Barbara Orioni

Different mechanisms of action of antimicrobial peptides: insights from fluorescence spectroscopy experiments and molecular dynamics simulations†

Most antimicrobial peptides exert their activity by interacting with bacterial membranes, thus perturbing their permeability. They are investigated as a possible solution to the insurgence of bacteria resistant to the presently available antibiotic drugs. However, several different models have been proposed for their mechanism of membrane perturbation, and the molecular details of this process are still debated. Here, we compare fluorescence spectroscopy experiments and molecular dynamics (MD) simulations regarding the association with lipid bilayers and lipid perturbation for two different amphiphilic helical antimicrobial peptides, PMAP‐23 and trichogin GA IV. PMAP‐23, a cationic peptide member of the cathelicidin family, is considered to induce membrane permeability according to the Shai‐Matsuzaki‐Huang “carpet” model, while trichogin GA IV is a neutral peptide, member of the peptaibol family. Although several lines of evidence suggest a “barrel‐stave” mechanism of pore formation for the latter peptide, its length is only half the normal thickness of a lipid bilayer. Both fluorescence spectroscopy experiments and MD simulations indicated that PMAP‐23 associates with membranes close to their surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. By contrast, trichogin GA IV can undergo a transition from a surface‐bound state to a transmembrane orientation. In the first arrangement, it does not cause any strong membrane perturbation, while in the second orientation it might be able to span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. Copyright

Monomer−Dimer Equilibrium in Glutathione Transferases: A Critical Re-Examination

Glutathione transferases (GSTs) are dimeric enzymes involved in cell detoxification versus many endogenous toxic compounds and xenobiotics. In addition, single monomers of GSTs appear to be involved in particular protein-protein interactions as in the case of the pi class GST that regulates the apoptotic process by means of a GST-c-Jun N-terminal kinase complex. Thus, the dimer-monomer transition of GSTs may have important physiological relevance, but many studies reached contrasting conclusions both about the modality and extension of this event and about the catalytic competence of a single subunit. This paper re-examines the monomer-dimer question in light of novel experiments and old observations. Recent papers claimed the existence of a predominant monomeric and active species among pi, alpha, and mu class GSTs at 20-40 nM dilution levels, reporting dissociation constants (K(d)) for dimeric GST of 5.1, 0.34, and 0.16 microM, respectively. However, we demonstrate here that only traces of monomers could be found at these concentrations since all these enzymes display K(d) values of <<1 nM, values thousands of times lower than those reported previously. Time-resolved and steady-state fluorescence anisotropy experiments, two-photon fluorescence correlation spectroscopy, kinetic studies, and docking simulations have been used to reach such conclusions. Our results also indicate that there is no clear evidence of the existence of a fully active monomer. Conversely, many data strongly support the idea that the monomeric form is scarcely active or fully inactive.

Membrane thickness and the mechanism of action of the short peptaibol trichogin GA IV

Trichogin GA IV (GAIV) is an antimicrobial peptide of the peptaibol family, like the extensively studied alamethicin (Alm). GAIV acts by perturbing membrane permeability. Previous data have shown that pore formation is related to GAIV aggregation and insertion in the hydrophobic core of the membrane. This behavior is similar to that of Alm and in agreement with a barrel-stave mechanism, in which transmembrane oriented peptides aggregate to form a channel. However, while the 19-amino acid long Alm has a length comparable to the membrane thickness, GAIV comprises only 10 amino acids, and its helix is about half the normal bilayer thickness. Here, we report the results of neutron reflectivity measurements, showing that GAIV inserts in the hydrophobic region of the membrane, causing a significant thinning of the bilayer. Molecular dynamics simulations of GAIV/membrane systems were also performed. For these studies we developed a novel approach for constructing the initial configuration, by embedding the short peptide in the hydrophobic core of the bilayer. These calculations indicated that in the transmembrane orientation GAIV interacts strongly with the polar phospholipid headgroups, drawing them towards its N- and C-termini, inducing membrane thinning and becoming able to span the bilayer. Finally, vesicle leakage experiments demonstrated that GAIV activity is significantly higher with thinner membranes, becoming similar to that of Alm when the bilayer thickness is comparable to its size. Overall, these data indicate that a barrel-stave mechanism of pore formation might be possible for GAIV and for similarly short peptaibols despite their relatively small size.

Fluctuations and the Rate-Limiting Step of Peptide-Induced Membrane Leakage

Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.

Esculentin 1 - 21: a linear antimicrobial peptide from frog skin with inhibitory effect on bovine mastitis-causing bacteria ‡

Mastitis, or inflammation of the mammary gland, is the most common and expensive illness of dairy cows throughout the world. Although stress and physical injuries may give rise to inflammation of the udders, infections by bacteria or other microorganisms remain the major cause, and infusion of antibiotics is the main treatment approach. However, the increased emergence of multidrug‐resistant pathogens and the production of milk contaminated with antibiotics has become a serious threat in the livestock. Hence, there is an urgent need for the discovery of new therapeutic agents with a new mode of action. Gene‐encoded AMPs, which represent the first line of defence in all living organisms, are considered as promising candidates for the development of new anti‐infective agents. This paper reports on the antibacterial activities in vitro and in an animal model, of the frog skin AMP esculentin 1–21 [Esc(1–21)], along with a plausible mode of action. Our data revealed that this peptide (i) is highly potent against the most common mastitis‐causing microbes (e.g. Streptococcus agalactiae); and (ii) is active in vivo, causing a visible regression of the clinical stage of mastitis in dairy cows, after 1 week of peptide treatment. Biophysical characterisation revealed that the peptide adopts an α‐helical structure in microbial mimicking membranes and is able to permeate the membrane of S. agalactiae in a dose‐dependent manner. Overall, these data suggest Esc(1–21) as an attractive AMP for the future design of new antibiotics to cure mastitis in cattle. Copyright

The thin line between cell-penetrating and antimicrobial peptides: the case of Pep-1 and Pep-1-K ‡

Cell‐penetrating peptides (CPPs) are cationic oligopeptides able to translocate across biological membranes without perturbing them, while antimicrobial peptides (AMPs) kill bacteria mainly by disrupting their membranes. The two peptide classes share several characteristics (charge, amphipathicity, helicity, and length), and therefore the molecular properties discriminating between the two different bioactivities are not clear. Pep‐1‐K (KKTWWKTWWTKWSQPKKKRKV) is a new AMP derived from the widely studied CPP Pep‐1 (KETWWETWWTEWSQPKKKRKV), or ‘Chariot’, known for its ability to carry large cargoes across biological membranes. Pep‐1‐K was obtained from Pep‐1 by substituting the three Glu residues with Lys, to increase its cationic character. Previous studies showed that these modifications endow Pep‐1‐K with a potent antimicrobial activity, with MICs in the low micromolar range. Here, we characterized the interaction of Pep‐1 and Pep‐1‐K with model membranes to understand the reason for the antimicrobial activity of Pep‐1‐K. The data show that this peptide causes vesicle aggregation, perturbs membrane order, and induces the leakage of ions, but not of larger solutes, while these effects were not observed for Pep‐1. These differences are likely due, at least in part, to the higher affinity of Pep‐1‐K toward anionic bilayers, which mimick the composition of bacterial membranes. Copyright