinfective agents combining antibiotic and immunostimulating properties. A detailed knowledge of their physicochemical, biological and pharmacological properties and of their impact on clinical settings is an important prerequisite to this end. The Trans2Care project offers an invaluable opportunity to share knowledge, technical expertise and laboratory facilities to achieve a better understanding of the biological features and therapeutic potential of AMPs

Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells.

Mammalian antimicrobial peptides provide rapid defense against infection by inactivating pathogens and by influencing the functions of cells involved in defense responses. Although the direct antibacterial properties of these peptides have been widely characterized, their multiple effects on host cells are only beginning to surface. Here we investigated the mechanistic and functional aspects of the interaction of the proline-rich antimicrobial peptide Bac7(1-35) with mammalian cells, as compared with a truncated analog, Bac7(5-35), lacking four critical N-terminal residues (RRIR) of the Bac7(1-35) sequence. By using confocal microscopy and flow cytometry, we showed that although the truncated analog Bac7(5-35) remains on the cell surface, Bac7(1-35) is rapidly taken up into 3T3 and U937 cells through a nontoxic energy- and temperature-dependent process. Cell biology-based assays using selective endocytosis inhibitors and spectroscopic and surface plasmon resonance studies of the interaction of Bac7(1-35) with phosphatidylcholine/cholesterol model membranes collectively suggest the concurrent contribution of macropinocytosis and direct membrane translocation. Structural studies with model membranes indicated that membrane-bound Bac7(5-35) is significantly more aggregated than Bac7(1-35) due to the absence of the N-terminal cationic cluster, thus providing an explanation for hampered cellular internalization of the truncated form. Further investigations aimed to reveal functional implications of intracellular uptake of Bac7(1-35) demonstrated that it correlates with enhanced S phase entry of 3T3 cells, indicating a novel function for this proline-rich peptide

Covalent grafting of titanium with a cathelicidin peptide produces an osteoblast compatible surface with antistaphylococcal activity

Bacterial infection of orthopaedic implants, often caused by Staphylococcus species, may ultimately lead to implant failure. The development of infection-resistant, osteoblast-compatible biomaterials could represent an effective strategy to prevent bacterial colonization of implants, reducing the need for antibiotics. In this study, the widely used biomaterial titanium was functionalized with BMAP27(1-18), an \uf061-helical cathelicidin antimicrobial peptide that retains potent staphylocidal activity when immobilized on agarose beads. A derivative bearing a short spacer with a free thiol at the N-terminus was coupled to silanized titanium disks via thiol-maleimide chemistry. Tethering was successful, as assessed by Contact angle, Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), and X-ray Photoelectron Spectroscopy (XPS), with an average surface mass density of 456 ng/cm2 and a layer thickness of 3 nm. The functionalized titanium displayed antimicrobial properties against a reference strain of Staphylococcus epidermidis with well-known biofilm forming capability. Reduction of bacterial counts and morphological alterations of adhering bacteria, upon 2h incubation, indicate a rapid contact-killing effect. The immobilized peptide was not toxic to osteoblasts, which adhered and spread better on functionalized titanium when co-cultured with bacteria, compared to non-coated surfaces. Results suggest that functionalization of titanium with BMAP27(1-18) could be promising for prevention of bacterial colonization in bone graft applications

Bovine Neutrophil Antibiotic Peptides and Their Precursors: Structure and Role in Innate Immunity

Four peptides were characterized in extracts of bovine neutrophil granules: an Arg-rich dodecapeptide, maintained in a cyclic structure by a disulfide bridge; a Trp-rich tridecapeptide named indo- licidin; and two 43- and 59 amino acids long peptides, named Bac5 and Bac7, with frequent repeats of the triplets Arg-Pro-Pro and Pro-Arg-Pro, respectively. The full length cDNA of the first three of these peptides was characterized recently. Sequence analysis showed that the prosequences of the predicted precursors of all the three peptides are highly identical and exhibited also a remarkable similarity to cathelin, a porcine inhibitor of cathepsin L. Purified proBacö actually proved in in vitro assays to inhibit cathepsin L, but not other cysteine proteinases such as cathepsin B. Unlike proBacö, proBac7 is selectively chemotactic to monocytes. Several fragments of Bac5 and Bac7 (from 6 to 35 residues) were synthesized by the Fmoc method. The results of antibacterial assays show that the N-terminal portion, the most cationic one in both Bac5 and Bac7, is essential for the antimicrobial activity and that the minimal length necessary to arrest the growth of susceptible bacteria is 18-20 residues

Evidence of an Antimicrobial-Immunomodulatory Role of Atlantic Salmon Cathelicidins during Infection with Yersinia ruckeri

Cathelicidins are a family of antimicrobial peptides that act as effector molecules of the innate immune system with broad-spectrum antimicrobial properties. These evolutionary conserved cationic host-defence peptides are integral components of the immune response of fish, which are generally believed to rely heavily on innate immune defences to invading pathogens. In this study we showed that Atlantic salmon cathelicidin 1 and 2 (asCATH1 and asCATH2) stimulated peripheral blood leukocytes increasing the transcription of the chemokine interleukin-8. Further, functional differences were identified between the two cathelicidins. In the presence of serum, asCATH1 displayed greatly diminished host haemolytic activity, while the constitutively expressed asCATH2 had no haemolytic activity with or without serum. These findings support our hypothesis that fish cathelicidins exert their primary antimicrobial action at the site of pathogen invasion such as epithelial surfaces. Further, we hypothesise that like their mammalian counterparts in the presence of serum they act as mediators of the innate and adaptive immune response via the release of cytokines thus indirectly protecting against a variety of pathogens. We highlight the importance of this immunomodulatory role from the involvement of asCATHs during an infection with the fish pathogen Yersinia ruckeri. While we were able to demonstrate in vitro that asCATH1 and 2, possessed direct microbicidal activity against the fish pathogen, Vibrio anguillarum, and a common gram negative bacterium, Escherichia coli, little or no bactericidal activity was found against Y. ruckeri. The contribution of either asCATH in the immune response or as a potential virulence factor during yersiniosis is highlighted from the increased expression of asCATH1 and 2 mRNA during an in vivo challenge with Y. ruckeri . We propose that Atlantic salmon cathelicidins participate in the interplay between the innate and adaptive immune systems via the release of cytokines enabling a more effective response to invading pathogens

The Potential of Surface-Immobilized Antimicrobial Peptides for the Enhancement of Orthopaedic Medical Devices: A Review

Due to the well-known phenomenon of antibiotic resistance, there is a constant need for antibiotics with novel mechanisms and different targets respect to those currently in use. In this regard, the antimicrobial peptides (AMPs) seem very promising by virtue of their bactericidal action, based on membrane permeabilization of susceptible microbes. Thanks to this feature, AMPs have a broad activity spectrum, including antibiotic-resistant strains, and microbial biofilms. Additionally, several AMPs display properties that can help tissue regeneration. A possible interesting field of application for AMPs is the development of antimicrobial coatings for implantable medical devices (e.g., orthopaedic prostheses) to prevent device-related infection. In this review, we will take note of the state of the art of AMP-based coatings for orthopaedic prostheses. We will review the most recent studies by focusing on covalently linked AMPs to titanium, their antimicrobial efficacy and plausible mode of action, and cytocompatibility. We will try to extrapolate some general rules for structure–activity (orientation, density) relationships, in order to identify the most suitable physical and chemical features of peptide candidates, and to optimize the coupling strategies to obtain antimicrobial surfaces with improved biological performance

The Potential of Surface-Immobilized Antimicrobial Peptides for the Enhancement of Orthopaedic Medical Devices: A Review

Due to the well-known phenomenon of antibiotic resistance, there is a constant need for antibiotics with novel mechanisms and different targets respect to those currently in use. In this regard, the antimicrobial peptides (AMPs) seem very promising by virtue of their bactericidal action, based on membrane permeabilization of susceptible microbes. Thanks to this feature, AMPs have a broad activity spectrum, including antibiotic-resistant strains, and microbial biofilms. Additionally, several AMPs display properties that can help tissue regeneration. A possible interesting field of application for AMPs is the development of antimicrobial coatings for implantable medical devices (e.g., orthopaedic prostheses) to prevent device-related infection. In this review, we will take note of the state of the art of AMP-based coatings for orthopaedic prostheses. We will review the most recent studies by focusing on covalently linked AMPs to titanium, their antimicrobial efficacy and plausible mode of action, and cytocompatibility. We will try to extrapolate some general rules for structure–activity (orientation, density) relationships, in order to identify the most suitable physical and chemical features of peptide candidates, and to optimize the coupling strategies to obtain antimicrobial surfaces with improved biological performance

Structural and Functional Diversity of Cathelicidins

Cathelicidins are a ubiquitous family of host defence peptides (HDPs) in vertebrate animals. Unlike other HDP families, they are defined by the common and relatively well conserved proregion rather than the mature active peptides, which are highly diverse and conform to at least five different structural groups. They seem to have fol-lowed a rather distinctive evolutionary path in their development. Cathelicidin-derived peptides play a relevant role in defending the host against microbial infection, by displaying both a broad-spectrum, direct antimicrobial activity and the capacity to modulate other host responses to infection and injury. Both types of effect depend on the structural type, which in turn affects the particular mode of action of each peptide. This chapter begins by briefly describing the discovery of cathelicidins before dis-cussing their molecular diversity and con-sidering their evolution. It then considers their expression and processing, the struc-ture-dependence of the distinct modes of action shown by different members, and briefly touches on th