Temporin G, an amphibian antimicrobial peptide against influenza and parainfluenza respiratory viruses: Insights into biological activity and mechanism of action.

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.

Binding of an antimicrobial peptide to bacterial cells: Interaction with different species, strains and cellular components.

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.

The Amphibian Antimicrobial Peptide Temporin B Inhibits In Vitro Herpes Simplex Virus 1 Infection.

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 in vitro 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.

Bacillomycin D and its combination with amphotericin B: promising antifungal compounds with powerful antibiofilm activity and wound-healing potency.

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.

Alpha-defensin increase in peripheral blood mononuclear cells from patients with hepatitis C virus chronic infection.

The alpha-defensin genes promoter regions contain a putative nuclear factors of activated T cells (NFAT)-binding site and it is known that hepatitis C virus (HCV) core protein activates the interleukin (IL)-2 gene transcription through the NFAT pathway. The aims of this study were to investigate if HCV affects the alpha-defensin expression in peripheral human mononuclear cells (PBMCs) and to evaluate the existence of a correlation between alpha-defensins and liver damage in patients with chronic hepatitis C. Ninety patients with chronic hepatitis C, 30 with chronic hepatitis B and 25 healthy controls were enrolled. Alpha-defensins were identified and quantified in PBMCs by mass spectrometry, enzyme-linked immunosorbent assay, antibacterial activity and mRNA levels. PBMCs from three patients and controls were stimulated with HCV core protein, hepatitis B virus core antigen and the alpha-defensin mRNAs level was quantified. We found that HCV core protein activates in vitro the alpha-defensin transcription. Alpha-defensin levels in patients with chronic hepatitis C (mean +/- SD = 1.103 +/- 0.765 ng/10(6) cells), chronic hepatitis B (0.53 +/- 0.15) and healthy controls (0.217 +/- 0.09) resulted significantly different (P < 0.001). In patients with chronic hepatitis C, the alpha-defensin levels and antibacterial activity correlate with the liver fibrosis. Our data suggest that HCV induces alpha-defensin expression. The high linear correlation of alpha-defensin levels with advancing fibrosis makes the measure of these peptides a reliable marker to evaluate fibrosis stage.

Temporins, anti-infective peptides with expanding properties.

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.

Structure-function relationships in bombinins H, antimicrobial peptides from Bombina skin secretions.

Skin secretions of amphibia of the Bombina genus contain two families of antimicrobial peptides, the bombinins (bombinin-like peptides) and the bombinins H (H for hydrophobic and hemolytic). The latter family includes a number of peptides containing a D-amino acid in the second position, in addition to their corresponding all L-isomers. The antimicrobial activity of three pairs of bombinin H isomers, H2/H4, H6/H7 and GH-1D/GH-1L, has been investigated. The first two pairs of peptides were actually isolated from the secretion, whereas the third was synthesized according to the sequence deduced from a gene coding for a bombinin-like peptide in Bombina orientalis.

Structure-function relationships of temporins, small antimicrobial peptides from amphibian skin.

Temporins, antimicrobial peptides of 10-13 residues, were isolated from secretions of Rana temporaria [Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M.L. & Barra, D. (1996) Eur. J. Biochem. 242, 788-792]. These molecules are specific to this amphibian species, which is also able to secrete on its skin other antimicrobial peptides similar to those found in different Rana species. The effect of temporins A, B and D (13 residues, net charge +2), and H (10 residues, net charge +1 and +2, respectively) against both artificial membranes of differing lipid composition and bacteria has been investigated in order to gain insight into their mechanisms of action. The results indicate that: the lytic activity of temporins is not greatly affected by the membrane composition; temporins A and B allow the leakage of large-size molecules from the bacterial cells; temporin H renders both the outer and inner membrane of bacteria permeable to hydrophobic substances of low molecular mass; and temporin D, although devoid of antibacterial activity, has a cytotoxic effect on erythrocytes. The results allow important conclusions to be drawn about the minimal structural requirements for lytic efficiency and specificity of temporins.

Expression and activity of cyclic and linear analogues of esculentin-1, an anti-microbial peptide from amphibian skin.

Esculentin-1 is a potent anti-microbial peptide present in minute amounts in skin secretions of Rana esculenta. It contains 46 amino-acid residues and a C-terminal disulfide bridge. We have explored the possibility of producing analogues of this peptide by recombinant expression in Escherichia coli of a fusion protein which is sequestered in inclusion bodies. The peptide of interest has been inserted at the N-terminus of the protein, from which it can be released by cyanogen bromide cleavage. The anti-microbial activities of the recombinant peptide as well as that of a mutant linear form devoid of the disulfide bridge are presented. The recombinant analogues retain the biological activity of the natural peptide, as tested with an inhibition zone assay against a variety of microorganisms. However, experiments on the rate of bacterial killing show that gram-negative bacteria are more sensitive to the peptides than the gram-positive bacterium, the effect of the cyclic peptide being in all cases faster than that of the linear molecule. Moreover, the activity against gram-negative bacteria for both peptides is not affected by salts, whereas the activity against Staphylococcus aureus is lost at high salt concentration.

Experimental infections of Rana esculenta with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora.

Frogs can be useful models for studying the mechanisms that may regulate their natural microbial flora. Their skin glands produce a secretion containing 20-30 different peptides, some antimicrobial some neurotrophic. As they often live in soil or silt that is rich in microbes, they can be expected to be able to prevent or eliminate infections in very short periods of time. The bacterium Aeromonas hydrophila is widely distributed in nature and is considered as part of the natural flora of frogs and many animals, including humans. From an alternative frog strain of A. hydrophila, Bo-3, we isolated a spontaneous and stable mutant (Bo-3N), resistant to nalidixic acid, here used to follow the host-microbe interactions in experimental infection of mouth and skin of Rana esculenta. The skin peptides had been previously isolated, sequenced and cloned. We showed that skin treatment with a glucocorticoid (GC) cream blocked de novo synthesis of these peptides and, simultaneously, prepropeptide mRNAs disappeared while IkappaBalpha was up-regulated. Experimental mouth infections with 20 million cells of A. hydrophila Bo-3N showed that a normal wild frog can eliminate the bacteria from the mouth within 15 min, while a frog pretreated with GC cream for 1 h could not reduce Bo-3N below 3500 colony-forming units (CFU)/5 microl 'saliva'. An in vitro comparison showed that frog blood or serum allowed bacteria to grow, while the skin secretion killed the bacteria within 10 min. Using different enzyme-linked immunosorbent assays (ELISAs) with rabbit anti-Bo-3 serum as a positive control, we were able to rule out immunoglobulin G (IgG) binding to A. hydrophila. An assay for immunoglobulin M (IgM) (or some other serum component) in frog serum showed binding to A. hydrophila only corresponding to a few per cent of the positive control. For skin infections we bathed the frogs for 10 min in an overnight culture of Bo-3N diluted to about 10(7) CFU/ml. Electrical stimulation after the bath showed, for the total secretion, a two to fourfold increase in the antibacterial activity, while a pretreatment with GC cream reduced the activity to about one-third of that of the non-bathed control frog. HPLC analysis of the peptide pattern confirmed these findings. The survival value of antimicrobial peptides have earlier been demonstrated in vivo and in vitro only in Drosophila. The present experiments are the first combined in vivo and in vitro demonstrations of the function of peptide antibiotics in a vertebrate. One such function is involved in the control of the natural flora.

Effect of glucocorticoids on the synthesis of antimicrobial peptides in amphibian skin.

Gene-encoded peptide antibiotics are widespread in insects, plants and vertebrates and confer protection against bacterial and fungal infections. NF-kappaB is an important transcription factor for many immunity-related mammalian proteins and also for insect immune genes. The activity of NF-kappaB is regulated by the interaction with an inhibitor, I kappaB. It was recently demonstrated that glucocorticoids induce the synthesis of I kappaB in human cell lines. So far, all genes for peptide antibiotics have promoter motifs with NF-kappaB binding sites, but its actual function in peptide regulation has been studied only in insects. Here we show that glucocorticoid treatment of the frog Rana esculenta inhibits the transcription of all genes encoding antibacterial peptides by inducing the synthesis of I kappaB alpha. These results suggest that also in vertebrates peptide-mediated innate immunity is controlled by NF-kappaB-regulated transcription.

Temporins, antimicrobial peptides from the European red frog Rana temporaria.

A cDNA library from the skin of Rana temporaria has been screened using a cDNA fragment probe that encodes the signal peptide of the precursor of esculentin from the skin secretion of Rana esculenta. With this approach, the cDNAs encoding the precursors of three peptides were isolated. Subsequently, the peptides predicted from the sequence of the cloned cDNAs as well as several structurally related peptides could be isolated from the skin secretion of R. temporaria. These peptides, which were named temporins, have a length of 10-13 residues and show some sequence similarity to hemolytic peptides isolated from Vespa venom [Argiolas, A. & Pisano, J. J. (1984) J. Biol. Chem. 259, 10106-10111]. Natural and synthetic temporins have antibacterial activity against gram-positive bacteria, but they are not hemolytic. Temporins are the smallest antibacterial peptides hitherto found in nature.