Search

Temporin family is one of the largest among antimicrobial peptides (AMPs), which act mainly by penetrating and disrupting the bacterial membranes. To further understand the relationship between the physical-chemical properties and their antimicrobial activity and selectivity, an analogue of Temporin L, [Nle1, dLeu9, dLys10]TL (Nle-Phe-Val-Pro-Trp-Phe-Lys-Phe-dLeu-dLys-Arg-Ile-Leu-CONH2) has been developed in the present work. The design strategy consisted of the addition of a norleucine residue at the N-terminus of the lead peptide sequence, [dLeu9, dLys10]TL, previously developed by our group. This modification promoted an increase of peptide hydrophobicity and, interestingly, more efficient activity against both Gram-positive and Gram-negative strains, without affecting human keratinocytes and red blood cells survival compared to the lead peptide. Thus, this novel compound was subjected to biophysical studies, which showed that the peptide [Nle1, dLeu9, dLys10]TL is unstructured in water, while it adopts β-type conformation in liposomes mimicking bacterial membranes, in contrast to its lead peptide forming α-helical aggregates. After its aggregation in the bacterial membrane, [Nle1, dLeu9, dLys10]TL induced membrane destabilization and deformation. In addition, the increase of peptide hydrophobicity did not cause a loss of anti-inflammatory activity of the peptide [Nle1, dLeu9, dLys10]TL in comparison with its lead peptide. In this study, our results demonstrated that positive net charge, optimum hydrophobic−hydrophilic balance, and chain length remain the most important parameters to be addressed while designing small cationic AMPs. [ABSTRACT FROM AUTHOR]

Temporin family is one of the largest among antimicrobial peptides (AMPs), which act mainly by penetrating and disrupting the bacterial membranes. To further understand the relationship between the physical-chemical properties and their antimicrobial activity and selectivity, an analogue of Temporin L, [Nle1, dLeu9, dLys10]TL (Nle-Phe-Val-Pro-Trp-Phe-Lys-Phe-dLeu-dLys-Arg-Ile-Leu-CONH2) has been developed in the present work. The design strategy consisted of the addition of a norleucine residue at the N-terminus of the lead peptide sequence, [dLeu9, dLys10]TL, previously developed by our group. This modification promoted an increase of peptide hydrophobicity and, interestingly, more efficient activity against both Gram-positive and Gram-negative strains, without affecting human keratinocytes and red blood cells survival compared to the lead peptide. Thus, this novel compound was subjected to biophysical studies, which showed that the peptide [Nle1, dLeu9, dLys10]TL is unstructured in water, while it adopts β-type conformation in liposomes mimicking bacterial membranes, in contrast to its lead peptide forming α-helical aggregates. After its aggregation in the bacterial membrane, [Nle1, dLeu9, dLys10]TL induced membrane destabilization and deformation. In addition, the increase of peptide hydrophobicity did not cause a loss of anti-inflammatory activity of the peptide [Nle1, dLeu9, dLys10]TL in comparison with its lead peptide. In this study, our results demonstrated that positive net charge, optimum hydrophobic−hydrophilic balance, and chain length remain the most important parameters to be addressed while designing small cationic AMPs.

Temporins are one of the largest families of antimicrobial peptides with both anti-inflammatory and antimicrobial activity. Herein, for a panel of cyclic temporin L isoform analogues, the antifungal and antibiofilm activities were determined against representative Candida strains, including C. albicans, C. glabrata, C. auris, C. parapsilosis and C. tropicalis. The outcomes indicated a significant anti-candida activity against planktonic and biofilm growth for four peptides (3, 7, 15 and 16). The absence of toxicity up to high concentrations and survival after infection were assessed in vivo by using Galleria mellonella larvae, and the correlation between conformation and cytotoxicity was investigated by fluorescence assays and circular dichroism (CD). By combining fluorescence spectroscopy, CD, dynamic light scattering, confocal and atomic force microscopy, the mode of action of four analogues was hypothesized. The results pinpointed that peptide 3 emerged as a non-toxic compound showing a potent antibiofilm activity and represents a promising compound for biomedical applications.

Temporins are a family of antimicrobial peptides (AMPs) isolated from frog skin, which are very short, weakly charged, and highly hydrophobic. They execute bactericidal activities in different ways from many other AMPs. This work investigated morphological changes of planar bilayer membranes composed of mixed zwitterionic and anionic phospholipids induced by temporin B and L (TB and TL) using all-atom and coarse-grained molecular dynamics simulations. We found that TB and TL fold to α-helices at the membrane surface and penetrate shallowly into the bilayer. These short AMPs have low propensity to induce membrane pore formation but possess high ability to extract lipids out. At relatively high peptide concentrations, the strong hydrophobicity of TB and TL promotes them to aggregate into clusters on the membrane surface. These aggregates attract a large amount of lipids out of the membrane to release compression induced by other dispersed peptides binding to the membrane. The extruded lipids mix evenly with the peptides in the cluster and form tubule-like protrusions. Certain water molecules follow the movement of lipids, which not only fill the cavities of the protrusion but also assist in maintaining the tubular structures. In contrast, the peptide-free leaflet remains intact. The present results unravel distinctive antimicrobial mechanisms of temporins disturbing membranes.

Interkingdom polymicrobial diseases are caused by different microorganisms that colonize the same niche, as in the case of yeast-bacteria coinfections. The latter are difficult to treat due the absence of any common therapeutic target for their elimination, both in animals and humans. Staphylococcus pseudintermedius and Malassezia pachydermatis belong to distinct kingdoms. They can colonize the same skin district or apparatus being the causative agents of fastidious pet animals’ pathologies. Here we analysed the antimicrobial properties of a panel of 11 peptides, derived from temporin L, against Malassezia pachydermatis. Only peptide 8 showed the best mycocidal activity at 6.25 μM. Prolonged application of peptide 8 did not cause M. pachydermatis drug-resistance. Peptide 8 was also able to inhibit the growth of Staphylococcus pseudintermedius, regardless of methicillin resistance, at 1.56 μM for methicillin-susceptible S. pseudintermedius (MSSP) and 6.25 μM for methicillin-resistant S. pseudintermedius (MRSP). Of interest, peptide 8 increased the susceptibility of MRSP to oxacillin. Oxacillin MIC value reduction was of about eight times when used in combination with peptide 8. Finally, the compound affected the vitality of bacteria embedded in S. pseudintermedius biofilm. In conclusion, peptide 8 might represent a valid therapeutic alternative in the treatment of interkingdom polymicrobial infections, also in the presence of methicillin-resistant bacteria.

Antimicrobial peptides (AMPs) are the promising future therapeutic candidates because of their multifunctional roles and unique mode of action against microbes. Despite several advantages, developing AMPs into therapeutic antibiotics is often associated with limitations, such as thermal and enzymatic stability, moderate antimicrobial activity and higher toxicity. We here report the synthesis of 2-fluoro- and 2,6-difluorophenyalanine, their introduction into naturally occurring antimicrobial peptide Temporin L (TL). We also report the antimicrobial and hemolytic activity of parent TL as well as the fluorinated variant in plasma and buffer conditions. Circular dichroism studies clearly show that fluorination reduces the helical propensity, thus accounting for lower activity. We further demonstrated that fluorinated TL can act as antimicrobial agents in creams and gels used for treating skin infections. Graphical Abstract: [ABSTRACT FROM AUTHOR]

[Display omitted] • L9βl-TL showed higher proteolytic stability, therapeutic index and activity against MRSA biofilm than Temporin-L (TL). • Though unordered in bacterial membrane-mimetic lipid vesicles, L9βl-TL depolarized these vesicles and live MRSA. • The results show the design of a TL-analogue with higher therapeutic potential by impairing its secondary structure. Leucine and glycine residues, at the 9th and 10th positions of helical domain of naturally occurring antimicrobial peptide (AMP), Temporin L were substituted with an unnatural amino acid, β-leucine (homovaline) to improve its serum protease stability, haemolytic/cytotoxic properties and reduce the size to some extent. The designed analogue, L9βl-TL showed either equal or improved antimicrobial activity to TL against different microorganisms including the resistant strains. Interestingly, L9βl-TL also exhibited lower haemolytic and cytotoxic activities against human red blood cells and 3T3 cells, respectively. Moreover, L9βl-TL showed antibacterial activity in presence of 25% (v/v) human serum and showed resistance against proteolytic cleavage in presence of it that suggested the serum protease stability of the TL-analogue. L9βl-TL exhibited un-ordered secondary structures in both bacterial and mammalian membrane mimetic lipid vesicles as compared to the helical structures of TL in these environments. However, tryptophan fluorescence studies demonstrated more selective interaction of L9βl-TL with bacterial membrane mimetic lipid vesicles in comparison to non-selective interactions of TL with both kinds of lipid vesicles. Membrane depolarization studies with live MRSA and bacterial membrane-mimetic lipid vesicles suggested a membrane-disrupting mode of action of L9βl-TL. L9βl-TL showed faster bactericidal mechanism compared to TL against MRSA. Interestingly, L9βl-TL was found as more potent than TL either in inhibiting biofilm formation or in eradicating the mature biofilm formed by MRSA. Overall, the present work demonstrates a simple and useful strategy to design of an analogue of TL, with minimal modifications while maintaining its antimicrobial activity with lesser toxicity and higher stability which could be attempted for other AMPs as well. [ABSTRACT FROM AUTHOR]

Abstract: The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro3]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6–13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro3]TL with the corresponding d isomers, known as helix breakers. Structure–activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro3]TL, without giving a toxic effect towards human cells. [Copyright &y& Elsevier]

Abstract: In order to enhance the membrane disruption of antimicrobial peptides both targeting and multivalent presentation approaches were explored. The antimicrobial peptides anoplin and temporin L were conjugated via click chemistry to vancomycin and to di- and tetravalent dendrimers. The vancomycin unit led to enhanced membrane disruption of large unilamellar vesicles (LUVs) displaying the vancomycin target lipid II, but only for temporin L and not for anoplin. The multivalent presentation led to enhanced LUV membrane disruption in the case of anoplin but not for temporin L. [Copyright &y& Elsevier]

Temporins are one of the largest families of antimicrobial peptides with both anti-inflammatory and antimicrobial activity. Herein, for a panel of cyclic temporin L isoform analogues, the antifungal and antibiofilm activities were determined against representative Candida strains, including C. albicans, C. glabrata, C. auris, C. parapsilosis and C. tropicalis. The outcomes indicated a significant anti-candida activity against planktonic and biofilm growth for four peptides (3, 7, 15 and 16). The absence of toxicity up to high concentrations and survival after infection were assessed in vivo by using Galleria mellonella larvae, and the correlation between conformation and cytotoxicity was investigated by fluorescence assays and circular dichroism (CD). By combining fluorescence spectroscopy, CD, dynamic light scattering, confocal and atomic force microscopy, the mode of action of four analogues was hypothesized. The results pinpointed that peptide 3 emerged as a non-toxic compound showing a potent antibiofilm activity and represents a promising compound for biomedical applications. [ABSTRACT FROM AUTHOR]

Over the years, the increasing acquisition of antibiotic resistance genes has led to the emergence of highly resistant bacterial strains and the loss of standard antibiotics' efficacy, including β-lactam/β-lactamase inhibitor combinations and the last line carbapenems. Klebsiella pneumoniae is considered one of the major exponents of a group of multidrug-resistant ESKAPE pathogens responsible for serious healthcare-associated infections. In this study, we proved the antimicrobial activity of two analogues of Temporin L against twenty carbapenemase-producing K. pneumoniae clinical isolates. According to the antibiotic susceptibility assay, all the K. pneumoniae strains were resistant to at least one other class of antibiotics, in addition to beta-lactams. Peptides 1B and C showed activity on all test strains, but the lipidated analogue C expressed the greater antimicrobial properties, with MIC values ranging from 6.25 to 25 µM. Furthermore, the peptide C showed bactericidal activity at MIC values. The results clearly highlight the great potential of antimicrobial peptides both as a new treatment option for difficult-to-treat infections and as a new strategy of drug-resistance control. [ABSTRACT FROM AUTHOR]

Temporins are a family of antimicrobial peptides (AMPs) isolated from frog skin, which are very short, weakly charged, and highly hydrophobic. They execute bactericidal activities in different ways from many other AMPs. This work investigated morphological changes of planar bilayer membranes composed of mixed zwitterionic and anionic phospholipids induced by temporin B and L (TB and TL) using all-atom and coarse-grained molecular dynamics simulations. We found that TB and TL fold to α-helices at the membrane surface and penetrate shallowly into the bilayer. These short AMPs have low propensity to induce membrane pore formation but possess high ability to extract lipids out. At relatively high peptide concentrations, the strong hydrophobicity of TB and TL promotes them to aggregate into clusters on the membrane surface. These aggregates attract a large amount of lipids out of the membrane to release compression induced by other dispersed peptides binding to the membrane. The extruded lipids mix evenly with the peptides in the cluster and form tubule-like protrusions. Certain water molecules follow the movement of lipids, which not only fill the cavities of the protrusion but also assist in maintaining the tubular structures. In contrast, the peptide-free leaflet remains intact. The present results unravel distinctive antimicrobial mechanisms of temporins disturbing membranes. [ABSTRACT FROM AUTHOR]

Interkingdom polymicrobial diseases are caused by different microorganisms that colonize the same niche, as in the case of yeast-bacteria coinfections. The latter are difficult to treat due the absence of any common therapeutic target for their elimination, both in animals and humans. Staphylococcus pseudintermedius and Malassezia pachydermatis belong to distinct kingdoms. They can colonize the same skin district or apparatus being the causative agents of fastidious pet animals' pathologies. Here we analysed the antimicrobial properties of a panel of 11 peptides, derived from temporin L, against Malassezia pachydermatis. Only peptide 8 showed the best mycocidal activity at 6.25 μM. Prolonged application of peptide 8 did not cause M. pachydermatis drug-resistance. Peptide 8 was also able to inhibit the growth of Staphylococcus pseudintermedius, regardless of methicillin resistance, at 1.56 μM for methicillin-susceptible S. pseudintermedius (MSSP) and 6.25 μM for methicillin-resistant S. pseudintermedius (MRSP). Of interest, peptide 8 increased the susceptibility of MRSP to oxacillin. Oxacillin MIC value reduction was of about eight times when used in combination with peptide 8. Finally, the compound affected the vitality of bacteria embedded in S. pseudintermedius biofilm. In conclusion, peptide 8 might represent a valid therapeutic alternative in the treatment of interkingdom polymicrobial infections, also in the presence of methicillin-resistant bacteria. [ABSTRACT FROM AUTHOR]

The comprehension of the mechanism of action of antimicrobial peptides is fundamental for the design of new antibiotics. Studies performed looking at the interaction of peptides with bacterial cells offer a faithful picture of what really happens in nature. In this work we focused on the interaction of the peptide Temporin L with E. coli cells, using a variety of biochemical and biophysical techniques that include: functional proteomics, docking, optical microscopy, TEM, DLS, SANS, fluorescence. We identified bacterial proteins specifically interacting with the peptides that belong to the divisome machinery; our data suggest that the GTPase FtsZ is the specific peptide target. Docking experiments supported the FtsZ-TL interaction; binding and enzymatic assays using recombinant FtsZ confirmed this hypothesis and revealed a competitive inhibition mechanism. Optical microscopy and TEM measurements demonstrated that, upon incubation with the peptide, bacterial cells are unable to divide forming long necklace-like cell filaments. Dynamic light scattering studies and Small Angle Neutron Scattering experiments performed on treated and untreated bacterial cells, indicated a change at the nanoscale level of the bacterial membrane. The peptide temporin L acts by a non-membrane-lytic mechanism of action, inhibiting the divisome machinery. Identification of targets of antimicrobial peptides is pivotal to the tailored design of new antimicrobials. Unlabelled Image • The mechanism of action of TL is investigated by a functional proteomic approach. • The morphology of bacterial cells upon interaction with TL is detected by SANS. • TL inhibits the cell division process, by binding to FtsZ. [ABSTRACT FROM AUTHOR]

Cytotoxic frog antimicrobial peptide Temporin L (TempL) is an attractive molecule for the design of lead antimicrobial agents due to its short size and versatile biological activities. However, noncytotoxic TempL variants with desirable biological activities have rarely been reported. TempL analogue Q3K,TempL is water-soluble and possesses a significant antiendotoxin property along with comparable cytotoxicity to TempL. A phenylalanine residue, located at the hydrophobic face of Q3K,TempL and the "d" position of its phenylalanine zipper sequence, was replaced with a cationic lysine residue. This analogue, Q3K,F8K,TempL, showed reduced hydrophobic moment and was noncytotoxic with lower antimicrobial activity. Interestingly, swapping between tryptophan at the fourth and serine at the sixth positions turned Q3K,F8K,TempL totally amphipathic as reflected by its helical wheel projection with clusters of hydrophobic and hydrophilic residues and the highest hydrophobic moment among these peptides. Surprisingly, this analogue, SW,Q3K,F8K,TempL, was as noncytotoxic as Q3K,F8K,TempL but showed augmented antimicrobial and antiendotoxin properties, comparable to that of TempL and Q3K,TempL. SW,Q3K,F8K,TempL exhibited appreciable survival of mice against P. aeruginosa infection and a lipopolysaccharide (LPS) challenge. Unlike TempL and Q3K,TempL, SW,Q3K,F8K,TempL adopted an unordered secondary structure in bacterial membrane mimetic lipid vesicles and did not permeabilize them or depolarize the bacterial membrane. Overall, the results demonstrate the design of a nontoxic TempL analogue that possesses clusters of hydrophobic and hydrophilic residues with impaired secondary structure and shows a nonmembrane-lytic mechanism and in vivo antiendotoxin and antimicrobial activities. This paradigm of design of antimicrobial peptide with clusters of hydrophobic and hydrophilic residues and high hydrophobic moment but low secondary structure could be attempted further.

A 13-residue frog antimicrobial peptide Temporin L (TempL) possesses versatile antimicrobial activities and is considered a lead molecule for the development of new antimicrobial agents. To find out the amino acid sequences that influence the anti-microbial property of TempL, a phenylalanine zipper-like sequence was identified in it which was not reported earlier. Several alanine-substituted analogs and a scrambled peptide having the same composition of TempL were designed for evaluating the role of this motif. To investigate whether leucine residues instead of phenylalanine residues at 'a' and/or 'd' position(s) of the heptad repeat sequence could alter its antimicrobial property, several TempL analogs were synthesized after replacing these phenylalanine residues with leucine residues. Replacing phenylalanine residues with alanine residues in the phenylalanine zipper sequence significantly compromised the anti-endotoxin property of TempL. This is evident from the higher production of tumor necrosis factor-α and interleukin-6 in lipopolysaccharide (LPS)-stimulated rat bone-marrow-derived macrophage cells in the presence of its alanine-substituted analogs than TempL itself. However, replacement of these phenylalanine residues with leucine residues significantly augmented anti-endotoxin property of TempL. A single alanine-substituted TempL analog (F8A-TempL) showed significantly reduced cytotoxicity but retained the antibacterial activity of TempL, while the two single leucine-substituted analogs (F5L-TempL and F8L-TempL), although exhibiting lower cytotoxicity, were able to retain the antibacterial activity of the parent peptide. The results demonstrate how minor amino acid substitutions in the identified phenylalanine zipper sequence in TempL could yield analogs with better antibacterial and/or anti-endotoxin properties with their plausible mechanism of action., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)