Carla Luzi

Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles

The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings.

Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

ABSTRACT Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptides affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used β-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and β-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-α levels, resulting in the highest survival rates.

pH-dependent disruption of Escherichia coli ATCC 25922 and model membranes by the human antimicrobial peptides hepcidin 20 and 25

The human hepcidin 25 (hep‐25) and its isoform hepcidin 20 (hep‐20) are histidine‐containing, cystein rich, β‐sheet structured peptides endowed with antimicrobial activity. We previously reported that, similar to other histidine‐containing peptides, the microbicidal effects of hep‐25 and hep‐20 are highly enhanced at acidic pH. In the present study, we investigated whether pH influences the mode of action of hep‐25 and hep‐20 on Escherichia coli American Type Culture Collection 25922 and model membranes. A striking release of β‐galactosidase by hepcidin‐treated E. coli was observed at pH 5.0, whereas no inner membrane permeabilization capacity was seen at pH 7.4, even at bactericidal concentrations. Similar results were obtained by flow cytometry when assessing the internalization of propidium iodide by hepcidin‐treated E. coli. Scanning electron microscope imaging revealed that both peptides induced the formation of numerous blebs on the surface of bacterial cells at acidic pH but not at neutral pH. Moreover, a phospholipid/polydiacetylene colourimetric vesicle assay revealed a more evident membrane damaging effect at pH 5.0 than at pH 7.4. The leakage of entrapped dextrans of increasing molecular size from liposomes was also assessed at pH 7.4. Consistent with the lack of β‐galactosidase release from whole E. coli observed at such a pH value, evident leakage of only the smallest 4‐kDa dextran (and not of dextrans of 20 or 70 kDa) was observed, indicating a poor ability of hepcidin peptides to permeabilize liposome vesicles at pH 7.4. Altogether, the data obtained in the present study using different approaches strongly suggest that the ability of hepcidins to perturb bacterial membranes is markedly pH‐dependent.

Natural D240G Toho-1 mutant conferring resistance to ceftazidime: biochemical characterization of CTX-M-43

OBJECTIVES The aim of this article is biochemical and kinetic characterization of CTX-M-43, a natural Asp-240-->Gly mutant of CTX-M-44 (ex Toho-1), from a clinical isolate of Acinetobacter baumannii isolated in a Bolivian hospital. METHODS Steady-state kinetic parameters (K(m) and k(cat)) were determined for a large pattern of substrates. Analysis of inactivators and transient inactivators was performed to determine the efficiency of acylation (k(+2)/K) and the deacylation constant (k(+3)). Molecular modelling of Michaelis complex of ceftazidime, cefotaxime and ceftibuten, obtained from molecular mechanics calculations, was carried out. RESULTS CTX-M-43 showed a general increase in affinity towards all cephalosporins tested, with respect to CTX-M-44. Carbapenems acted as inactivators with a good acylation efficiency for meropenem and ertapenem and significant deacylation constant for imipenem. MICs of imipenem obtained at a higher bacterial inoculum of recombinant Escherichia coli were increased. CONCLUSIONS Kinetic data and molecular modelling of Michaelis complex confirmed that Asp-240-->Gly allows a better accommodation of the bulky C7beta aminothiazol-oxyimino substituent, resulting in a general increase in the enzyme affinity towards oxyimino cephalosporins. The ascertained potentialities of CTX-M-type enzymes, supported by the kinetic data and the behaviour of the recombinant E. coli at different bacterial inocula towards carbapenems, make a possible evolution of those enzymes towards a carbapenemase activity plausible.

Folding propensity of anoplin: A molecular dynamics study of the native peptide and four mutated isoforms

Anoplin, a cationic decapeptide amide GLLKRIKTLL‐NH2 derived from venom sac of the solitary wasp Anoplius samariensis has been investigated through Molecular Dynamics. The wild‐type (WT) and four isoforms were simulated both in water and in the membrane‐mimicking solvent trifluoroethanol (TFE). In water all the investigated species, found to be in rapid equilibrium between different conformational states, can be considered as unfolded. On the other hand, in TFE all the systems enhance their rigidity and, in general, show α‐helix as the main folded conformation. Interestingly, a semi‐quantitative thermodynamic analysis has suggested that the folding driving force is not always the same being in some cases (e.g., the WT Anoplin) of entropic nature and in other cases of energetic nature.

Apoptotic effects of bovine apo‐lactoferrin on HeLa tumor cells

Lactoferrin (Lf), a cationic iron‐binding glycoprotein of 80 kDa present in body secretions, is known as a compound with marked antimicrobial activity. In the present study, the apoptotic effect of iron‐free bovine lactoferrin (apo‐bLf) on human epithelial cancer (HeLa) cells was examined in association with reactive oxygen species and glutathione (GSH) levels. Apoptotic effect of iron‐free bovine lactoferrin inhibited the growth of HeLa cells after 48 hours of treatment while the diferric‐bLf was ineffective in the concentration range tested (from 1 to 12.5 μM). Western blot analysis showed that key apoptotic regulators including Bax, Bcl‐2, Sirt1, Mcl‐1, and PARP‐1 were modulated by 1.25 μM of apo‐bLf. In the same cell line, apo‐bLf induced apoptosis together with poly (ADP‐ribose) polymerase cleavage, caspase activation, and a significant drop of NAD+. In addition, apo‐bLf–treated HeLa cells showed a marked increase of reactive oxygen species level and a significant GSH depletion. On the whole, apo‐bLf triggered apoptosis of HeLa cells upon oxygen radicals burst and GSH decrease.

P‐113 Peptide: New experimental evidences on its biological activity and conformational insights from molecular dynamics simulations

In this article, we report novel and additional results, both experimental and computational, obtained in our laboratories on the peptide P‐113. In particular, our experimental results indicate that this peptide is endowed with a high target‐cell selectivity towards yeast species, suggesting its potential development as a new drug against oral microbial infections. To provide additional structural insights, we performed several Molecular Dynamics simulations in different conditions. Results suggest that P‐113 is a rather compact species presumably because of its highly charged state as emerged from the dramatic increase of internal fluctuation occurring upon point‐mutation. The peptide turns out to adopt, in water, a beta‐hairpin‐like conformation and, in a more hydrophobic environment, is found to be in a (probably slow) equilibrium between α‐helix and hairpin conformations. Complexation with Zn2+ induces a drastic mechanical stabilization, which prevents any conformational organization of the peptide into a biologically active state.

Cerium oxide nanoparticles as potential antibiotic adjuvant. Effects of CeO2 nanoparticles on bacterial outer membrane permeability

BACKGROUND Therapeutic options against Multi Drug Resistant (MDR) pathogens are limited and the overall strategy would be the development of adjuvants able to enhance the activity of therapeutically available antibiotics. Non-specific outer membrane permeabilizer, like metal-oxide nanoparticles, can be used to increase the activity of antibiotics in drug-resistant pathogens. The study aims to investigate the effect of cerium oxide nanoparticles (CeO2 NPs) on bacterial outer membrane permeability and their application in increasing the antibacterial activity of antibiotics against MDR pathogens. METHODS The ability of CeO2 NPs to permeabilize Gram-negative bacterial outer membrane was investigated by calcein-loaded liposomes. The extent of the damage was evaluated using lipid vesicles loaded with FITC-dextran probes. The effect on bacterial outer membrane was evaluated by measuring the coefficient of permeability at increasing concentrations of CeO2 NPs. The interaction between CeO2 NPs and beta-lactams was evaluated by chequerboard assay against a Klebsiella pneumoniae clinical isolate expressing high levels of resistance against those antibiotics. RESULTS Calcein leakage increases as NPs concentrations increase while no leakage was observed in FITC-dextran loaded liposomes. In Escherichia coli the outer membrane permeability coefficient increases in presence of CeO2 NPs. The antibacterial activity of beta-lactam antibiotics against K. pneumoniae was enhanced when combined with NPs. CONCLUSIONS CeO2 NPs increases the effectiveness of antimicrobials which activity is compromised by drug resistance mechanisms. The synergistic effect is the result of the interaction of NPs with the bacterial outer membrane. The low toxicity of CeO2 NPs makes them attractive as antibiotic adjuvants against MDR pathogens.

Crabrolin, a natural antimicrobial peptide: structural properties

A joint application of experimental and computational approaches has revealed the exceptionally high attitude of crabrolin, a 13‐residue peptide with sequence FLPLILRKIVTAL‐NH2, to adopt alpha‐helix conformation not only in membrane‐mimicking solvents but also in the presence of a not negligible amount of water. Our study shows that this propensity essentially resides in the intrinsic thermodynamic stability of alpha‐helix conformation whose kinetic stability is drastically reduced in water solvent. Our analysis suggests that this is due to two effects enhanced by water: a more local effect consisting of the demolition of intra‐peptide H‐bonds, essential for the alpha‐helix formation, and a bulk – electrostatic – effect favoring conformational states more polar than alpha‐helix. Copyright