The herpes simplex virus 1 (HSV-1) is widespread in the population, and in most cases its infection is asymptomatic. The currently available anti-HSV-1 drugs are acyclovir and its derivatives, ...although long-term therapy with these agents can lead to drug resistance. Thus, the discovery of novel antiherpetic compounds deserves additional effort. Naturally occurring antimicrobial peptides (AMPs) represent an interesting class of molecules with potential antiviral properties. To the best of our knowledge, this study is the first demonstration of the
anti-HSV-1 activity of temporin B (TB), a short membrane-active amphibian AMP. In particular, when HSV-1 was preincubated with 20 μg/ml TB, significant antiviral activity was observed (a 5-log reduction of the virus titer). Such an effect was due to the disruption of the viral envelope, as demonstrated by transmission electron microscopy. Moreover, TB partially affected different stages of the HSV-1 life cycle, including the attachment and the entry of the virus into the host cell, as well as the subsequent postinfection phase. Furthermore, its efficacy was confirmed on human epithelial cells, suggesting TB as a novel approach for the prevention and/or treatment of HSV-1 infections.
AIMS: In this study, we evaluated the ability of the lipopeptide bacillomycin D and the antifungal drug amphotericin B as well as their combination, to inhibit Candida albicans biofilm formation and ...to accelerate keratinocyte cell migration. METHODS AND RESULTS: The antibiofilm activity of bacillomycin D and its combination with amphotericin B was carried out by crystal violet colorimetric method. Our results have shown that, when combined together at low concentrations nontoxic to mammalian cells, corresponding to 1/32 MIC (0·39 μg ml⁻¹) and 1/4 MIC (0·06 μg ml⁻¹) for bacillomycin D and amphotericin B, respectively, a clear antibiofilm activity is manifested (95% inhibition of biofilm formation) along with a clear inhibition of germ tube formation. Moreover, the effect of both drugs on preformed biofilm of C. albicans strain was determined using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction assay. The combination of the two antifungal compounds at 0·39 and 1 μg ml⁻¹ for bacillomycin D and amphotericin B, respectively, resulted in a clear enhancement of biofilm eradication compared to the results obtained with each drug alone. Furthermore, this combination was found to promote the closure of a gap produced in a monolayer of human keratinocytes. CONCLUSIONS: Bacillomycin D and its combination with amphotericin B display impressive anti‐biofilm and wound‐healing activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of the lipopeptide bacillomycin D and the antifungal drug amphotericin B in medical devices may offer a promising alternative for topical treatment of Candida‐associated infections in the setting of a wound.
Antimicrobial peptides are effector molecules of the innate immune response of all pluricellular organisms, providing them with first-line defence against pathogens. Amphibian skin secretions ...represent one of the richest natural sources for such peptide antibiotics, and temporins, a large family of antimicrobial peptides from frog skin, are among the smallest ones found in nature to date. Their functional role and modes of action have been described, along with their interesting and unique properties. These properties make temporins good molecules for an in-depth understanding of host defence peptides in general. Furthermore, they are attractive templates for the future design of new therapeutics against infectious diseases with new modes of action, urgently needed due to the increasing resistance of microorganisms to the available drugs.
Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have ...characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores (“carpet” mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.
Temporins are a group of closely related short antimicrobial peptides from frog skin. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, plays important ...roles in the activity of temporins. Earlier studies have found that LPS induces oligomerization of temporin-1Tb (TB) thus preventing its translocation across the outer membrane and, as a result, reduces its activity on gram-negative bacteria. On the other hand, temporin-1Tl (TL) exhibits higher activity, presumably because of lack of such oligomerization. A synergistic mechanism was proposed, involving TL and TB in overcoming the LPS-mediated barrier. Here, to gain insights into interactions of TL and TB within LPS, we investigated the structures and interactions of TL, TB, and TL+TB in LPS micelles, using NMR and fluorescence spectroscopy. In the context of LPS, TL assumes a novel antiparallel dimeric helical structure sustained by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. By contrast, independent TB has populations of helical and aggregated conformations in LPS. The LPS-induced aggregated states of TB are largely destabilized in the presence of TL. Saturation transfer difference NMR studies have delineated residues of TL and TB in close contact with LPS and enhanced interactions of these two peptides with LPS, when combined together. Fluorescence resonance energy transfer and (31)P NMR have pointed out the proximity of TL and TB in LPS and conformational changes of LPS, respectively. Importantly, these results provide the first structural insights into the mode of action and synergism of antimicrobial peptides at the level of the LPS-outer membrane.
Treatment of respiratory viral infections remains a global health concern, mainly due to the inefficacy of available drugs. Therefore, the discovery of novel antiviral compounds is needed; in this ...context, antimicrobial peptides (AMPs) like temporins hold great promise. Here, we discovered that the harmless temporin G (TG) significantly inhibited the early life‐cycle phases of influenza virus. The in vitro hemagglutinating test revealed the existence of TG interaction with the viral hemagglutinin (HA) protein. Furthermore, the hemolysis inhibition assay and the molecular docking studies confirmed a TG/HA complex formation at the level of the conserved hydrophobic stem groove of HA. Remarkably, these findings highlight the ability of TG to block the conformational rearrangements of HA2 subunit, which are essential for the viral envelope fusion with intracellular endocytic vesicles, thereby neutralizing the virus entry into the host cell. In comparison, in the case of parainfluenza virus, which penetrates host cells upon a membrane‐fusion process, addition of TG to infected cells provoked ~1.2 log reduction of viral titer released in the supernatant. Nevertheless, at the same condition, an immunofluorescent assay showed that the expression of viral hemagglutinin/neuraminidase protein was not significantly reduced. This suggested a peptide‐mediated block of some late steps of viral replication and therefore the impairment of the extracellular release of viral particles. Overall, our results are the first demonstration of the ability of an AMP to interfere with the replication of respiratory viruses with a different mechanism of cell entry and will open a new avenue for the development of novel therapeutic approaches against a large variety of respiratory viruses, including the recent SARS‐CoV2.
In this study we designed and synthesized a new library of antimicrobial peptides correlated to Pro3,DLeu9TL 1, a temporin L derivative devoid of cytolytic effects in vitro, and investigated the ...correlation between the α-helical content of the compounds and their antibacterial, cytotoxic and hemolytic activities. We systematically replaced Gly in position 10 of reference peptide with several amino acids. Structure-activity relationship studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with CD spectroscopy analyses. NMR analysis was also accomplished for compound 10. As well, the most promising peptides were additionally evaluated for their activity against some clinical strains isolated from human skin and for their mechanism of action by studying the kinetics of membrane perturbation of some representative microbial strains. We identified novel analogues with interesting properties that make them attractive lead compounds for potential topical applications.
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•Novel antimicrobial temporin L analogues were designed and synthesized.•Their conformation was correlated with antibacterial, cytotoxic and hemolytic activities.•Replacement of glycine with other residues yielded interesting derivatives.•SAR information were enriched by antimicrobial and cytotoxicity assays.•CD and NMR spectroscopy analyses supported the relevance of certain compounds.
The antimicrobial peptide esculentin 1–21 (Esc 1–21) is a shorter synthetic version of the 46-residue peptide occurring in the
Rana esculenta skin secretion. Here we propose an integrated proteomic ...and transcriptomic approach to interpret the biological effects of this peptide on
Saccharomyces cerevisiae. We further investigated the response to this peptide by correlating the results of the transcriptome and proteome analysis with phenotypic effects. The results show that
S. cerevisiae adapts to Esc 1–21 using the High Osmolarity Glycerol (HOG) pathway involved in osmotic tolerance and cell wall maintenance. Comparative proteomics reveals that Esc 1–21 causes downregulation of enzymes of the lower glycolytic pathway and in genes involved in spindle body formation and remodelling of cell-wall synthesis. Moreover the peptide induces downexpression of protein actin within 45 min and cells pre-treated with peptide show less sensitivity to osmotic stress and increased sensitivity to heat shock stress. The results obtained with the two different methodologies are in agreement at the cellular process levels. A combined approach may help elucidate the main aspects related to the effects of this peptide on the eukaryotic cell. The employment of different technologies may reveal the potential and limitations of each adapted approach in a prospective application for drug screening.
Objective: Non-invasive testing often does not identify coronary artery disease (CAD) in diabetic subjects. This study was designed in order to examine the prevalence of CAD in a cohort of ...asymptomatic type 2 diabetic patients at high cardiovascular risk and negative nuclear imaging, using multi-slice computed tomography (MSCT) angiography.
Methods: In total, 770 type 2 diabetic patients were screened from January 2008 through July 2010. Of these, 132 Caucasians with diabetic nephropathy and asymptomatic for angina were eligible for a cross-sectional study. Patients underwent MSCT after ischaemia was excluded by myocardial Single Photon Emission Computed Tomography (SPECT) at rest and after dynamic exercise. When obstructive plaques were found (≥50% lumen narrowing), patients were sent to conventional coronary angiography (CCA).
Results: Six subjects were not included in the analysis because of motion artefacts. MSCT was positive for CAD in 114 patients (90%). Within patients with positive MSCT, 60 (48% of all) showed one or more obstructive plaques. CCA confirmed significant stenosis (≥50%) in 48 of these 60 patients (80%). Some 21 (35%) showed stenosis ≥75% and were submitted to the revascularisation procedure.
Conclusion: MSCT seems to better identify CAD than myocardial SPECT in asymptomatic patients with type 2 diabetes and diabetic nephropathy.
ABSTRACT
Secretions of amphibian skin glands contain numerous antimicrobial peptides that play a crucial role in the defense against microorganisms. The location of these glands on the surface of the ...animal makes them a useful model for in vivo studies of the relationships between the innate immune system and the natural flora. Here, we present the results of a study showing that in Rana esculenta the total antimicrobial activity of skin secretion is modulated by the presence of the natural flora. Frogs kept in a sterile environment do not produce antimicrobial peptides. This finding represents the first in vivo demonstration of the induction of defense peptides in a vertebrate. We also present data on the morphological changes in skin glands of animals kept in sterile conditions or treated with glucocorticoids. Cells from glands regenerated under normal conditions, but not those from “sterile” frogs, contain κB‐binding activity in the cytoplasm.
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